Finally, the first city in the Americas (that we know of) grew about 5,100 years ago, about ten miles from the Pacific Ocean in what is now Peru
I honestly had no idea what the first city of the americas would be and i would not have guessed it to be in the location of peru.
ilk production was a state-controlled industry and there were severe penalties including execution for leaking information about the process.
silk is crazy for the amount of money and control china made off an animal is crazy it shows how the right materials and thinking can make you endless amounts of profit.
While Hierakonpolis was becoming a significant city, an 8,000 to 9,000 year long period known as the African Humid Period or "Green Sahara" was just beginning to come to an end.
i get that the world is always changing when it comes tto temperatures in areas but when you this of a dessert that is really all you think that landscape ever could have been.
Villages, then towns, and finally cities allowed populations to boom, so it could be very reasonably argued that these innovations increased the number of people who could live in a particular region.
this allowed for growth in people and specialty work which greatened craftsman ship and allowed us to prosper more than ever before.
The Indus Valley Civilization's cotton was not only used to clothe its people, but was central to its economy, with cotton textiles exported to Mesopotamia
I find it incredible how cotton wasn't just for clothes but a huge part of their economy.
One advantage Egypt had in its later history, which was not shared by other early cultures, was that it was protected from invasion by geography
unlike other early civilization that had to constantly defend themselves Egypt sea and deserts kind of acted like natural barriers which protected them.
Wild silkworms, which were abundant in the region, were fed on mulberry leaves and then their cocoons were harvested and boiled to extract silk threads
I wonder how they figured out boiling cocoons would give them silk threads?
While there is no clear evidence of palaces, kings, or warfare, Caral had monuments and sunken plazas that seem to have been used for community rituals.
I believe it’s interesting that Caral didn't have clear rulers or places but still built big monuments and irrigation systems. It shows that their society was more about team work.
The Indus culture established a uniform system of weights and seals, which helped standardize trade.
This is cool because the Indus had standard system of weights and seals. It shows how organized their trades were.
Egypt's farmers grew wheat, lentils, chickpeas, and flax (for linen). An kin-based elite emerged in early communities like Hierakonpolis, in upper Egypt
It is so intresting how farming wasn’t just about food but also about making materials like linen from flax.
Synthèse du webinaire : "Créer une communauté d'action : comment se lancer ?"
Résumé
Ce document de synthèse analyse les principaux enseignements du webinaire organisé par le Réseau National des Maisons des Associations (RNMA) et La Fonda, centré sur la création et l'animation de "communautés d'action".
Fondée sur la méthodologie de la stratégie d'impact collectif, cette approche vise à structurer la coopération entre divers acteurs territoriaux pour répondre de manière concertée et transversale à des enjeux complexes.
Les points clés sont les suivants :
1. Une Méthodologie Structurante :
La démarche s'appuie sur des phases claires (impulsion, structuration, pérennisation) et cinq conditions de réussite, dont la plus cruciale est l'existence d'une "fonction de soutien".
Cette fonction, véritable colonne vertébrale de la coopération, est chargée d'animer, de coordonner et de faciliter le travail collectif.
2. Deux Expérimentations Riches d'Enseignements :
Pendant deux ans, des expérimentations ont été menées à Morlaix (portée par le Résam sur la transition écologique et alimentaire) et à Mulhouse (portée par le Carré des associations sur la jeunesse).
Ces deux cas pratiques démontrent la capacité de la méthode à fédérer des acteurs hétérogènes (associations, citoyens, collectivités, institutions) et à produire des résultats concrets, allant de l'amélioration des parcours pour les jeunes à la création de projets sur la sécurité sociale de l'alimentation.
3. Des Résultats Tangibles au-delà des Projets :
L'impact majeur réside dans la transformation des modes de collaboration.
Les expérimentations ont permis de renforcer l'interconnaissance, d'instaurer des relations plus horizontales, et d'ancrer la méthode au sein de politiques publiques locales (Contrat de Ville, Contrat Territorial Global de la CAF à Mulhouse).
4. Le Défi Central du Financement : La pérennisation de ces dynamiques se heurte à un obstacle majeur : le financement de la fonction de soutien.
Les financeurs publics privilégient traditionnellement les "actions concrètes" au détriment du temps d'animation et de coordination, pourtant essentiel à la réussite et à la durabilité des coopérations.
5. Des Perspectives d'Essaimage :
Le webinaire a confirmé un fort intérêt pour la méthode. Les participants ont exprimé des besoins clairs en matière d'outils, de formation et d'échanges entre pairs.
Le RNMA et La Fonda envisagent la création d'une "communauté d'apprentissage" pour accompagner les territoires désireux de se lancer.
En conclusion, la création de communautés d'action représente une voie prometteuse pour renforcer l'impact des initiatives locales.
Sa réussite dépend cependant d'un changement de paradigme, notamment de la part des financeurs, pour reconnaître et soutenir le travail indispensable de mise en lien et d'animation territoriale.
I. Le Cadre Méthodologique : La Stratégie d'Impact Collectif
La démarche de création de communautés d'action s'ancre dans l'approche de la stratégie d'impact collectif, présentée comme un cadre permettant de structurer et de pérenniser la coopération sur un territoire.
A. Définition et Promesse
L'impact collectif est défini comme la capacité à mettre en cohérence des actions et à favoriser des dynamiques concertées pour apporter des réponses adaptées et transversales à des enjeux complexes et partagés.
La promesse de cette approche est de générer des réponses de meilleure qualité, car elles sont construites en commun autour d'objectifs partagés.
Le postulat de départ est que les enjeux territoriaux (du quartier à l'international) sont multifactoriels et appellent à la coopération.
L'objectif n'est pas de multiplier les projets, mais de relier ce qui existe déjà pour aligner les actions vers un changement souhaité, défini collectivement.
B. Les Étapes et Conditions de Réussite
La mise en œuvre d'une stratégie d'impact collectif suit trois grandes phases :
1. Impulsion : Définition et partage de l'enjeu.
2. Structuration : Mise en commun et coordination des moyens et des ressources.
3. Pérennisation : Ancrage de la dynamique dans la durée.
Cinq conditions sont identifiées comme nécessaires à la réussite de la démarche :
• Une vision partagée des enjeux.
• Des actions qui se complètent mutuellement autour d'un plan d'action partagé.
• Une approche de l'évaluation pensée dès le début et conduite en commun.
• Une communication continue entre les acteurs.
• L'existence d'une "fonction de soutien" dédiée.
C. La "Fonction de Soutien" : Clé de Voûte de la Coopération
La "fonction de soutien" est l'acteur ou la structure en charge d'animer et de faire vivre la coopération.
Elle est décrite comme la colonne vertébrale de la démarche.
Missions Clés :
Postures et Rôles :
• Écoute : Pour comprendre les besoins et favoriser l'inclusion.
• Coordinateur : Pour structurer les échanges et veiller au bon déroulement des actions.
• Animateur : Adopter une "posture haute" pour affirmer une position dans certaines décisions, ou une "posture basse" pour favoriser la prise d'initiative des membres.
Cette fonction assure également un important travail "caché" entre les réunions : préparation des ordres du jour, rédaction des comptes-rendus et des documents stratégiques.
II. Retours d'Expérience : Les Communautés d'Action de Morlaix et Mulhouse
Deux territoires ont expérimenté cette méthode pendant deux ans, avec l'accompagnement du RNMA et de La Fonda.
A. L'Expérimentation de Morlaix (Résam) : La Transition Alimentaire
• Structure porteuse : Le Résam (Réseau d'échange et de service aux associations du Pays de Morlaix), une maison des associations associative à gouvernance partagée.
• Genèse du Projet : Le projet est né d'une demande des associations locales autour des transitions écologiques et d'un besoin de l'équipe du Résam de renouveler ses pratiques d'animation territoriale. Le contexte local, marqué par une pénurie d'eau inédite, a renforcé la pertinence du sujet.
• Déroulement et Acteurs : Sur une thématique large des "transitions écologiques", la communauté d'action a progressivement affiné son objet pour se concentrer sur l'autonomisation alimentaire du territoire respectueuse du vivant.
La dynamique a rassemblé un groupe hétérogène de citoyens, associations, entrepreneurs, techniciens de collectivités et élus, animé via des journées complètes de travail tous les deux mois. Un comité de pilotage a été formé avec le Résam, le Pôle ESS local et l'Ulamir CPIE.
• Difficultés Rencontrées :
◦ Le décalage de temporalité avec les politiques publiques locales, notamment le Plan Alimentaire Territorial en cours d'élaboration.
• Réussites et Résultats :
◦ Une forte coordination et des habitudes de travail communes installées entre les trois structures de la fonction de soutien.
◦ Une démarche perçue comme novatrice sur le territoire, réussissant à structurer un dialogue entre des acteurs variés.
◦ Une forte dimension humaine, avec un plaisir partagé des participants à se retrouver.
◦ La validation d'une charte commune après un travail collectif approfondi.
◦ Le lancement d'une expérimentation sur la sécurité sociale de l'alimentation.
◦ Une reconnaissance croissante de la "communauté d'action" sur le territoire.
B. L'Expérimentation de Mulhouse (Carré des associations) : L'Accompagnement de la Jeunesse
• Structure porteuse : Le Carré des associations, une maison des associations municipale, intégrée à la Direction cohésion sociale et vie des quartiers de la Ville de Mulhouse.
• Genèse du Projet : Le constat d'un territoire très dynamique mais peinant à pérenniser ses expérimentations et à faire travailler ses acteurs ensemble.
La ville de Mulhouse, très jeune et marquée par de fortes disparités sociales, a vu dans la méthode un moyen de créer du lien et d'améliorer l'accompagnement des initiatives.
• Déroulement et Acteurs : Partie d'un enjeu général d'accompagnement de projet, la communauté d'action s'est rapidement focalisée sur la jeunesse.
Elle a rassemblé des partenaires institutionnels variés : services de la Ville, État (politique de la ville), CAF, Alsace Active, Unicité, Mission Locale, etc.
Un principe clé a été l'adoption d'une posture horizontale, où la Ville n'est pas un décideur mais un partenaire égal aux autres.
• Difficultés Rencontrées :
◦ Le départ de certains partenaires (État, France Active) par manque de temps ou d'alignement avec la thématique.
◦ L'émergence récente de la question de la rémunération du temps de travail investi par les partenaires associatifs, posant un défi pour la pérennisation.
• Réussites et Résultats :
◦ L'amélioration de la connaissance mutuelle et la transformation des relations hiérarchiques en coopération horizontale.
◦ La méthode a nourri les pratiques de chaque structure partenaire (projets sociaux des centres sociaux, par exemple).
◦ Des passerelles concrètes ont été créées, fluidifiant les parcours des jeunes entre les différentes structures (ex: lien entre le budget participatif de la ville et les jeunes suivis par Unicité).
◦ La méthode a été inscrite dans des cadres structurants comme le nouveau Contrat de Ville et le Contrat Territorial Global (CTG) de la CAF.
◦ Un poste pour la fonction de soutien est envisagé dans le cadre du CTG, offrant une voie de pérennisation.
III. Outils et Méthodes Clés
Deux outils structurants utilisés lors des expérimentations ont été présentés.
A. Le Scénario Idéal : Construire une Vision Partagée
Cet outil de prospective a été utilisé en phase d'impulsion pour définir une vision et une problématique communes.
• Objectif : Aider le groupe à se projeter dans un futur souhaitable pour identifier les enjeux prioritaires et se mettre d'accord sur un vocabulaire commun.
• Processus :
B. La Cartographie des Acteurs : Rendre Visible et Relier l'Existant
Cet outil, utilisé en phase de structuration, vise à capitaliser sur l'existant plutôt qu'à créer de nouveaux dispositifs.
• Objectif : Rendre lisible comment les acteurs présents répondent déjà à l'enjeu partagé, identifier les complémentarités et les "trous dans la raquette".
• Processus :
• Utilité : La cartographie devient une boussole pour les professionnels, facilitant l'orientation des publics entre les différentes structures et renforçant la coopération opérationnelle.
IV. Enjeux et Perspectives pour l'Essaimage
La fin du webinaire a ouvert une discussion sur la transférabilité de la démarche.
A. Les Besoins des Acteurs de Terrain
Les participants ont exprimé un vif intérêt et ont formulé plusieurs besoins pour se lancer :
• Accès aux outils : Le site ressource en préparation par le RNMA est attendu.
• Formation : Des temps dédiés pour s'approprier la méthode de manière plus approfondie.
• Échange entre pairs et codéveloppement : Un espace pour partager les expériences, les difficultés et les réussites dans la durée.
B. Le Défi du Financement de la "Fonction de Soutien" C'est l'enjeu principal qui a émergé. Les financements publics sont souvent fléchés vers des actions visibles et quantifiables, rendant difficile la valorisation du temps d'ingénierie, de coordination et d'animation.
Or, sans ce temps dédié, les dynamiques coopératives peinent à se maintenir.
Les intervenants ont qualifié la reconnaissance de ce travail de "combat politique" à mener, tant par les associations que par les collectivités, pour démontrer que l'animation territoriale est une condition essentielle de l'impact des politiques publiques.
C. Vers une Communauté d'Apprentissage
En réponse aux besoins exprimés, le RNMA et La Fonda proposent de lancer une communauté d'apprentissage.
L'objectif serait de créer un espace d'échange de pratiques et de soutien pour les structures souhaitant impulser des communautés d'action sur leur territoire, que ce soit dans le cadre de Guide'Asso, de politiques alimentaires ou d'autres thématiques.
Des rencontres futures, notamment avec des acteurs de la philanthropie comme la Fondation de France, sont envisagées pour faire avancer la réflexion sur le financement de ces transformations systémiques.
Rotating crops and using fields year-round allowed Mohenjo-Daro to grow to about 60,000 and Harappa to reach 40,000 by about 4,500 years ago. Several other less-well-known cities may have reached similar sizes.
knowing that thy need to rotate crops to have plentiful harvest is crazy we know now today that it deals with having to put nitrogen and other things back it the soil from crops that rob it like corn.
The Indus culture established a uniform system of weights and seals, which helped standardize trade.
I find it interesting how they made a standardized trade system so early on in history
(Ethel Waters, Bing Crosby)
The list here overlooks several other pivotal early women in jazz worth mentioning:
Marion Harris ** (1897 – 1944) - widely recognized as the first woman to record in front of a big band with “When I Hear That Jazz Band Play” with the (Edward T. ) King's Orchestra on Victor Records (1917). 
** Alberta Hunter** (1895—1984) - began singing at the Panama Club and then Chicago’s Dreamland Ballroom in 1917; soon after performed with King Joe Oliver; recorded with Louis Armstrong and Sidney Bechet in 1924.
** Ada “Bricktop” Smith ** (1894-1984) - a key figure in Chicago and later Paris, known for getting Duke Ellington and Claude Hopkins booked into the Baron’s Exclusive Club.
Cora “Lovie” Austin (1887–1972) — Chicago bandleader, pianist, composer, and arranger; led the Blues Serenaders (with Kid Ory and Johnny Dodds) in the 1920s and became one of the most respected session musicians of her era.
La Démarche d'Observation Locale de la Vie Associative (OLVA)
Résumé
La démarche d'Observation Locale de la Vie Associative (OLVA), pilotée par le Réseau National des Maisons des Associations (RNMA), est une initiative structurée visant à combler le manque de données sur le tissu associatif à l'échelle territoriale.
Face à une statistique publique jugée insuffisante et mal adaptée aux réalités associatives, l'OLVA propose une méthodologie rigoureuse pour objectiver les réalités, mesurer les évolutions et permettre des comparaisons entre territoires.
La finalité est de "connaître pour agir" : transformer la connaissance produite en actions concrètes, en politiques publiques éclairées et en un accompagnement plus pertinent pour les associations.
La démarche repose sur deux piliers :
une enquête quantitative (l'observation) via un questionnaire standardisé d'une cinquantaine de questions, et
la création d'une dynamique partenariale (l'observatoire) associant acteurs associatifs et publics.
Ce processus se décline en trois phases : la préparation (définition des enjeux locaux, formation), la diffusion de l'enquête (mobilisation des partenaires), et l'analyse des données suivie d'actions post-enquête.
Le RNMA fournit un accompagnement complet incluant des formations, des outils (questionnaire, plateforme en ligne), l'analyse statistique et la mise en réseau des quelque 50 observatoires actifs.
Les livrables finaux incluent une infographie synthétique, une analyse des associations employeuses et un rapport d'enquête détaillé, conçus pour aider à la décision, renforcer la reconnaissance du secteur et mettre en débat les enjeux de la vie associative.
1. Contexte et Origine de la Démarche
1.1. Le Réseau National des Maisons des Associations (RNMA)
Le RNMA est une tête de réseau regroupant un peu plus de 100 structures membres réparties sur le territoire français.
Sa spécificité réside dans l'hybridité de ses membres, qui incluent des Maisons des Associations sous statut associatif et des services "Vie Associative" de collectivités publiques.
Cette diversité est considérée comme une richesse pour le réseau.
Les missions principales du RNMA sont :
• Faire réseau : Créer des synergies entre les membres via des rencontres nationales et des projets communs. • Coordonner des projets : L'OLVA est un exemple de projet né d'un enjeu commun identifié : le besoin de connaissance de la vie associative locale. D'autres chantiers portent sur l'engagement ou le métier d'accompagnateur. • Être un relais : Porter les enjeux et problématiques des membres et des associations qu'ils accompagnent auprès des partenaires publics et privés.
1.2. La Genèse de l'OLVA
La démarche OLVA est née d'un constat fondamental : la méconnaissance de la vie associative à l'échelle d'un territoire. La statistique publique est jugée "assez faible" pour décrire ce secteur, étant principalement conçue pour le monde économique.
Pour répondre à ce besoin, le RNMA s'est inspiré dès 2006 des travaux de Viviane Chardononog, chercheuse au CNRS qui produit régulièrement des "paysages associatifs français".
La méthodologie d'observation nationale a été adaptée et déclinée à l'échelle locale de manière progressive, par des tests puis la création d'outils.
Depuis 2021, une évolution majeure a été la mise en place d'un système de lancement d'observations synchronisées, créant des "vagues" d'observatoires. Cette approche facilite l'accompagnement et génère une dynamique d'échange et de partage entre les territoires participants.
2. Les Objectifs Stratégiques de l'OLVA
La démarche poursuit un objectif principal : "Connaître pour agir". La connaissance n'est pas une fin en soi, mais un levier pour transformer les réalités et répondre aux besoins identifiés.
2.1. Objectifs Liés à l'Observation
1. Objectiver les réalités : Dépasser les "ressentis" sur la structuration du tissu associatif en produisant des chiffres et des données factuelles.
2. Mesurer les évolutions : Après une première enquête qui établit une "photo statique", les observations suivantes permettent de comparer les "millésimes" et de suivre les dynamiques sur le long terme.
3. Se comparer : La méthodologie commune appliquée sur une cinquantaine de territoires permet de situer les réalités locales par rapport à d'autres territoires et aux tendances nationales décrites par Viviane Chardononog.
2.2. Objectifs Liés à l'Action
• Aider à la décision : Fournir des données pour orienter les politiques publiques des collectivités et les stratégies d'accompagnement des structures d'appui associatives.
• Communiquer sur le secteur : Lutter contre les préjugés sur le monde associatif en partageant des réalités objectivées.
• Favoriser la reconnaissance : Mettre en lumière la transversalité et la globalité de la vie associative, souvent cloisonnée par secteurs (sport, culture, etc.) au sein des collectivités. L'enquête permet d'aborder les enjeux de manière transversale.
• Créer du commun : Les temps de restitution de l'enquête sont des occasions pour des associations de secteurs différents de se rencontrer, de partager des difficultés communes et de créer une "espèce de commun de la vie associative".
• Mettre en débat : Utiliser les résultats de l'enquête pour identifier les difficultés, se poser les bonnes questions et rassembler les acteurs autour de la table.
3. Méthodologie et Déroulement
La démarche OLVA s'articule autour de deux axes complémentaires : l'observation (l'enquête elle-même) et l'observatoire (la dynamique partenariale).
3.1. L'Enquête (L'Observation) Il s'agit d'une enquête principalement quantitative, avec la possibilité d'y associer un volet qualitatif (par exemple, via un partenariat universitaire).
Le Questionnaire : * • Format : Environ 50 questions. * • Temps de remplissage : Environ 20 minutes.
• Structure en 4 parties :
Ce questionnaire "tronc commun" peut être complété par des questions spécifiques définies en fonction des enjeux du territoire.
3.2. La Dynamique Partenariale (L'Observatoire)
C'est un aspect essentiel qui consiste à associer d'autres acteurs du territoire concernés par l'enquête (fédérations associatives, acteurs publics, etc.). Cette collaboration vise à : • Garantir une large diffusion du questionnaire au-delà des contacts habituels. • Permettre une action post-enquête en impliquant dès le départ ceux qui pourront répondre aux besoins exprimés. • Favoriser l'interconnexion des acteurs du territoire.
3.3. Les Trois Phases de la Démarche
Phase
Description des Actions
Préparation de l'enquête
2. Diffusion et recueil
3. Analyse et post-enquête
4. Moyens et Accompagnement du RNMA
4.1. Moyens Humains Requis
Le RNMA identifie trois fonctions clés à mobiliser pour mener à bien une démarche OLVA :
1. Pilotage : Gestion de projet (planification, suivi, moyens).
2. Animation : Mobilisation des partenaires, diffusion de l'enquête, communication. Cette fonction peut être assurée en interne ou via des stagiaires ou services civiques.
4.2. Appuis Fournis par le RNMA
• Accompagnement et Formations :
• Outillage : Fourniture du questionnaire tronc commun, mise en ligne, plateforme de collecte de données et outils issus de l'expérience des autres territoires.
• Analyse de données : Exploitation des données existantes (créations d'associations, associations employeuses) et analyse complète des données collectées.
• Mise en réseau et valorisation : ◦ Organisation de journées d'échange de pratiques deux fois par an pour les 50 observatoires. ◦ Valorisation des travaux locaux via des colloques, articles, etc.
4.3. Livrables
Trois types de documents sont produits à l'issue de l'analyse :
5. Partenariats et Enjeux Territoriaux (Synthèse des échanges)
5.1. Articulation avec Guid'Asso
5.2. Relations avec les Collectivités et la Charte des Engagements Réciproques
5.3. Échelle d'Action et Compétences * • L'observation se fait souvent à l'échelle d'une commune ou d'une communauté de communes. * • Une difficulté est identifiée à l'échelle des communautés de communes : la "vie associative" reste souvent une compétence communale, ce qui peut limiter la capacité des intercommunalités à porter de tels projets. * • Cependant, lorsque le portage est associatif (par une Maison des Associations), le bassin de vie ou la communauté de communes est plus régulièrement l'échelle privilégiée.
6. Considérations Pratiques
Synthèse du webinaire : Face à la marchandisation des associations
Résumé
Ce document de synthèse analyse les conclusions du webinaire intitulé "Face à la marchandisation des associations", deuxième épisode du cycle "Écrire collectivement le scénario de renforcement du monde associatif".
Il met en lumière une transformation profonde du secteur associatif français, caractérisée par un glissement d'un modèle de partenariat avec la puissance publique vers une logique de marché concurrentiel.
Les principaux moteurs de cette "marchandisation" sont doubles : * l'adoption du New Public Management depuis les années 1980, qui impose une culture du résultat et de l'évaluation quantitative, et * les règles de concurrence du marché unique européen, qui considèrent par défaut les associations comme des entreprises.
Cette dynamique se matérialise par le recul de la subvention de fonctionnement au profit de la commande publique (appels à projet, marchés publics), transformant les associations en prestataires de services et les privant de leur capacité d'initiative et d'interpellation politique.
Ce phénomène est exacerbé par l'essor de l'entrepreneuriat social, qui brouille les frontières entre lucrativité et non-lucrativité, et par la financiarisation du secteur via des outils comme l'investissement et les contrats à impact social.
Le cas du secteur de la petite enfance illustre concrètement comment des acteurs lucratifs pénètrent des champs historiquement associatifs, utilisant des montages complexes ("associations écran") pour capter des fonds publics.
Face à ce constat, plusieurs pistes de "démarchandisation" sont proposées.
Elles incluent la nécessité de redonner un pouvoir politique à la subvention, d'explorer des modèles de financement alternatifs inspirés de la Sécurité Sociale de l'Alimentation ou du modèle belge de l'éducation permanente, et de "reconscientiser" les élus et techniciens des collectivités à la spécificité du fait associatif.
La reconquête sémantique, notamment sur la notion d'évaluation (utilité sociale contre mesure d'impact), et le renforcement des réseaux et du plaidoyer collectif apparaissent comme des stratégies cruciales pour défendre un modèle associatif fondé sur l'intérêt général, la citoyenneté et la démocratie.
1. Introduction et Contexte du Webinaire
Le webinaire s'inscrit dans un cycle de réflexion organisé en partenariat par le Réseau National des Maisons des Associations (Rnma) et le Collectif des associations citoyennes (CAC).
Il vise à explorer collectivement les scénarios de renforcement du monde associatif face aux dynamiques d'affaiblissement actuelles.
Cet épisode se concentre spécifiquement sur le phénomène de marchandisation, ses constats, ses réalités de terrain et les pistes pour y faire face.
Intervenants principaux :
• Marianne Langlais : Coordinatrice de l'Observatoire citoyen de la marchandisation des associations (CAC).
• Frédéric Bilde : Président de l'ACEPP Sud-Ouest (Association des Collectifs Enfants Parents Professionnels).
• Thomas Lowers : Administrateur du Rnma et Directeur de la Maison des Associations de Roubaix.
Le webinaire s'est articulé autour de deux temps forts : une analyse du processus de marchandisation et une exploration des pistes de "démarchandisation".
2. Analyse Approfondie du Phénomène de Marchandisation
A. Un Cadre Théorique : Les Quatre Vecteurs d'Affaiblissement
Marianne Langlais a introduit l'analyse en présentant quatre vecteurs interdépendants qui affaiblissent le monde associatif, identifiés par le CAC :
1. La Marchandisation : La transformation des modes de financement, avec le passage de la subvention à la commande publique et l'augmentation des ressources marchandes.
2. La Managérialisation : L'adoption de techniques de management issues de l'entreprise lucrative, perçues comme le seul modèle légitime, au détriment des formes d'organisation collective propres aux associations.
3. L'Instrumentalisation : Les associations sont de plus en plus vues comme de simples prestataires de services chargés de mettre en œuvre des politiques publiques pensées sans elles, perdant leur rôle d'initiative et de création de droits.
4. La Mise à Mal des Libertés Associatives :
Conséquence des trois autres vecteurs, le financement public est de plus en plus perçu comme devant s'accompagner d'une "soumission à la politique gouvernementale", rendant illégitime toute parole politique critique de la part d'une association vue comme prestataire.
B. Origines et Mécanismes de la Marchandisation
La marchandisation est le résultat de deux tendances de fond :
• Le New Public Management (années 1980) : Cette approche a introduit dans les services publics, et par ricochet dans les associations, une "culture du résultat, de la performance, de l'évaluation quantitative et du management par objectif", en important les outils du secteur privé lucratif.
• Les Règles du Marché Unique Européen : Elles imposent le marché comme règle générale. Dans ce cadre, les associations sont considérées comme des entreprises et les subventions comme des aides d'État susceptibles de fausser la concurrence. Bien que des exemptions existent, elles restent l'exception.
Ce cadre a engendré un mécanisme central : le recul de la subvention au profit de la commande publique. Cette mutation modifie radicalement le rapport aux pouvoirs publics :
Mode de Financement
Caractéristiques Clés
Subvention
Commande Publique
C. L'Entrepreneuriat Social et la Financiarisation
Le tournant néolibéral est également accompagné et nourri par des concepts qui brouillent les frontières entre lucrativité et non-lucrativité. • L'Entrepreneuriat Social : Terme importé du monde anglo-saxon dans les années 1980, il promeut l'utilisation de compétences entrepreneuriales et de techniques de management du privé pour résoudre des problèmes sociaux.
Marianne Langlais souligne que ce concept, sans statut juridique propre, "invisibilise les associations" et "affaiblit la portée politique que peuvent représenter les associations".
Elle cite Jean-Marc Borello (Groupe SOS) pour qui le modèle associatif, "arquebouté sur le principe non lucratif apparaît à présent inadapté".
• La Financiarisation : Ce flou favorise l'arrivée de l'investissement à impact social, qui promet de "faire du bien et du profit en même temps". * ◦ Le Contrat à Impact Social (CIS) est un exemple frappant. Ce mécanisme financier engage un investisseur privé, une association et la puissance publique.
L'investisseur finance un programme associatif, et la puissance publique le rembourse avec intérêts en fonction de l'atteinte d'indicateurs de performance prédéfinis. * ◦ Conséquence : Les actions associatives sont transformées en produits financiers, et la mesure d'impact social, souvent monétarisée (coûts évités), devient le nouveau mode d'évaluation, supplantant la notion d'utilité sociale.
D. Illustration Sectorielle : Le Cas de la Petite Enfance Frédéric Bilde a illustré concrètement ce processus dans le secteur de la petite enfance.
• Historique : Le secteur s'est initialement développé sur un modèle associatif et citoyen, avec des crèches parentales (ACEPP) fondées sur une démarche ascendante, partant des besoins locaux. • Le tournant : Les directives européennes (Bolkestein, 2006) ont ouvert le secteur à la concurrence et aux acteurs privés lucratifs. L'État français a choisi de maintenir les services à la petite enfance dans ce champ concurrentiel.
• Conséquences directes :
◦ Nouveau langage : Les notions de "rentabilité", "taux de remplissage", "optimisation" et "coût de revient" priment sur le "bien-être de l'enfant" et le "soutien aux parents".
◦ Pervertissement du modèle associatif : Des groupes privés lucratifs créent des "associations écran" pour rassurer les municipalités et remporter des délégations de service public (DSP).
Ces structures sont ensuite vidées de leur substance, les bénéfices étant "aspirés" vers le groupe via des centrales d'achat, des frais de gestion ou des loyers versés à des sociétés civiles immobilières (SCI) appartenant au même groupe.
3. Pistes de "Démarchandisation" et Stratégies de Renforcement
Face à ce diagnostic, les intervenants ont proposé plusieurs axes d'action pour inverser la tendance.
A. Redonner un Pouvoir Politique à la Subvention
La piste principale est de réaffirmer la légitimité de la subvention de fonctionnement comme mode de financement principal des associations.
Il est jugé indispensable qu'une partie de l'impôt finance les associations qui contribuent à l'intérêt général. Toutefois, cette subvention doit être repensée pour la "sortir du discrétionnaire politique" afin de garantir la fonction d'interpellation des associations.
B. Explorer des Modèles de Financement Alternatifs
Plusieurs modèles inspirants sont explorés :
1. La Sécurité Sociale de l'Alimentation : Ce projet propose un accès universel à une alimentation de qualité via une allocation et un conventionnement de producteurs, le tout géré par des caisses locales démocratiques. Transposé aux associations, ce modèle pourrait permettre de repenser la redistribution de l'argent public en dehors des logiques de marché.
2. Le Modèle Belge de l'Éducation Permanente : Un décret en Wallonie garantit un financement pérenne et à long terme pour les associations de ce secteur.
Crucialement, l'article 1 de ce décret "garantit la fonction d'interpellation des associations". Ce modèle promeut également l'auto-évaluation, une alternative à l'évaluation par la mesure d'impact.
C. Renforcer le Dialogue et la Coconstruction Une stratégie clé consiste à "reconscientiser" la puissance publique, notamment les élus locaux.
• Changement de regard : Thomas Lowers observe que beaucoup d'élus locaux récents, issus d'un monde où la logique de marché est une "évidence", n'envisagent plus d'alternative.
Il est donc crucial de démontrer la valeur et le sérieux du monde associatif, qui n'est pas "un coût à gérer" mais un partenaire.
• Formation : L'utilisation de dispositifs comme le Certificat de Formation à la Gestion Associative (CFGA) pour former également les élus est une piste pour créer une culture commune.
• Défendre l'interpellation : Léa Gallois (Institut Alinsky) a insisté sur la nécessité de faire reconnaître la fonction d'interpellation comme "riche pour la démocratie locale" et de créer des "fonds d'interpellation" pour la soutenir matériellement.
D. S'Organiser Collectivement et Mener la "Bataille des Mots"
• Force du collectif : Frédéric Bilde a souligné que "seul, on n'y arrivera pas". La mobilisation au sein de fédérations et de réseaux est essentielle pour peser dans les décisions politiques.
• Bataille sémantique : La discussion a mis en exergue l'importance de déconstruire le langage de la marchandisation. L'évaluation est un terrain de lutte central.
Il s'agit de défendre une évaluation de l'utilité sociale (qui montre la spécificité associative) contre la mesure de l'impact social (qui réduit l'action à une valeur monétaire et est le "cheval de Troie de la financiarisation").
4. Points Clés de la Discussion
• Le Mécénat : Il n'est pas vu comme une solution miracle. Il est souvent ponctuel, orienté vers des "pépites" médiatiques et peut lui-même s'inscrire dans une logique d'investissement ("venture philanthropy") qui s'éloigne du don.
• Règles Européennes : Le rapport du CESE sur le financement des associations est cité comme une ressource clé pour argumenter en faveur d'une sortie des associations du cadre concurrentiel du marché unique.
• Statuts et Agréments : La discussion a montré comment les exigences administratives et la standardisation des projets (via les agréments) peuvent dénaturer des initiatives de terrain innovantes, transformant une logique ascendante en une logique descendante.
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Hi All! Our aim is to read all of Part I (so Chapters 1 & 2; up to page 50) to discuss in class on Thursday, Oct 2nd. I'll add annotations as I re-read the chapters this week, and you do too please!
Synthèse du Soutien de l'État à la Vie Associative : Du National au Local
Résumé
Cette note de synthèse analyse les formes et les logiques du soutien de l'État à la vie associative en France, en se basant sur une recherche menée par Mathilde Rtinassi et Emmanuel Porte.
L'étude révèle une action publique éclatée et peu coordonnée, dépourvue d'une politique unifiée.
Le soutien financier, bien que stable en volume global de subventions (environ 8,5 milliards d'euros), est perçu comme étant en baisse en raison de sa répartition sur un nombre croissant d'associations, ce qui diminue le montant moyen par structure.
La recherche identifie quatre grands objectifs poursuivis par l'État : la consolidation des structures, l'articulation du secteur, l'observation du monde associatif, et la reconnaissance de sa légitimité. Ces objectifs sont inégalement poursuivis selon les ministères, menant à une typologie de quatre formes de soutien distinctes :
1. Soutien partiel et institué : Une relation de compagnonnage de longue date, mais ne couvrant qu'une partie des objectifs.
2. Soutien multiforme : Le modèle le plus complet, couvrant les quatre objectifs, porté par des acteurs comme la CNAF ou la DJEPVA.
3. Soutien par la reconnaissance : Limité aux procédures réglementaires (agréments, labels), avec une relation distante.
4. Soutien par relation intéressée : Le modèle le plus répandu, où l'association est principalement un instrument pour le déploiement des politiques publiques, souvent via des appels d'offres.
Un constat majeur est la corrélation directe entre la qualité du dialogue entre les acteurs publics et les associations et la richesse des formes de soutien.
Enfin, la production de connaissance sur le secteur associatif reste le "parent pauvre" de l'action publique nationale, et les "têtes de réseau" jouent un rôle opérationnel indispensable que l'État ne peut assumer seul.
1. Contexte et Méthodologie de la Recherche
La recherche intitulée "Les soutiens national à la vie associative : enquête exploratoire sur une action publique éclatée" a été initiée mi-2019 pour répondre à deux constats principaux :
• L'illisibilité du soutien à la vie associative : Des rapports antérieurs (Inspection Générale, 2009 et 2016) soulignaient la complexité et la confusion des dispositifs, labels et acronymes (CRIB, PAVA, PIVA), rendant l'écosystème difficile à naviguer pour les associations. Une citation d'un rapport illustre ce point : "il existe des crib (...) qui sont des Pavas (...), des cribes non Pava, des Pavas non crib..."
• Une connaissance parcellaire des soutiens ministériels : Au-delà du rôle historique du ministère de la Jeunesse, la manière dont les autres ministères soutiennent le secteur associatif restait mal connue, alors que chacun interagit avec lui (ex: fiscalité pour le ministère de l'Économie, gestion du greffe pour l'Intérieur). Méthodologie
L'étude repose sur une approche purement qualitative :
• 42 entretiens semi-directifs ont été réalisés avec :
◦ Des agents de 15 ministères et organismes d'État.
◦ 19 têtes de réseau associatives.
◦ 1 responsable politique.
• Une analyse documentaire approfondie (doctrines de financement, appels à projets, rapports d'activité) a été menée pour aller au-delà du discours officiel.
Le terrain de recherche a été complexe, marqué par des difficultés d'accès aux ministères et une interruption de 14 mois due à la crise du Covid-19.
2. Analyse du Soutien Financier : Nuances et Réalités
Contrairement à l'idée reçue d'une baisse généralisée des financements, l'analyse des données (notamment le "Jaune" budgétaire associatif) apporte des nuances importantes :
• Stabilité des subventions : Le montant global des subventions versées par l'État aux associations est relativement stable, s'élevant à environ 8,5 milliards d'euros.
• Augmentation du volume global des moyens : Le volume financier total attribué aux associations augmente, mais une part croissante de ces moyens n'est pas versée sous forme de subventions (ex: marchés publics).
• Étalement sur le tissu associatif : L'État finance un nombre croissant d'associations.
Par conséquent, même avec un budget global stable, le montant moyen et médian par association est en baisse.
Ce phénomène d'étalement est le principal facteur expliquant la perception d'une diminution des financements et de la part des subventions.
3. Les Quatre Grands Objectifs du Soutien de l'État
L'analyse des entretiens et des documents a permis d'identifier quatre objectifs principaux que l'État poursuit, de manière plus ou moins explicite, à travers son soutien au monde associatif.
Objectif Description Exemples d'Actions Consolidation
Vise à pérenniser la structure associative en la rendant robuste sur le long terme pour qu'elle puisse répondre aux besoins sociaux et aux politiques publiques. * - Soutien financier (subventions, etc.)<br>- Accompagnement à la professionnalisation<br>- Renforcement du modèle socio-économique et de la gouvernance<br>- Accueil, information et orientation<br>-
Formation des membres et salariés
Articulation / Maillage Concerne la circulation de l'information, le partage de bonnes pratiques et la facilitation des coopérations entre associations, et avec d'autres acteurs (publics, privés). * - Mise en réseau des acteurs<br> * - Organisation d'échanges de pratiques entre pairs<br> * - Facilitation de l'essaimage d'expérimentations
Observation / Objectivation
Recouvre la production de connaissances sur le secteur associatif pour éclairer l'action publique. Cet objectif est souvent le moins prioritaire.
Reconnaissance / Légitimation Englobe toutes les procédures de reconnaissance officielle des structures, de leurs projets ou de leurs activités, leur conférant une légitimité à agir au nom de l'intérêt général.
Ces objectifs sont souvent interconnectés. Par exemple, la reconnaissance (via un agrément) facilite l'accès aux financements, contribuant ainsi à la consolidation de l'association.
4. Typologie des Formes de Soutien National
En croisant les objectifs poursuivis, la nature du dialogue et le type de relation entre l'État et les associations, la recherche a établi une typologie de quatre modèles de soutien. Type de Soutien Description et Relation Objectifs Couverts
Exemples et Caractéristiques
La politique publique est fortement adossée aux têtes de réseau, qui deviennent des partenaires incontournables.
Consolidation (financière) et Articulation/Maillage.
Ministère de la Culture (pratiques amateurs) : Les fédérations sont des "coquilles vides" potentielles sans le soutien de l'État, et l'État ne peut agir sans elles.<br>
Délégation à la Sécurité Routière avec l'association Prévention Routière.<br>
Tendance à créer un "monopole" d'interlocuteurs.
2. Soutien multiforme
Le "champion" du soutien : Le modèle le plus complet et diversifié.
La relation est basée sur la co-construction et un dialogue riche.
Il existe une culture "militante" en faveur du monde associatif au sein de ces administrations.
Les 4 objectifs sont couverts, y compris l'Observation.
3. Soutien par la reconnaissance
Relation procédurale et distante : Le soutien est quasi-exclusivement articulé autour d'une procédure réglementaire (agrément, label).
Principalement la Reconnaissance/Légitimation.
4. Soutien par relation intéressée
Relation instrumentale : Le soutien est secondaire par rapport à l'objectif principal du ministère, qui est le déploiement de sa politique publique. Les associations sont vues comme des prestataires.
Principalement la Consolidation (uniquement pour qu'elles puissent "tenir" et mettre en œuvre la politique).
5. Le Rôle Crucial des Têtes de Réseau
La recherche souligne que le soutien de l'État ne pourrait exister sans le rôle opérationnel des têtes de réseau, qui agissent comme le bras armé d'un "État stratège" mais souvent démuni de capacités d'action directe.
Leurs contributions principales sont :
• Représentation et Plaidoyer : Faire remonter les besoins des territoires et défendre les intérêts du secteur.
• Identification des problématiques : Aider à l'émergence de nouvelles questions sociales ou d'innovations.
• Mutualisation d'expertise : Produire de la connaissance utile pour leurs membres et non-membres.
Les têtes de réseau sont traversées par un débat sur leur approche territoriale :
• Logique d'équité : Viser un soutien équitable pour toutes les structures membres, souvent via une centralité budgétaire.
• Logique d'adaptation : Soutenir les structures de manière "ad hoc" en fonction des spécificités territoriales, ce qui implique une gouvernance moins pyramidale mais pose des défis de connaissance des contextes locaux et de risque de concurrence interne.
6. Perspectives Locales et Divergences
L'intervention de Luciana complète la perspective nationale en soulignant l'importance du contexte territorial, souvent absent des discours ministériels (à l'exception de l'ANCT et de la DJEPVA).
• Le territoire comme construction sociale : Au-delà de l'espace administratif, le territoire est façonné par les relations entre les acteurs. Les politiques nationales sont appropriées différemment selon les spécificités locales.
• Lisibilité des politiques publiques locales : La multiplication des dispositifs et le traitement en silo par secteur d'activité créent un manque de transversalité.
La présence d'un élu référent, d'un service dédié ou d'un observatoire local de la vie associative (OLVA) peut renforcer le dialogue et la cohérence de l'action publique locale.
7. Conclusions et Points Soulevés en Discussion
Constats Clés
1. Absence d'une politique unifiée : Il n'existe pas de politique de soutien à la vie associative intégrée au niveau de l'État. La coordination est faible, y compris au sein d'un même ministère.
2. La qualité du dialogue est déterminante : Plus le dialogue est riche et orienté vers la co-construction, plus le soutien est diversifié et complet.
3. L'Observation, parent pauvre de l'action publique :
La production de connaissance sur le secteur associatif est souvent considérée comme secondaire ou un "impensé" au niveau national, alors qu'elle est un levier puissant au niveau local.
Discussion avec les Participants
• Financements et clientélisme : Les participants ont évoqué un ressenti de clientélisme.
La recherche met plutôt en évidence un "effet de monopole", où les ministères préfèrent dialoguer avec un interlocuteur unique ou principal.
• La charge administrative : Il a été souligné que les associations passent un temps considérable à remplir des dossiers pour des dispositifs multiples et chronophages, au détriment de l'action de terrain.
Cela renvoie à la nécessité de simplifier les procédures et de privilégier des financements pluriannuels (CPO) plutôt que des appels à projets annuels.
Document de Synthèse : Webinaire "Décrypter la recherche - Épisode 1"
Synthèse
Ce document de synthèse résume les points clés du webinaire "Décrypter la recherche - Épisode 1", organisé par le Réseau National des Maisons des Associations (RNMA).
La discussion centrale a exploré la dichotomie conceptuelle de l'association, vue soit comme une "entreprise sociale", soit comme une "action collective", dans le contexte des débats actuels sur une potentielle révision de la loi française sur l'Économie Sociale et Solidaire (ESS) de 2014.
La présentation principale, assurée par Luciana Riero, doctorante au sein du RNMA, a exposé les résultats d'une cartographie scientifique ("science mapping") de la recherche internationale.
Cette analyse révèle la structuration du champ académique autour de deux traditions dominantes :
1. L'approche de l'entrepreneuriat social, prédominante dans le monde anglo-saxon, qui valorise l'activité entrepreneuriale et une vision plus individualiste, centrée sur le profil de l'entrepreneur social.
2. L'approche du secteur à but non lucratif ("non-profit"), plus proche de la tradition européenne et française, qui analyse les associations dans leur relation avec l'action publique et en tant qu'expression de l'action collective.
Le choix entre ces deux prismes d'analyse n'est pas neutre et a des conséquences directes sur la perception des associations, notamment en matière de gouvernance démocratique, d'économisation du social et de la dimension politique de l'ESS.
Le débat sur la révision de la loi ESS française cristallise ces tensions, opposant une vision d'une ESS palliative et isomorphique aux modèles d'entreprises capitalistes, à une vision d'une ESS alternative et émancipatrice.
Enfin, la position du RNMA est de défendre la spécificité de l'association comme action collective et de plaider pour que toute évolution législative renforce de manière opérationnelle la coopération et la co-construction sur les territoires.
1. Introduction au Webinaire et à la Démarche de Recherche
Contexte et Objectifs
Le webinaire constitue le premier épisode d'un cycle intitulé "Décrypter la recherche", visant à créer un dialogue semestriel (tous les 4 à 6 mois) entre le monde de la recherche et les acteurs associatifs.
L'objectif n'est pas de prendre position pour ou contre la révision de la loi ESS, mais d'utiliser les apports de la recherche pour fournir des "clés de lecture" sur les conséquences des évolutions en cours pour les associations et les territoires.
Le RNMA et la Recherche
Le RNMA entretient des liens historiques avec le monde de la recherche, notamment depuis les années 2000 avec des collaborations sur les observatoires locaux de la vie associative (avec des chercheurs comme Vivian Tchernonog ou Lionel Prouto).
Cette démarche vise à rendre la recherche opérationnelle pour aider les associations à mieux comprendre leur fonctionnement et à faire évoluer leurs pratiques.
La Thèse de Luciana Riero
Luciana Riero est doctorante au sein du RNMA via un dispositif CIFRE. Sa thèse s'intitule "La qualification et interprétation des relations entre les associations et le territoire".
Son objectif est d'identifier, de qualifier et de mesurer ces relations, ainsi que de démontrer les liens de causalité entre les caractéristiques socio-économiques des territoires et les caractéristiques organisationnelles des associations.
2. Cartographie de la Recherche Internationale sur les Associations et le Territoire
Méthodologie du "Science Mapping"
La présentation s'appuie sur une cartographie scientifique, une méthode d'analyse bibliométrique quantitative qui permet de visualiser l'état des connaissances sur un sujet. La démarche est inductive, sans hypothèses a priori.
• Corpus : 2 857 articles scientifiques issus de la base de données Web of Science.
• Mots-clés de recherche : Croisement de termes liés aux associations ("nonprofit organization", "social enterprise", "voluntary sector") et de termes liés au territoire ("spatial", "urban", "local", "development").
• Constat initial : Une augmentation des publications sur ce thème est observée depuis 2011. Les couples de mots-clés les plus fréquents sont "social enterprise + development" et "nonprofit organization + development", suggérant que la notion de développement est plus prégnante dans la littérature internationale que celle de territoire.
Résultats Clés de la Cartographie L'analyse révèle une structuration de la recherche mondiale autour de deux grands pôles intellectuels :
Cluster Thématique
Description
Pôle 1 : L'Entreprise Sociale Ce courant est centré sur l'entreprise sociale et l'activité entrepreneuriale. Il analyse l'émergence de ce concept, souvent dans une perspective internationale comparative (Europe vs. États-Unis), et son ancrage dans la théorie des organisations hybrides.
Pôle 2 : Le Secteur à But Non Lucratif Ce courant est centré sur les organisations à but non lucratif, la philanthropie et le bénévolat. L'analyse porte principalement sur les relations entre ces organisations et l'État (l'action publique), questionnant les phénomènes d'institutionnalisation, de banalisation ou de marchandisation du secteur.
Opposition Conceptuelle : Approches Américaine et Européenne de l'Entreprise Sociale
• Aux États-Unis, deux écoles de pensée coexistent :
1. L'école de la recette marchande : Considère comme entreprise sociale toute organisation, quel que soit son statut, qui déploie une activité économique marchande au profit d'une finalité sociale.
2. L'école de l'innovation sociale : Met l'accent sur la figure de l'entrepreneur social (son dynamisme, sa créativité, son leadership) comme facteur déterminant. * ◦ Conclusion : Une approche à dominante
• En Europe, les travaux du réseau EMES (Emergence of Social Enterprise) ont fondé le concept sur un idéal-type reposant sur trois dimensions clés : 1. Un projet économique : Activité continue de production de biens ou services. 2. Une mission sociale : Objectif explicite de service à la communauté et distribution limitée des profits. 3. Une gouvernance participative : Association des différentes parties prenantes. * ◦ Conclusion : Une approche à dominante
3. Conséquences et Enjeux des Approches Théoriques
Le choix d'analyser les associations par le prisme de l'entreprise sociale ou de l'action collective a des implications profondes.
• Gouvernance Démocratique : L'économie sociale repose sur le principe "une personne, une voix" et la double qualité des membres (bénéficiaires et sociétaires).
L'approche par l'entreprise sociale, notamment dans ses formes comme le "social business", rend ces frontières plus floues, le critère de démocratie interne n'étant pas toujours explicite.
La gouvernance participative constitue une "dimension cruciale des ruptures possibles" entre les deux modèles.
• Économisation du Social : L'approche par l'entreprise sociale peut accentuer la lecture d'un "déplacement d'une production publique vers une production privée".
• Lien avec l'Action Publique : La recherche sur le secteur non lucratif met en débat la relation avec la puissance publique, oscillant entre des perspectives d'isomorphisme (tendance des associations à adopter les modes d'organisation des entreprises capitalistes) et de co-construction des politiques publiques.
Positionnement de la Thèse : Face à ces constats, la recherche de Luciana Riero s'inscrira dans la continuité des approches francophones en termes d'action collective et d'auto-organisation.
Elle renforcera une vision du territoire comme une construction sociale, en interaction avec l'action collective, se démarquant ainsi d'une vision internationale qui le perçoit souvent comme une simple donnée administrative (urbain/rural).
4. Mise en Perspective : La Loi ESS et ses Débats
Avertissement : Les points suivants sont basés sur les travaux d'acteurs comme ESS France, le RTES ou des chercheurs comme Timothée Duverger, et non sur l'expertise directe de la présentatrice.
La Loi de 2014 : Un Double Projet
L'article 1 de la loi de 2014 définit l'ESS non seulement comme un "mode d'entreprendre", mais aussi comme un "mode de développement économique", ce qui le distingue d'un simple projet d'entreprise pour en faire un projet politique de société.
Ce développement est précisé comme étant local et durable (via les PTCE, article 9).
Points Clés du Débat sur la Révision
À l'approche des 10 ans de la loi, plusieurs enjeux sont débattus :
• Agrément ESUS (Entreprise Solidaire d'Utilité Sociale) : Des acteurs proposent de le réviser pour imposer un meilleur partage de la valeur, renforcer le contrôle et ajouter une obligation de reporting d'utilité sociale et environnementale.
• Moyens financiers : Les fonds créés sont jugés souvent "faibles et mêlés à une politique de soutien de l'économie d'impact aux contours un peu flous".
• Rapprochement avec la RSE : La loi PACTE de 2019 a créé les "sociétés à mission", invitant les entreprises conventionnelles à se doter d'une "raison d'être". Ce mouvement, auquel des structures de l'ESS ont participé, questionne les frontières et les spécificités de l'ESS.
• Lien ESS et Territoire : Un axe de développement serait d'assumer pleinement le rôle des régions comme "chefs de file" de l'ESS et de reconnaître celui des départements comme premiers financeurs.
Enjeux Théoriques du Débat Français
Le débat académique français gravite autour d'une tension fondamentale :
• Une ESS alternative et émancipatrice vs. une ESS palliative et d'isomorphisme.
• La montée en puissance des thématiques de l'entreprise sociale, de l'entrepreneuriat social, voire du "social business", tend à masquer l'hétérogénéité de l'ESS et à réduire son projet politique à une simple finalité sociale.
Conclusion : L'approche adoptée pour analyser les associations (entreprise sociale ou action collective) influence directement la dimension politique du débat sur l'avenir de l'ESS.
5. Contributions des Participants et Conclusion
Précisions Techniques et conceptuelles
• Distinction entre activité économique et marchande (Colin Blard, avocat) : Une spécificité française cruciale est que toute activité économique n'est pas marchande.
La "plus-value sociale ajoutée" (liée à la notion d'utilité sociale) permet à une association de développer une activité économique tout en conservant son statut non lucratif et sa non-sujétion aux impôts commerciaux. Cette notion d'utilité sociale est intrinsèquement liée au territoire, notamment via l'analyse de la concurrence.
• Évolution historique du secteur : Un participant avec 20 ans d'expérience dans l'ESS a rappelé que la raréfaction des financements publics depuis les années 1980 a poussé les associations vers une hybridation de leurs ressources et une professionnalisation, avec l'émergence de compétences issues du monde de l'entreprise.
Ce mouvement a renforcé leur autonomie économique mais a aussi instauré une logique de mise en concurrence via les appels à projets.
Positionnement et Plaidoyer du RNMA
En conclusion, Thomas (RNMA) a exposé la position du réseau :
• La loi de 2014 a été positive pour la reconnaissance de l'ESS, mais ses effets transformateurs sur les territoires sont restés limités.
• Le RNMA défend la spécificité de l'association comme "action collective" au sein de l'ESS.
• Plaidoyer : Si une révision de la loi a lieu, le RNMA plaidera pour qu'elle intègre une traduction opérationnelle forte des principes de coopération et de co-construction multi-acteurs.
L'enjeu est de reconnaître et d'outiller l'apport des associations aux transitions des territoires et de la société.
Le Burnout Parental : Synthèse de la Conférence d'Isabelle Roskam
Résumé
Ce document de synthèse analyse les points clés de la conférence d'Isabelle Roskam, Professeure de psychologie du développement, sur le phénomène du burnout parental.
La parentalité au 21e siècle est soumise à des pressions sociétales intenses et inédites, transformant une expérience traditionnellement perçue comme joyeuse en une source potentielle de souffrance profonde.
Le burnout parental est un syndrome clinique spécifique, distinct de la dépression et du burnout professionnel, caractérisé par un épuisement physique et émotionnel extrême, une distanciation affective avec ses enfants, et une perte de plaisir dans le rôle parental.
Il est la conséquence d'un déséquilibre prolongé entre les stresseurs (personnels, familiaux, situationnels) et les ressources disponibles pour y faire face.
Avec une prévalence touchant jusqu'à 8 % des parents dans des pays comme la France et la Belgique, ce trouble constitue un problème de santé publique majeur.
Ses conséquences sont graves, incluant des problèmes de santé pour le parent, des idées suicidaires, ainsi que des actes de négligence et de violence envers les enfants.
Les solutions proposées sont à la fois individuelles et collectives.
Au niveau individuel, il s'agit de restaurer l'équilibre en réduisant les stresseurs et en augmentant les ressources, via l'écoute, la prévention et des thérapies ciblées.
Au niveau collectif, une prise de conscience est nécessaire pour relâcher la pression vers une parentalité parfaite, mieux soutenir les parents et recréer un "village" solidaire pour briser l'isolement parental.
1. Le Contexte Moderne : Pourquoi Être Parent Est Devenu si Exigeant
Isabelle Roskam postule que la parentalité contemporaine est fondamentalement différente de celle des générations précédentes. Plusieurs changements sociétaux majeurs survenus dans la seconde moitié du 20e siècle ont intensifié la pression sur les parents.
• Évolution des rôles de genre : Le modèle traditionnel (mère au foyer, père pourvoyeur de ressources) a laissé place à une attente de double performance pour les femmes et à une redéfinition du rôle des pères.
Cela a introduit de nouveaux défis, notamment celui de la coparentalité, qui exige un ajustement constant entre les parents sur les valeurs et méthodes éducatives.
• Montée de l'individualisme : Les sociétés occidentales valorisent l'épanouissement personnel, les désirs et les aspirations individuelles.
Devenir parent crée une injonction contradictoire : il faut faire passer les besoins de l'enfant avant les siens.
Cela génère un conflit interne permanent entre la culpabilité de prendre du temps pour soi et la frustration de se dédier entièrement à ses enfants.
• L'avènement de la contraception : Le concept de l'enfant choisi a transformé la parentalité en un projet de vie conscient.
Cet engagement volontaire augmente la valeur attribuée à l'enfant et au rôle parental, mais induit aussi une pression sociale forte : "tu les as voulus, tu dois assumer", rendant difficile l'expression de la souffrance.
• Changement du statut de l'enfant : En un siècle, la société est passée d'une relative indifférence envers l'enfant (considéré parfois comme une force de travail) à une préoccupation intense pour l'optimisation de son développement (cognitif, émotionnel, social). Rien n'est trop beau ou trop cher pour l'enfant, qui est devenu une valeur centrale.
• La Convention internationale des droits de l'enfant (1989) : Ce texte a formalisé ce nouveau statut en définissant l'enfant comme un sujet de droits. Cela a bouleversé les dynamiques familiales :
◦ L'enfant a désormais droit au chapitre, peut négocier et décider pour sa vie.
◦ Les parents ont le devoir de lui offrir tout le nécessaire pour atteindre son plein potentiel.
◦ L'État a le rôle d'aider les parents (crèches, allocations) mais aussi de surveiller les familles, mettant fin au "règne du pater familias" et instaurant un monitoring social pouvant aller jusqu'au retrait de l'enfant.
• Développement des sciences psychologiques : La diffusion massive de connaissances sur l'éducation, via la littérature de vulgarisation et les réseaux sociaux, a créé une pression immense pour devenir un "bon parent" et appliquer les principes de la parentalité positive, générant une anxiété de performance et une peur de l'erreur.
2. Le Burnout Parental : Définition, Symptômes et Marqueurs Biologiques
Le burnout parental est un syndrome spécifique qui ne doit pas être confondu avec la dépression ou un simple état de fatigue.
Les Symptômes Clés
1. Épuisement intense : Un épuisement physique et émotionnel qui se manifeste exclusivement dans la sphère parentale. Le parent n'a plus aucune énergie pour s'occuper de ses enfants, mais peut en conserver pour d'autres activités (travail, amis).
2. Distanciation émotionnelle : Le parent fonctionne en "pilotage automatique". Il assure les tâches essentielles (conduire à l'école, nourrir) mais n'a plus les ressources pour se connecter émotionnellement à ses enfants.
3. Perte de plaisir dans le rôle parental : Les interactions avec les enfants, autrefois sources de joie, deviennent une corvée.
4. Contraste avec le "parent d'avant" : Le parent en burnout a conscience de ce changement radical. Il était souvent un parent très investi, voire perfectionniste, avant de s'effondrer.
Témoignage marquant : "Ce mot 'maman' je ne le supporte plus. La première fois que votre bébé vous dit maman, c’est le plus beau jour de votre vie et aujourd’hui, ce n’est plus un mot que je suis heureuse d’entendre. Vraiment, c’est devenu un mot de torture."
Distinction avec la Dépression et le Burnout Professionnel
• Contextualisation : Le burnout parental est spécifique à la sphère familiale. Une personne peut être en burnout parental et trouver refuge dans son travail, et inversement.
• Transversalité : La dépression est un trouble transversal qui affecte toutes les sphères de la vie. Une personne déprimée n'aura ni l'envie ni l'énergie pour ses enfants, son travail ou ses loisirs.
La Preuve Biologique : Le Cortisol Des études scientifiques ont mesuré le taux de cortisol (l'hormone du stress) accumulé dans les cheveux des parents. Les résultats démontrent une souffrance physiologique réelle et mesurable.
Groupe de personnes
Niveau de stress (mesuré par le cortisol capillaire)
Parents en burnout
Très élevé
Victimes de violences conjugales
Élevé
Patients souffrant de douleurs chroniques sévères
Élevé
Parents épanouis
Modéré (plus élevé qu'un non-parent)
Étudiant en période d'examens
Modéré
Cette hiérarchie montre que le stress chronique subi par les parents en burnout est physiologiquement supérieur à celui de populations connues pour leur détresse extrême.
L'excès de cortisol est toxique et explique de nombreux problèmes de santé physique (migraines, troubles digestifs, douleurs) rapportés par ces parents.
3. Le Mécanisme du Burnout : Le Modèle de la Balance
Le burnout parental est le résultat d'un déséquilibre chronique entre les stresseurs et les ressources.
Il survient lorsque les stresseurs sont trop nombreux ou trop intenses, pendant trop longtemps, sans ressources suffisantes pour les compenser.
• Stresseurs Parentaux : Tout ce qui augmente la charge et la difficulté d'être parent.
• Ressources Parentales : Tout ce qui aide à faire face aux stresseurs.
Le burnout n'est pas nécessairement causé par un seul gros stresseur, mais souvent par une accumulation de petits stresseurs quotidiens qui font pencher la balance du mauvais côté.
4. Prévalence et Conséquences Graves
Le burnout parental doit être pris au sérieux pour deux raisons majeures : sa prévalence élevée et la gravité de ses conséquences.
Prévalence : Un Problème de Santé Publique
Une étude menée dans 42 pays révèle que les pays occidentaux sont les plus touchés.
• France et Belgique : Des taux de prévalence de 6 à 8 %.
• À l'échelle de la France : Cela représente environ 900 000 parents en souffrance.
Conséquences
Le burnout parental a des répercussions dévastatrices sur l'ensemble de l'écosystème familial.
• Pour le parent :
◦ Problèmes de santé physique exacerbés par le cortisol.
◦ Idées suicidaires très fréquentes. Contrairement au burnout professionnel où l'on peut démissionner ou se mettre en arrêt maladie, il n'y a pas de porte de sortie à la parentalité. Le suicide est parfois perçu comme la seule issue.
• Pour l'enfant :
◦ Négligence : Le parent n'a plus l'énergie de s'occuper adéquatement de l'enfant (aide aux devoirs, surveillance).
◦ Violence : La violence peut être verbale ("ma vie serait tellement plus simple si tu n'étais pas là") ou physique. Le parent, à bout, peut avoir des pulsions violentes qu'il peine à contrôler.
• Pour le couple :
◦ Flambée des conflits conjugaux.
◦ Idées de divorce ou de séparation. La garde alternée peut être envisagée non pas à cause de la fin de l'amour, mais comme une stratégie de survie pour pouvoir "souffler une semaine sur deux".
5. Pistes de Solution : Agir à l'Échelle Individuelle et Collective
Solutions Individuelles
1. Écouter et valider la souffrance : La première étape est de briser le tabou et de permettre au parent d'exprimer sa souffrance sans jugement.
2. Prévention : Des programmes comme "Parents sur le fil" visent à aider les parents à relâcher la pression qu'ils s'imposent.
3. Restaurer l'équilibre de la balance : Identifier les stresseurs pour les réduire et identifier/activer des ressources pour les augmenter.
4. Traitement spécialisé : Pour les cas avancés, des thérapies de groupe ont prouvé leur efficacité, réduisant le taux de cortisol de 52 % en huit semaines et le ramenant à un niveau proche de celui des parents épanouis.
Solutions Collectives
Le burnout parental étant en partie un phénomène de société, la réponse doit aussi être collective.
1. Prendre garde aux pressions normatives : Il faut questionner les injonctions à la perfection véhiculées par les réseaux sociaux et certains professionnels.
2. Adopter la bienveillance envers les parents : Les professionnels (pédiatres, enseignants) doivent considérer le bien-être du parent autant que celui de l'enfant.
L'analogie de l'avion est parlante : "mettre son propre masque à oxygène avant d'aider son enfant". Un parent qui s'épuise ne peut plus prendre soin de son enfant.
3. Repenser la parentalité positive : Ce concept doit être vu comme un "phare" qui donne une direction, et non comme un but inatteignable.
Une parentalité "suffisamment bonne" est plus saine pour le parent et pour l'enfant, qui a besoin de se construire face à des adultes imparfaits.
4. Combattre l'isolement parental : La parentalité est devenue une activité solitaire. Il est crucial de recréer du lien et de la solidarité.
◦ Distinguer le soutien formel et informel : Les pays occidentaux offrent beaucoup de soutien formel (services de l'État, associations), mais ont perdu le soutien informel (famille élargie, voisinage). Or, ce dernier est essentiel pour le soutien émotionnel.
◦ Retrouver l'esprit du village : Il faut réhabiliter l'idée qu'il est normal et nécessaire de partager les tâches et les responsabilités parentales au sein d'une communauté.
Comme le dit le proverbe africain : "Pour élever un enfant, il faut tout un village".
https://www.youtube.com/watch?v=r_cQsQ49uHk
Résumé de la vidéo [00:00:06][^1^][1] - [00:24:15][^2^][2]:
Cette vidéo présente la 8ème Journée Départementale de la Parentalité à Agde en 2022, avec une conférence d'Isabelle Roskam. Elle aborde le burn-out parental et les défis de la parentalité au 21e siècle, en mettant l'accent sur les pressions sociétales et les attentes envers les parents.
Points forts: + [00:00:06][^3^][3] Introduction d'Isabelle Roskam * Présentation de son parcours professionnel * Expérience en psychologie du développement et recherche sur le burn-out parental * Auteur d'ouvrages sur la parentalité + [00:01:47][^4^][4] La parentalité et les émotions positives * La perception culturelle de la parentalité associée au bonheur * Les défis et le stress liés à l'éducation des enfants * La difficulté d'exprimer les aspects négatifs de la parentalité + [00:07:05][^5^][5] Parentalité comme un travail exigeant * Comparaison de la parentalité à un emploi sans possibilité de démission * L'évolution des rôles de genre et les défis de la coparentalité * L'impact des valeurs individualistes sur la parentalité + [00:10:38][^6^][6] Changements sociétaux affectant la parentalité * L'influence de la contraception et le concept de l'enfant choisi * L'évolution du statut de l'enfant et les droits de l'enfant * Les responsabilités parentales décrites dans la Convention internationale des droits de l'enfant + [00:16:57][^7^][7] Développement des sciences psychologiques et éducation * Pression sur les parents à travers les médias et les professionnels * L'importance de l'engagement parental et les recommandations sur la bonne parentalité * La nouvelle pression historique sur les parents et leurs responsabilités + [00:19:02][^8^][8] Le glissement vers le burn-out parental * La différence entre la pression sociétale et le burn-out parental * Description du burn-out parental et ses symptômes * L'importance de l'investissement parental et le contraste avec le burn-out Résumé de la vidéo [00:24:17][^1^][1] - [00:44:58][^2^][2]:
La conférence aborde le burn-out parental, ses symptômes, ses causes et ses conséquences sur les parents et les enfants. Elle souligne l'importance de l'équilibre entre les stresseurs et les ressources disponibles pour les parents, et propose des solutions pour prévenir et traiter le burn-out parental.
Points forts: + [00:24:17][^3^][3] Symptômes du burn-out parental * Témoignage d'une mère épuisée par les demandes constantes de ses enfants * Différenciation entre burn-out parental et dépression + [00:26:39][^4^][4] Causes du stress parental * Impact du stress sur la santé physique des parents * Comparaison des niveaux de cortisol chez les parents en burn-out et d'autres groupes stressés + [00:30:00][^5^][5] Prévalence du burn-out parental * Statistiques montrant une prévalence élevée dans les pays occidentaux * Discussion sur l'importance de s'occuper du burn-out parental comme un problème de santé publique + [00:38:01][^6^][6] Conséquences et traitement * Effets néfastes sur la santé des parents et le bien-être des enfants * Approches de prévention et de traitement efficaces pour réduire le stress parental Résumé de la vidéo [00:45:00][^1^][1] - [01:06:42][^2^][2]:
La conférence d'Isabelle Roskam aborde les défis de la parentalité moderne, contrastant avec les pratiques des années 80. Elle souligne la pression sur les parents pour répondre aux besoins académiques, émotionnels, nutritionnels et sociaux des enfants, tout en évitant la surstimulation et en favorisant une alimentation saine. Roskam discute de l'isolement croissant des parents dans une société individualiste et plaide pour un retour à la solidarité communautaire, rappelant le proverbe africain selon lequel il faut tout un village pour élever un enfant.
Points forts: + [00:45:00][^3^][3] Contraste entre la parentalité en 1982 et 2019 * Pression pour répondre à tous les besoins des enfants * Différences dans les attentes et les pratiques éducatives * Humour pour souligner les changements sociétaux + [00:46:25][^4^][4] Parentalité solitaire dans la société moderne * Individualisme et réticence à demander de l'aide * Importance de partager les responsabilités parentales * Nécessité de soutien communautaire et informel + [00:50:11][^5^][5] Réflexion sur les sociétés collectivistes * Comparaison avec les modèles éducatifs où l'enfant est élevé par la communauté * Discussion sur l'adaptation des sociétés occidentales à ces modèles * Soutien formel et informel et leur impact sur la parentalité + [00:59:31][^6^][6] Équilibre personnel et parentalité * Gestion du stress parental et importance de maintenir une identité diversifiée * Rôle du travail et de la carrière dans la prévention de l'épuisement parental * Influence du nombre d'enfants et de la dynamique familiale sur le bien-être parental
Jackson Laboratories Strain #001800
DOI: 10.3390/kinasesphosphatases3040020
Resource: RRID:IMSR_JAX:001800
Curator: @dhovakimyan1
SciCrunch record: RRID:IMSR_JAX:001800
Plasmid_84329
DOI: 10.3390/kinasesphosphatases3040020
Resource: RRID:Addgene_84329
Curator: @scibot
SciCrunch record: RRID:Addgene_84329
RRID:SCR_012553
DOI: 10.3390/kinasesphosphatases3040020
Resource: Kinexus Bioinformatics Corporation (RRID:SCR_012553)
Curator: @scibot
SciCrunch record: RRID:SCR_012553
RRID:SCR_018712
DOI: 10.1016/j.celrep.2025.116318
Resource: QuantStudio 3 Real Time PCR System (RRID:SCR_018712)
Curator: @scibot
SciCrunch record: RRID:SCR_018712
La Faculté de Punir : Analyse des Transformations du Châtiment des Enfants
Résumé
Ce document de synthèse analyse l'évolution de la "faculté de punir" appliquée aux enfants, en s'appuyant sur les travaux de Didier Fassin.
Il met en lumière un paradoxe central : alors que les sociétés occidentales sont devenues de plus en plus punitives envers les adultes depuis la seconde moitié du XXe siècle, elles se sont montrées de plus en plus protectrices à l'égard des enfants.
Cette protection a évolué à travers une série de redéfinitions successives des mauvais traitements, passant d'un cadre légal et familial (la "correction modérée") à des cadres médical (le "syndrome de l'enfant battu"), de santé publique (la "maltraitance infantile") et enfin psychologique et sexuel (les "abus sexuels").
Cette tendance globale à la protection a conduit à une prohibition croissante des châtiments corporels, bien que de manière inégale à travers le monde, avec des exceptions notables comme les États-Unis.
Cependant, l'analyse révèle que cette protection est elle-même vulnérable.
L'État, en revendiquant le monopole de la faculté de punir, restreint le droit de correction dans la sphère privée tout en se réservant le droit de châtier les mineurs dans des cadres spécifiques.
Cette vulnérabilité se manifeste de deux manières principales :
En définitive, bien que la punition des enfants ait reculé dans la sphère privée, la protection qui leur est accordée par la loi reste fragile et soumise aux pressions politiques qui associent délinquance et immigration, remettant ainsi en cause le statut d'exception du mineur.
1. La Nature et la Légitimité de la Punition
Punir, dans son acception la plus générale, consiste à infliger une forme de souffrance (physique ou psychique) à une personne supposée avoir violé une loi, une morale ou une norme.
Cette action est réputée légitime socialement car elle vise à corriger une infraction et à rétablir l'ordre. La légitimité du châtiment peut revêtir plusieurs dimensions :
• Légale : Lorsqu'elle est prononcée par une autorité judiciaire au terme d'un procès.
Ce système formel est lui-même historiquement, culturellement et politiquement déterminé.
• Sociale : En dehors du cadre légal, dans des "mondes sociaux" qui définissent leurs propres règles (ex: institution scolaire, milieux mafieux).
L'analyse se concentre principalement sur une évolution paradoxale : alors que l'appareil punitif de l'État est devenu plus sévère ("punitif"), la sphère familiale l'est devenue de moins en moins.
La légitimité du châtiment s'est renforcée dans l'espace public tout en se restreignant dans l'espace privé, notamment concernant les enfants.
2. La Généalogie du Châtiment de l'Enfant : De la Puissance Paternelle à la Protection
2.1. De la "Patria Potestas" à la "Correction Modérée" L'histoire du châtiment des enfants est marquée par une longue évolution depuis le pouvoir quasi absolu du père dans l'Empire romain.
• La Patria Potestas : Ce pouvoir du père sur ses enfants, allant jusqu'au droit de vie et de mort, était quasi absolu dans le cadre légal romain.
Il était cependant limité en pratique par des facteurs sociaux (mariage tardif, espérance de vie réduite) et une évolution progressive du droit romain lui-même.
• L'Évolution vers la Protection : Au fil des époques (médiévale, classique, Lumières), une obligation de protection de l'enfant s'est associée à la puissance paternelle, qui a également été étendue à la mère.
• La "Correction Modérée" : Au XIXe siècle, comme le note Philippe Antoine Merlin en 1813, le "droit de vie et de mort fut réduit à une simple correction, à un châtiment modéré".
Toutefois, les critères de cette modération n'étant pas définis, la législation protégeait peu l'enfant contre la violence parentale.
2.2. L'Intervention de l'État : Les Lois de la Fin du XIXe Siècle
La fin du XIXe siècle en France marque un tournant avec l'intervention de l'État dans la sphère familiale pour protéger les enfants.
• Loi du 24 juillet 1889 ("sur la protection des enfants maltraités et abandonnés") :
Elle prévoit la déchéance de la puissance paternelle pour les parents condamnés pour des crimes ou délits sur leurs enfants, ou dont le comportement (ivrognerie, mauvais traitements) compromet la santé, la sécurité ou la moralité des enfants.
• Loi du 19 avril 1898 ("sur la répression des violences...") :
Elle précise les infractions (coups, privation d'aliments) et alourdit les peines si les auteurs sont les parents ou une personne ayant autorité sur l'enfant, pouvant aller jusqu'aux travaux forcés à perpétuité.
Selon l'historien Georges Vigarello, ces lois s'inscrivent dans une triple transformation de la société :
Ces lois participaient ainsi à une double logique : la disciplinarisation des pauvres et la moralisation des enfants perçus comme de potentiels futurs délinquants.
2.3. La Reconfiguration des Mauvais Traitements : Une Triple Redéfinition
Au XXe siècle, les mauvais traitements ont connu trois redéfinitions successives, les inscrivant dans de nouveaux champs de savoir et d'action.
2.3.1. Le Cadre Médical : Le "Syndrome de l'Enfant Battu"
La médecine a joué un rôle crucial dans l'identification des violences familiales.
• Ambroise Tardieu (1860) : Ce médecin légiste français fut le premier à décrire le tableau clinique de sévices et mauvais traitements (hématomes multiples, séquelles de fractures), mais son travail eut peu d'écho à l'époque.
• Frederic Silverman (1953) : Ce radiologue pédiatrique américain identifie l'association de fractures multiples comme un signe de traumatismes.
En 1962, avec des collègues, il nomme ce tableau le Battered child syndrome ("syndrome de l'enfant battu"), ce qui initiera des lois imposant le signalement des mauvais traitements.
• Aujourd'hui : Le terme de "traumatisme non accidentel" (non-accidental trauma) est préféré pour inclure d'autres formes comme le "syndrome du bébé secoué".
2.3.2. Le Cadre de la Santé Publique : La "Maltraitance Infantile"
Le passage à la santé publique a changé l'échelle d'analyse : du cas individuel à la population, du diagnostic à la prévention.
• Nouveau concept : Le terme "maltraitance infantile" (child maltreatment) apparaît en France dans le dernier quart du XXe siècle, porté par la pédiatrie sociale.
• Changement d'échelle : Les médias se focalisent sur les cas extrêmes (infanticides), invisibilisant la "banalité des mauvais traitements".
Les données épidémiologiques révèlent une prévalence bien plus élevée que ce que les hospitalisations suggèrent.
Indicateur de Maltraitance Infantile
Données (États-Unis)
Données (France)
Enfants référés pour suspicion (avant 5 ans)
Proportion d'enfants victimes (violence/négligence) 19,2 % (enquête nationale 2010) - Taux basé sur les hospitalisations (formes graves) - 0,11 % (fortement sous-estimé) Taux de violence physique (enquêtes pop.) - Entre 4 % et 16 % (travaux internationaux) Taux de violence psychologique (enquêtes pop.) - Entre 4 % et 10 % (travaux internationaux)
2.3.3. La Reconnaissance Tardive : Les Abus Sexuels
Les abus sexuels sur enfants ont été occultés ou niés pendant la majeure partie du XXe siècle par différentes sphères :
• La médecine : A longtemps interprété les infections génitales chez les enfants comme des conséquences de la promiscuité ou du manque d'hygiène, désexualisant les pratiques.
• Le politique : Des parlementaires masculins se sont opposés aux réformes féministes.
• Le judiciaire : Le traitement dubitatif et inquisiteur des plaintes était dissuasif pour les victimes.
• La psychanalyse : A pu être instrumentalisée pour conforter le "négationnisme ambiant" en réduisant les accusations à des fantasmes.
Ce n'est qu'au début du XXIe siècle que le sujet entre dans l'espace public, révélant une ampleur considérable.
Données sur les Abus Sexuels
Méta-analyse (Europe/Amérique du Nord)
Rapport CIIVISE 2023 (France)
Garçons/Hommes
2,6% (sans contact) / 4% (avec contact)
1,5 million d'hommes victimes (6,4%)
Filles/Femmes
6,7% (sans contact) / 12,7% (avec contact)
3,9 millions de femmes victimes (14,5%)
Plus d'un adulte sur 10
Contexte
8% des cas avant 5 ans
Inceste dans plus de 8 cas sur 10
8,5 ans en moyenne
3. La Prohibition Mondiale des Châtiments Corporels : Une Progression Inégale
Parallèlement à la reconfiguration des violences en mauvais traitements, un mouvement mondial d'interdiction des châtiments corporels a émergé.
3.1. L'Interdiction dans la Sphère Familiale
• Pionnière : La Suède a été le premier pays au monde à inscrire cette interdiction dans sa législation en 1979.
• Progression : En 2000, 11 pays avaient suivi. En 2022, on en comptait 66.
• La France : Condamnée par le Conseil de l'Europe en 2015, la France est devenue le 56e pays à adopter une telle législation le 2 juillet 2019 avec la loi "relative à l'interdiction des violences éducatives ordinaires".
• Pays Réfractaires : En 2022, 133 pays ne l'avaient pas fait, parmi lesquels les États-Unis, le Canada, l'Australie, la Russie, l'Inde et le Royaume-Uni.
3.2. L'Exceptionnalisme Américain : La Persistance des Punitions à l'École
L'interdiction des châtiments corporels en milieu scolaire est plus répandue : 136 pays l'avaient adoptée en 2022. L'exception la plus marquante est celle des États-Unis.
• Légalité : La pratique reste autorisée dans les établissements privés (sauf 4 États) et dans les établissements publics de 17 États, principalement dans le Sud.
• Validation par la Cour Suprême : L'affaire Ingraham v. Wright (1977) a validé cette pratique.
La Cour a jugé que le 8e amendement de la Constitution (interdisant les "punitions cruelles et inhabituelles") ne s'appliquait pas aux élèves car il avait été conçu pour les criminels.
• La Dimension Raciale Invisibilisée : L'analyse souligne que cette affaire présente une dimension raciale cruciale mais ignorée :
"malgré un corpus considérable de recherche en sciences sociales établissant à la fois l'inefficacité des châtiments corporels et les dommages sociaux très graves qui peuvent en résulter, le système judiciaire s'obstine à récuser toute mise en cause de la constitutionnalité de cette forme de punition." - Dina PoKempner Sacks
3.3. Au-delà du Châtiment Corporel : Les Nouvelles Formes de Discipline Scolaire
La suppression des punitions physiques ne doit pas occulter la persistance d'autres formes de sanctions, qui affectent de manière disproportionnée les élèves des catégories défavorisées et des minorités ethnoraciales.
• Sanctions classiques : Heures de retenue, exclusions définitives.
• Sanctions moins visibles : Humiliations, stigmatisations.
• Nouveaux motifs : En France, le principe de laïcité tel que défini par la loi du 15 mars 2004 a créé de nouveaux motifs de sanction.
Pour l'année 2022-2023, 3 881 signalements ont été transmis, dont la moitié pour des tenues comme des jupes ou robes longues.
4. La Vulnérabilité de la Protection : Le Mineur face à l'État Punitif
4.1. Le Double Principe : L'Enfant comme Objet de Protection et Sujet de Droit
La protection accrue des mineurs repose sur une double argumentation, en apparence contradictoire mais qui se renforce mutuellement :
1. L'enfant comme objet de protection : En raison de son "manque de maturité physique et intellectuelle", il a besoin d'une protection spéciale (Déclaration des Droits de l'Enfant, 1959).
2. L'enfant comme sujet de droit : Il possède des droits fondamentaux au même titre qu'un adulte, en vertu de sa "dignité et de la valeur de la personne humaine" (Charte des Nations-Unies, 1945).
Ce double principe fonde l'abolition des châtiments corporels.
Cependant, il révèle surtout que l'État devient le maître du jeu, revendiquant le monopole de la faculté de punir et s'autorisant lui-même à infliger des châtiments aux enfants dans des cas précis (ex: tribunaux militaires israéliens jugeant des enfants palestiniens dès 12 ans).
4.2. La Justice Pénale des Mineurs en France : Du Souci de Protection au Durcissement Sécuritaire
L'histoire de la justice des mineurs en France illustre une tension permanente entre protection et punition.
• L'Ordonnance du 2 février 1945 : Marque un "âge presque révolu" où la protection primait sur la punition.
• Le Durcissement depuis les années 1990 : Sous l'effet du populisme pénal et de l'instrumentalisation de faits divers, la tendance s'est inversée.
◦ Érosion des principes de 1945 : Le législateur a progressivement restreint la présomption de non-discernement (avant 13 ans) et l'excuse de minorité, multiplié les lieux d'enfermement et rapproché la justice des mineurs de celle des adultes.
◦ Exemples de mesures : Création de la rétention judiciaire pour les moins de 13 ans (1994), création des centres éducatifs fermés (2002), abaissement temporaire de l'âge de responsabilité pénale à 10 ans.
• La Réponse des Magistrats : Les données statistiques sur la période 2000-2019 montrent un tableau contrasté.
◦ La délinquance juvénile est stable, voire en diminution.
◦ Le taux de réponse pénale augmente fortement (de 78% à 93%).
◦ Les magistrats privilégient les alternatives aux poursuites.
◦ Lorsqu'ils condamnent à la prison, les peines sont plus longues (quantum moyen passant de 5,5 à 9 mois) et les détentions provisoires également (de 3,4 à 7,1 mois).
◦ Les mesures éducatives sont en recul d'un quart, tandis que les sanctions éducatives sont multipliées par six.
En somme, les magistrats semblent vouloir moins condamner, mais le faire plus lourdement, tout en privilégiant des sanctions à vocation éducative plutôt que des mesures de pur accompagnement.
4.3. Les Mineurs Non Accompagnés (MNA) : Entre Protection et Répression
Le cas des mineurs étrangers non accompagnés (MNA) illustre une autre forme de mise en cause de la protection. Ici, la tension n'est pas entre protection et punition, mais entre protection et répression.
• Cadre Juridique : En principe, les MNA ne peuvent se voir opposer leur absence de titre de séjour et doivent être pris en charge par l'Aide Sociale à l'Enfance (ASE), relevant de la protection.
L'évaluation de leur minorité par les conseils départementaux est donc cruciale.
• Réalité de Terrain (Enquête dans les Hautes-Alpes) :
L'enquête montre que le protocole légal n'est souvent pas respecté.
◦ Refoulements illégaux à la frontière.
◦ Politique de "déminorisation" par les services départementaux, soumise à la pression politique sur les coûts. Dans le département étudié, le taux de reconnaissance est passé de 46% en 2017 à 4% en 2019.
◦ Logique de suspicion systématique durant les entretiens d'évaluation, où tout élément du récit peut être retourné contre le jeune pour contester sa minorité.
• Conséquences : Le rejet de leur minorité condamne ces jeunes à la précarité, l'errance et l'exposition à de multiples violences, voire les pousse vers des activités illicites pour survivre.
5. Conclusion : La Vulnérabilité de l'Exception Mineure
L'analyse des transformations du châtiment des enfants révèle une dynamique complexe.
Si la société a évolué vers une plus grande protection des mineurs dans la sphère privée, cette protection est loin d'être absolue et reste éminemment vulnérable.
Deux logiques distinctes mais convergentes sont à l'œuvre :
Dans les deux cas, l'exception dont les mineurs peuvent théoriquement se prévaloir est remise en cause par des politiques exigeant plus de sévérité.
Le rapprochement opéré dans le débat public entre criminalité et immigration ne fait que renforcer cette tendance, menaçant de faire converger le traitement des mineurs délinquants et des mineurs étrangers vers un même horizon répressif et punitif.
quant au Royaume-Uni il a été condamné par la Cour européenne des droits de l'homme le 23 septembre 00:28:21 1998 dans le cadre de l'affaire a versus United Kingdom pour ne pas avoir respecté l'article le 3 de la Convention européenne des droits de l'homme qui stipule que je cite nul ne peut être soumis à la torture ni à des peines ou 00:28:34 traitements inhumains ou dégradants un tribunal anglais avait en effet considéré non coupable un homme qui pour le corriger avait frappé son Beaufils de 9 ans avec une baguette en laissant sur ses cuiss plusieurs marques qui avait fait l'objet d'un signalement 00:28:47 par l'instituteur et avait été attesté par le certificat d'un pédiatre
les mineurs eux ne peuvent pas légalement se voir opposer 00:51:59 leur absence de titre de séjour en France ils sont censés être pris en charge par les services départementaux de l'aide sociale à l'enfance en d'autres termes leur traitement ne relève pas de la répression comme c'est 00:52:12 le cas pour les étrangers majeurs mais de la protection au titre de leur minorité la détermination de cette dernière par le Conseil départemental s'avère donc crucial savoir s'ils ont 00:52:24 plus ou moins de 18 ans il s'agit d'évaluer sur la base des documents présentés par la personne se déclarant mineur et au terme d'un entretien avec elle si elle est bien âgée de Mo- 18 ans 00:52:36 dans ce cas elle peut-être placée par l'autorité judiciaire soit dans un foyer soit confié à 1/3 dans le cas contraire elle est considérée comme adulte avec le risque 00:52:49 d'être expulsée si elle ne parvient pas à obtenir un titre de séjour par exemple dans le cadre de l'asile tel est donc le cadre juridique
à partir du milieu des 00:45:24 années 1990 la tendance inverse avec un durcissement de la législation chaque fois qu'une majorité de droite revient au pouvoir et une correction seulement partielle lorsque c'est la gauche qui 00:45:35 gouverne ainsi en 1994 on institue la rétention judiciaire autrement dit la garde à vue pour les moins de 13 ans en 1996 on permet la comparution immédiate et la comparution devant le juge des 00:45:49 enfants sans instruction préalable en 2002 on crée les centres éducatifs fermés ainsi que les établissements pénitentiaires pour mineurs et on abaisse l'âge de la responsabilité pénale de 13 à 10 ans 00:46:01 autorisant des sanctions beaucoup plus tôt dans la vie le code de la justice pénale des mineurs rétablira en fait en 2021 la limite de 13 ans en 2007 les exception 00:46:13 permettant de ne pas appliquer l'excuse de minorité pour les les mineurs de plus de 16 ans sont élargies ces dispositions seront toutefois abreugé en 2014 la pleine excuse de minorité se trouvant 00:46:25 alors rétablie en 2007 encore on supprime l'atténuation de la peine pour les mineurs de 16 ans en cas deuxèe récidif s'il commett un délit avec violence ou agression sexuelle en 00:46:38 2011 les tribunaux correctionnels pour mineurs sont créés pour juger les délits punis de plus de 3 ans d'emprisonnement en récidive par des adolescents de plus de 16 ans ils seront 00:46:50 cependant supprimé en 2016 en 2019 on permet d'appliquer au mineurs de plus de 13 la détention à domicile sous surveillance électronique progressivement ainsi avec 00:47:02 ces balancements que je vous ai indiqué le législateur érode le principe de protection de l'ordonnance de 1945 restreint les effets de la 00:47:14 présomption de non discernement et de l'excuse de minorité multiplie les lieux d'enfermement et les possibilités de peine correspondantes et rapproche la justice pénale des mineurs de la justice 00:47:27 pénale des adultes et vous aurez certainement remarqué que c'est un débat qui aujourd'hui est à nouveau sur la table
Résumé de la vidéo [00:00:00][^1^][1] - [00:25:06][^2^][2]:
Cette vidéo présente une conférence de Didier Fassin sur la faculté de punir, explorant les aspects légaux, sociaux et historiques de la punition. Fassin discute de la légitimité de la punition dans différents contextes, y compris la justice formelle, l'éducation et la famille.
Points forts: + [00:00:29][^3^][3] Définition de la punition * Infliger une souffrance pour violation de la loi, morale ou norme * Peut être physique ou psychique, légale ou informelle + [00:04:00][^4^][4] Évolution de la punition des enfants * Tendance vers moins de punition dans la famille malgré un État plus punitif * La légitimité du châtiment parental diminue + [00:07:12][^5^][5] Protection légale des enfants en France * Lois de 1889 et 1898 pour protéger les enfants maltraités et abandonnés * Introduction de la déchéance de la puissance paternelle + [00:12:00][^6^][6] Redéfinitions des mauvais traitements * Inclusion dans la chaîne pénale et reconnaissance médicale et publique * Évolution vers la prévention et la sensibilisation aux abus sexuels Résumé de la vidéo [00:25:08][^1^][1] - [00:49:11][^2^][2]:
Cette partie de la vidéo aborde l'évolution de la législation sur les châtiments corporels, en particulier dans le contexte familial et scolaire. Elle met en lumière les changements dans les lois et les attitudes envers la discipline physique des enfants, en soulignant les différences entre les pays et les évolutions au fil du temps.
Points forts: + [00:25:08][^3^][3] L'histoire des châtiments corporels * Suppression progressive des châtiments corporels dans de nombreux pays * La France a adopté une législation interdisant les violences éducatives ordinaires en 2019 * Évolution des attitudes et des lois concernant la discipline physique des enfants + [00:28:00][^4^][4] La situation aux États-Unis et au Royaume-Uni * Les États-Unis maintiennent le droit de corriger physiquement les enfants * Le Royaume-Uni a été condamné pour ne pas avoir respecté les droits de l'homme en matière de châtiments corporels * Discussion sur la dimension raciale de la violence scolaire aux États-Unis + [00:33:04][^5^][5] L'exceptionnalisme américain dans la discipline scolaire * Les punitions physiques restent autorisées dans certains États américains * La Cour suprême des États-Unis a validé ces pratiques en 1977 * Analyse de la persistance de ces pratiques malgré leur inefficacité et leurs dommages sociaux + [00:39:01][^6^][6] Le monopole de l'État sur la faculté de punir * L'État revendique le monopole de l'usage légitime de la faculté de punir * Discussion sur la protection des enfants et la responsabilité pénale des mineurs * Évolution de la justice pénale des mineurs vers plus de sévérité depuis les années 1990 Résumé de la vidéo [00:49:13][^1^][1] - [01:01:00][^2^][2]:
Cette partie de la vidéo aborde la justice pénale des mineurs en France, en mettant l'accent sur la tension entre protection et punition. Didier Fassin discute de l'augmentation des réponses pénales dans un contexte de délinquance juvénile stable ou en baisse, et de la pression politique et policière sur les magistrats. Il souligne également la situation particulière des mineurs non accompagnés (MNA) et les défis liés à leur évaluation et protection.
Points forts: + [00:49:13][^3^][3] Justice pénale des mineurs * Augmentation des réponses pénales malgré la stabilité de la délinquance * Préférence pour les alternatives aux poursuites plutôt que l'emprisonnement * Pressions politiques et policières influençant les décisions judiciaires + [00:50:53][^4^][4] Mineurs non accompagnés (MNA) * Difficultés d'évaluation de l'âge et de la minorité * Protection offerte par la convention internationale des droits de l'enfant * Enjeux politiques et administratifs liés à la prise en charge des MNA + [00:52:57][^5^][5] Enquête sur le traitement des MNA * Protocole souvent non suivi pour l'évaluation des MNA * Pression politique sur les personnels évaluant la minorité * Conséquences graves du rejet de la minorité pour les jeunes + [00:59:47][^6^][6] Protection des mineurs et politiques de sévérité * Vulnérabilité de la protection accordée aux mineurs * Tension entre protection et répression pour les MNA * Impact des politiques sur la vie des jeunes rejetés
Résumé de la vidéo [00:00:01][^1^][1] - [00:23:31][^2^][2]:
Cette vidéo présente le jeu Hexagone utilisé dans un lycée pour discuter de la laïcité.
L'activité ludique encourage les élèves à débattre et à réfléchir de manière critique sur le sujet.
Le professeur guide les élèves à travers des discussions constructives, en soulignant l'importance de la laïcité comme cadre permettant la liberté de conscience et de culte.
Points forts: + [00:00:41][^3^][3] Introduction du jeu Hexagone * Utilisé pour engager les élèves dans le débat sur la laïcité * Encourage l'autonomie et l'esprit critique * Les élèves travaillent en équipe pour répondre à des questions + [00:06:00][^4^][4] La laïcité dans l'éducation * Un concept mal compris par les jeunes * Le débat permet d'explorer différentes perspectives * Importance de discuter pour construire une compréhension commune + [00:11:00][^5^][5] Liberté de culte et espaces neutres * Discussion sur la possibilité d'avoir des salles de prière dans les écoles * La laïcité doit préserver la liberté de culte tout en maintenant la neutralité * Les élèves apprennent à naviguer entre les tensions et les ambiguïtés de la laïcité + [00:14:00][^6^][6] La laïcité constructive vs restrictive * La laïcité ne signifie pas cacher sa religion * Il est essentiel de discuter et de clarifier les termes flous * La laïcité devrait être un cadre pour la liberté plutôt que pour la contrainte Résumé de la vidéo [00:23:33][^1^][1] - [00:31:46][^2^][2]:
Cette partie de la vidéo discute de l'utilisation du jeu "L'Hexagone" dans les lycées pour aborder le sujet de la laïcité. L'intervenant explique comment il intègre cette activité ludique dans ses cours pour faciliter l'apprentissage des élèves sur des sujets complexes, en utilisant des hexagones pour organiser et relier des concepts.
Points forts: + [00:23:33][^3^][3] Utilisation en classe * Peut être utilisée comme activité d'introduction ou de conclusion * Aide à réactiver les préacquis et à visualiser les enjeux * Encourage la discussion et la négociation entre élèves + [00:25:08][^4^][4] Préparation du matériel * Les élèves découpent et collent les hexagones eux-mêmes * Activité manuelle qui stimule la réflexion et le débat * Nécessite une gestion du temps et une organisation en classe + [00:29:47][^5^][5] Encouragement de l'innovation pédagogique * Pas de prérequis spécifiques pour proposer cette activité * Importance de la formation des enseignants à de nouvelles méthodes * Partage et mutualisation des ressources entre collègues
L'École et les Valeurs de la République : Synthèse de la Journée d'Études
Résumé
Ce document de synthèse analyse les thèmes, arguments et données clés présentés lors de la journée d'études intitulée "L'École, un territoire vivant au cœur des valeurs de la République".
Organisée par l'INSPÉ de l'Académie de Lille, cette journée s'est déroulée dans un contexte marqué par l'attentat d'Arras, conférant une acuité particulière aux débats.
Les interventions soulignent unanimement la mission première de l'École, inscrite dans le Code de l'éducation, de faire partager les valeurs de la République.
Cette mission s'ancre dans un héritage historique profond, allant des Lumières aux lois Jules Ferry, et vise à former des citoyens émancipés par la raison et le savoir.
Une analyse sémantique et juridique révèle que la notion de "valeurs de la République" est d'usage récent, tant dans le discours public que dans les textes de loi, avec une augmentation significative depuis les années 1980.
Ces valeurs ne sont pas figées ; elles évoluent et s'enrichissent, comme en témoigne l'intégration de la lutte contre les discriminations.
Le droit ne leur donne pas de définition constitutionnelle, et leur mention prédomine dans le Code de l'éducation et le droit des étrangers.
Sur le plan pédagogique, un consensus émerge sur la nécessité de dépasser une "pédagogie de la prescription" pour atteindre une "pédagogie de la conviction".
Cette "approche citoyenne" refuse l'inculcation et promeut la pensée critique, l'expérimentation des valeurs au quotidien et la coopération.
L'objectif est de permettre aux élèves non seulement de connaître les valeurs, mais de les "éprouver" et d'en ressentir le bénéfice, transformant l'école en un "écosystème de valeurs".
Enfin, les discussions mettent en lumière les défis contemporains : le poids croissant qui pèse sur l'institution scolaire, le communautarisme, le relativisme et la nécessité de ne pas nier le réel tout en présentant les valeurs comme un idéal à conquérir.
L'écart entre la valeur et le réel est présenté non comme un échec, mais comme l'espace même de l'engagement citoyen.
1. La Mission Fondamentale de l'École dans un Contexte de Crise
Les propos introductifs des différents intervenants ont unanimement rappelé le rôle central et fondateur de l'École dans la transmission des valeurs républicaines, une mission rendue encore plus cruciale par le contexte contemporain.
1.1 Un Fondement Juridique et Historique
La mission de l'École est clairement définie par l'article L111-1 du Code de l'éducation, cité à plusieurs reprises, qui stipule que "la nation fixe comme mission première à l’école de faire partager aux élèves les valeurs de la République".
Cette mission n'est pas un simple "supplément d'âme" mais une obligation professionnelle qui constitue l'armature du projet républicain.
Les intervenants ont inscrit cette mission dans une profondeur historique :
• Les Lumières et la Révolution : Alain Frugère a évoqué l'esprit des Lumières (Molière), le projet d'instruction publique de Condorcet (1792) qui établit la primauté des savoirs issus de la recherche sur les opinions et les croyances, et le "pari de la raison émancipatrice".
• Le 19ème siècle : Madame Looher a rappelé le projet des républicains de la Troisième République (Gambetta, Ferdinand Buisson) de stabiliser le régime grâce à l'éducation, aboutissant aux lois Jules Ferry de 1881-82 qui instaurent un enseignement fondé sur la gratuité, l'obligation et la laïcité.
1.2 Le Poids du Contexte Actuel
La journée d'études, bien que planifiée de longue date, a été profondément marquée par l'assassinat de Dominique Bernard à Arras.
Cet événement a donné une "coloration tout à fait particulière" aux réflexions, comme l'a souligné Sébastien Jaibovski.
Ce contexte met en lumière plusieurs tensions :
• Le Poids sur l'Institution : Sébastien Jaibovski a soulevé la question du "poids qui aujourd'hui est très important, peut-être trop important" que la société fait peser sur l'École et ses enseignants.
• La Conquête Permanente : Il a également insisté sur le fait que "les valeurs ne sont jamais acquises mais elles sont toujours à être conquises et à conquérir".
• Les Défis Sociétaux : Alain Frugère a mentionné "le repli sur soi, le communautarisme, l'intolérance voire la haine" comme des défis quotidiens, tandis que Mathieu Clouet a listé les inégalités sociales, les effets de l'économie médiatique et le relativisme.
2. Analyse de la Notion de "Valeurs de la République"
L'intervention d'Ismaël Ferrat, professeur des universités, a offert une analyse lexicale et juridique détaillée, démontrant que la notion de "valeurs de la République" est à la fois complexe, évolutive et d'émergence récente.
2.1 Une Apparition Récente dans le Discours Public et Juridique
Contrairement à une idée reçue, l'usage du syntagme "valeurs de la République" est un phénomène récent.
• Dans les publications : Une analyse des corpus de textes numérisés (Google Books) et des archives du journal Le Monde montre une quasi-absence du terme jusqu'aux années 1980, suivie d'une "explosion" de son usage à partir de 1989.
• Dans le droit : L'occurrence du terme dans les codes juridiques français est très faible au début des années 2000 et connaît une forte poussée à partir de 2016.
Cette augmentation est principalement due à deux codes :
2.2 Une Définition Juridique Absente et Évolutive
L'analyse juridique révèle un paradoxe : bien que la notion soit de plus en plus utilisée, elle reste juridiquement insaisissable.
• Absence de définition constitutionnelle : Aucun texte constitutionnel ne définit précisément ce que sont les valeurs de la République. Le Conseil constitutionnel n'a produit aucune étude sur le sujet.
• L'avis du Conseil d'État : Saisi lors du projet de loi "séparatisme", le Conseil d'État a jugé la notion de "valeurs" trop large pour être un principe de droit généralisable, lui préférant celle de "principes républicains".
• Des valeurs évolutives : La liste des valeurs n'est pas figée. La lutte contre les discriminations, par exemple, est une valeur aujourd'hui considérée comme une évidence, alors que le premier article du Code pénal sur ce sujet ne date que de 1994.
3. L'Approche Pédagogique : De la Prescription à la Conviction
Mathieu Clouet, représentant l'équipe académique Valeurs de la République, a développé le concept d'une "approche citoyenne des valeurs à l'école", qui se distingue par son refus de l'inculcation au profit d'une adhésion réfléchie.
3.1 Refuser l'Inculcation, Viser la Conviction
L'objectif n'est pas seulement de faire connaître les valeurs, mais de les "faire partager".
• Pédagogie de la conviction : "Nous ne pouvons pas nous contenter d'une pédagogie de la prescription, il nous faut trouver la voix d'une pédagogie de la conviction."
• Appel à la raison : Cette approche repose sur l'éducation à la liberté, fait appel à la pensée critique et apprend aux élèves à interroger les valeurs elles-mêmes.
• Les trois dimensions de la valeur : Elle doit prendre en compte les dimensions
intellectuelle (contenus),
psycho-affective (ressenti) et
conative (action).
3.2 L'École comme "Écosystème de Valeurs"
Pour que les valeurs aient du prix aux yeux des élèves, ils doivent les "éprouver", c'est-à-dire en ressentir le bénéfice et en tester la réalité.
• Le rôle des savoirs : La transmission des connaissances participe à l'éducation aux valeurs. Citant Catherine Kintzler, Mathieu Clouet parle de la "puissance libératrice des enseignements" : maîtriser un savoir est une expérience concrète de la liberté.
• L'expérience vécue : L'éducation aux valeurs passe aussi par la coopération, la prise de responsabilité et les pratiques participatives. L'école doit être un lieu où les valeurs sont incarnées au quotidien pour éviter les écarts entre le discours et la réalité.
• Inverser la focale : Il est suggéré de replacer les faits négatifs (discriminations, racisme) dans la perspective plus large de la lutte pour l'égalité.
L'exemple de l'affaire Dreyfus est utilisé pour montrer que la France de l'époque n'était pas seulement celle de l'antisémitisme, mais aussi le seul pays d'Europe où des intellectuels se sont levés pour défendre un Juif.
4. Étude de Cas : l'Enseignement de la Laïcité
Ismaël Ferrat a illustré les enjeux de la transmission des valeurs à travers l'exemple de la laïcité, en analysant son traitement dans les programmes scolaires.
Période
Occurrence du mot "Laïcité" dans les programmes (élémentaire/collège)
Contexte et Enjeux
Années 1970-1980
Quasiment absente La laïcité est considérée comme une évidence, une "non-notion" sur le plan pédagogique.
Années 1990-2000
Forte augmentation
L'émergence est liée à la nécessité d'expliquer les règles, notamment suite à l'affaire du voile de Creil (1989) et la circulaire Bayrou (1994) sur les signes religieux ostensibles.
Depuis 2013 (Loi Peillon) Présence stabilisée à un niveau élevé
Un élève scolarisé aujourd'hui rencontre la notion environ 13 fois entre le primaire et le collège.
L'enjeu pédagogique est double :
Les résultats sont probants : une étude du Knesco montre que 90 % des élèves de 3e et 80 % des lycéens en terminale ont déjà abordé la laïcité en cours et maîtrisent globalement bien la notion. Cela démontre l'efficacité du travail mené en classe.
5. Conclusion : La Valeur comme Engagement et "Refus du Réel" La journée d'études se conclut sur une vision exigeante mais volontariste de la mission de l'École.
La transmission des valeurs de la République n'est pas l'imposition d'un dogme, mais une invitation à participer à un projet collectif de "perpétuelle réinvention démocratique".
Comme l'a formulé Mathieu Clouet, il faut se souvenir qu'"une valeur ça n'est pas seulement un reflet du réel, une valeur c'est aussi un refus du réel".
L'écart entre l'idéal prôné par la valeur (l'égalité, la fraternité) et les imperfections de la société n'est pas un signe d'échec.
Au contraire, "c'est précisément dans cet écart que nous pouvons trouver les moyens d'apporter aux élèves que nous encadrons la volonté d'agir et de s'engager dans la République française".
L'approche citoyenne des valeurs est donc, en définitive, une preuve de l'engagement citoyen de l'ensemble de la communauté éducative.
reply to u/GrandRevolutionary99 at https://reddit.com/r/stationery/comments/1nrkuqf/i_need_help_to_create_my_own_letterhead_for_my/
Typewriter enthusiasts often use 100% cotton or high linen content papers with weights in the 32 pound range for 8.5x11. This gives you some nice tactile feel, but will also feed into most typewriters, even with a solid backing sheet. If you want to do thicker card stocks, then you might opt for a bigger standard typewriter which generally have larger diameter platens and more easily handle much thicker paper (they were meant for doing carbon packs up up to 10 sheets or more.)
When it comes to the look of your letters, you can generally choose between silk (clean, crisp imprints), nylon (almost as clean as silk, but with more "grain"), and cotton typewriter ribbon (which leaves a very grainy/old timey and "typewriter-y" imprint). Comparisons here.
I've got a small fleet of typewriters and prefer to use the pica sizes for personal correspondence. I also tend toward the cursive or Vogue typefaces for those as well.
In the US, a lot of stationers have pre-cut paper and envelopes for 6-3/8" x 8-1/2" paper which is a good size sheet for quick notes. My typewriter pen pal Tom Hanks' most recent letter to me was on a custom page of 7.125 x 10.25" and had space for design at the top and bottom with some reasonable space in the middle. If you do custom designs, be sure to order a box or two of plain stock to use as second, third, etc. pages behind your first page if you tend to write over your first page.
Naturally custom designing your own can be fun as well, but get a few samples of the size and weight you want and try them out before ordering in quantity.
Lenore Fenton can give you tips on making carbon copies of your letters if you want to keep them for your own files while sending out the originals: https://www.youtube.com/watch?v=JUJfCfqgsX0
Searching r/typewriters for stationery, letterhead, paper, etc. might give you some ideas as well.
Otro punto clave de las PINNs es su adaptabilidad al ruido (George Em Karniadakis 2021) donde se ha demostrado que incluso a pesar de que los problemas no están perfectamente bien planteados o si existen parámetros desconocidos la red puede producir resultados significativos. Siendo ambas situaciones mencionadas comúnes en el ámbito científico. Complementado a las PINNs, la arquitectura DeepONet aprende operadores (mapeos entre espacios de funciones) en lugar de solo aproximar funciones a diferencia de las PINNs tradicionales, que predicen soluciones específicas para condiciones fijas, DeepONet es capaz de generalizar a nuevas condiciones iniciales y de frontera sin reentrenamiento, gracias a su estructura de red dual (branch-trunk).
Esto parece más intro o justifiación que conclusiones. Entiendo que lo escribes para introducir a los siguientes párrafos que sí se ven como conclusiones pero usaste 9 líneas, prácticamente un párrafo para ello - entonces, si consideras necesario mencionarlo, resúmelo a 3 líneas máximo.
Modelo contra resultados de Alessio Borgi
Revisando el código y los gráficos, creo que faltan los resultados de Borgi, y porqué separar las comparaciones en 3 subecciones: Borgi, numérico y analítico? puedes hacer 4 filas para comparar todas cualitativamente a la vez.
Here is a secret about college success that not many people know: successful students seek help. They use resources. And they do that as often as necessary to get what they need.
This reading helps me personally by letting me know, Its ok to ask for help. i failed in high school because I was afraid to ask for help. I'm going to use all the help and recourses to make my collage experience a success
There is actually a name for this condition: imposter syndrome. Students who feel like an imposter are worried that they don’t belong, that someone will “expose them for being a fake.” This feeling is pretty common for anyone who finds themselves in a new environment and is not sure if they have what it takes to succeed. Trust the professionals who work with first-year college students: you do have what it takes, and you will succeed. Just give yourself time to get adjusted to everything.
This stands out to me because it is a feeling I've been having since starting school. I've been away from school since dropping out of high school when i was 16. It is reassuring to me to know that its not just me, that its normal I guess. it's saying its normal and I do have what it takes to succeed!
learning in college is your responsibility.
This line is a reminder that I can use in my college career and which I think will be helpful. In this chapter of our studies, we are not spoon-fed anymore that everything will be taught to us one by one. We need to work harder for us to learn.
The relationships you build with your professors will be some of the most important ones you create during your college career.
For me this is an important matter. Having a good relationship with my professors helps me learn a lot more without the pressure of just wanting to pass the course but rather learn a lot from that course and that professor.
This concern often goes with imposter syndrome. Students who worry about making a mistake don’t like to answer questions in class, volunteer for a challenging assignment, and even ask for help from others. Instead of avoiding situations where you may fail, embrace the process of learning, which includes—is even dependent on—making mistakes. The more you practice courage in these situations and focus on what you are going to learn from failing, the more confident you become about your abilities.
I know that I get very nervous and am scared to make mistakes. I have always wanted to try and perfect things no matter what, I believe that is the "perfectionist" in me. I am also drawn to being a people pleaser so I tend to get super nervous because I don't wanna mess up. I think this is the best advice I can get from here because I feel that as soon as I start to embrace the process of learning I will become more confident in myself and my learning ability.
Taking responsibility for your learning will take some time if you are not used to being in the driver’s seat. However, if you have any difficulty making this adjustment, you can and should reach out for help along the way.
This is so important because it is very true. Back in high school we had teachers and others telling us we had to get things done, we had to be in class, and we had to go to school. Now it is our turn to be adults and be disciple making sure we get to class and do the work we have to do. This is not just school now this is part of our careers and so this is a crucial time in our lives to be very in check with reality.
By the middle of the semester, you’ll likely feel much more confident and a little more relaxed. Your grades are improving because you started going to tutoring and using better study strategies. You are looking ahead, even beyond the first semester, to start planning your courses for the next term. If you are working while in college, you may also find that you have a rhythm down for balancing it all; additionally, your time management skills have likely improved.
This reinforces well researched patterns of human adaptability.
Learning happens mostly outside of class and on your own. Faculty are responsible for assigning material and covering the most essential ideas; you are responsible for tracking and monitoring your learning progress.
When reading the differences between high school and college it completely made me rethink my mindset. I went into my first classes thinking that the professor was going to guide us step by step until we were ready to do the assignments, but it was quite the opposite. Because of this, I will use this to help benefit myself for success while in this new step for school.
It comes with its own language and customs, some of which can be confusing or confounding at first. Just like traveling to a foreign country, it is best if you prepare by learning what words mean and what you are expected to say and do in certain situations.
I know it's just the start of the chapter put this specific part of the text really stood out to me. Coming to college feels outer worldly to me, I moved here recently so I pretty much know no one here so it feels extremely foreign to me. I feel as if this passage is important because there's many people entering a new scene like this so calling it out can give a spark of some sort showing that they're not the only person being introduced to this chapter in their life.
Many of the college expectations that have been outlined so far may not be considered common knowledge, which is one reason that so many colleges and universities have classes that help students learn what they need to know to succeed. The term, which was coined by sociologists,7 describes unspoken, unwritten, or unacknowledged (hence, hidden) rules that students are expected to follow that can affect their learning.
The part about the hidden curriculum was really useful. I didn’t know that there were so many things students are expected to do before, during, and after class that professors don’t always say. The tips I will take moving forward are to read before class, take good notes, and review after. This will help me stay organized and do good.
The relationships you build with your professors will be some of the most important ones you create during your college career. You will rely on them to help you find internships, write letters of recommendation, nominate you for honors or awards, and serve as references for jobs.
This stood out to me because it allows me to see my professor as an important tool and mentor. Knowing I can turn to my professor for guidance and knowing that they are there to help further my career gives me piece of mind. Knowing they are helping me with future opportunities and scholarships and references. Building these relationships are very important early on.
Consider, for example, Black Americans, whose darker skin is often not recognized by hand soap and water dispensers in public spaces. This is not a natural limitation of technology—it is a consequence of designers choosing a sensor technology that must necessarily be calibrated for particular skin tones, and then calibrating it for white skin. Design justice argues, then, that some designs, when they cannot be universal, should simply not be made. And if they can be universal, then they should be made in ways that 1) center power inequalities, 2) center the voices of all directly impacted by the design outcomes, 3) prioritize impact on communities over designers’ intents, 4) view designers as facilitators rather than designers, 5) ensure designs are sustainable and community led, and 6) build upon and amplify the solutions that communities have already found.
I never thought about the fact that there are specific designs that have the power to exclude people, but given the broad scope of design, this makes sense and I agree with the sentiment of design justice. One example I can think of when it comes to design justice is video games having fewer playable characters of darker skin tones. Although games have slowly been moving towards representing women, many do not have the option to change skin tones. This is many times an afterthought. This widened my perspective on the idea that companies should hire designers of different groups to be inclusive of all.
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
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We thank the reviewer for their positive comments regarding the research article titled "The Ketogenic Diet Metabolite 1 β-Hydroxybutyrate Promotes Mitochondrial Elongation via Deacetylation and Improves Autism-like Behaviour in Zebrafish" by Uddin GM and colleagues. We appreciate your input, and we will address these comments as indicated below with specific responses to each point raised by reviewers.
The main changes in the updated manuscript are as follows:
We have revised the introduction to now incorporate additional background information on mitochondria, NAD, and mitochondrial dynamics and function. This addition aims to provide readers with a broader understanding of the mitochondrial context in relation to our study.
Furthermore, we recognize that previous studies have explored mitochondrial function in the context of the ketogenic diet. While our specific investigation centered on mitochondrial morphology, we acknowledge the importance of comprehensively investigating mitochondrial function. To this end, we have added new data showing how BHB impacts mitochondrial oxidative phosphorylation in HeLa cells (Sup Fig 2), and how both BHB and NMN impact oxygen consumption/glycolysis in zebrafish (Fig 7).
We have also added new behaviour analysis of the zebrafish (Fig 6), and have re-framed the discussion around neurodevelopment generally, rather than ASD specifically.
Finally, we have now included a section in our manuscript that discusses the limitations of our study. These limitations can be further investigated to explore and characterize the full mechanistic potential behind the effects of the ketogenic diet and/or NMN on mitochondrial dynamics.
This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *
*Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Uddin GM and colleagues presented a research article entitled 'The Ketogenic Diet Metabolite 1 β-Hydroxybutyrate Promotes Mitochondrial Elongation via Deacetylation and Improves Autism-like Behaviour in Zebrafish'. Roles of ketogenic diet (KD) and NAD+ precursors in health promotion and longevity, as well as on the alleviation of a broad range of diseases are evident. However, their roles in autism are not well done, which is the novelty of the current study. Addressing below questions will improve the quality of the paper.
Major concerns 1. In the introduction section, a broad overview of the roles of ketogenic diet (KD) in neurodegenerative disease (and ageing, if possible) should be provided. E.g., the authors should summarize exciting progress on the use of KD to treat Alzheimer's disease in animal models (PMID: 23276384). *
Response: Thank you for your valuable suggestion. While it is true that the KD appears to be beneficial in neurodegenerative (and other disease) models, our focus in this paper is looking at neurodevelopment, rather than all potential benefits of the KD. Nonetheless, we have addressed this comment by incorporating a brief overview of the roles of the KD in neurodegenerative diseases, including Alzheimer's disease (AD), in the introduction section of the manuscript. Specifically, we have summarized the exciting progress made in utilizing KD to treat AD in animal models, as highlighted in the suggested study. This addition helps to provide a better overview of the potential therapeutic effects of KD in neurodegenerative diseases and strengthens the introduction section of the manuscript.
Response: This information and the reference are now added to the discussion.
*In the introduction, a more detailed introduction of NAD+ and its roles in mitochondrial homeostasis (especially mitophagy and the mitochondrial fusion-fission balance) should be included (PMID: 24813611; PMID: 30742114; PMID: 31577933). *
Response: Although our paper focused primarily on mitochondrial fission and fusion, we have incorporated a new paragraph in the introduction to provide a more detailed introduction detailing NAD+ and its roles in mitochondrial homeostasis, specifically highlighting mitophagy. We have included the suggested references.
Response: Thank you for the comment. We agree that exploring the detailed mechanism of how the ketogenic diet (KD) affects NAD+ is an interesting question that will have important implications once answered. However, fully elucidating the mechanism of action would require a more comprehensive investigation, which is beyond the scope of this current project. We have now added this as a future direction in the manuscript.
*Fig. 1: in the NAD+ field, the normal used NR/NMN concentrations are normally high like to use 500 µM to 2-5 mM (as the NAD+ levels in cells are high). In addition to use 50 µM, the authors are strongly to have a dose-dependent study (50 µM, 500µM, 1, 2, 5 mM), and see changes of mitochondrial funciton and parameters. In this condition, NAD+ levels should be also checked. *
Response: We have added new supplemental data showing the initial dose response of the effects of BHB and NMN on mitochondrial morphology, which led us to choosing the relevant doses for the remainder of the paper. Our objective was not to investigate the broad impacts of different NMN concentrations on mitochondrial function and parameters, or NAD+ levels. As such, we have only focused on doses where we see effects on mitochondrial morphology.
*Fig. 2: a comprehensive characterization of mitochondrial fusion-fission should be performed. In addition to the protein evaluated, changes on other key fusion-fission proteins, like Bax, Bak, Mfn-1, Mfn-2, etc should be performed (PMID: 17035996; PMID: 24813611). *
Response: We agree that looking at other key proteins involved in mediating mitochondrial fission and fusion could provide additional insight. Indeed, given the changes in global acetylation that we see, it is expected that some other proteins may also be regulated in this way. However, there are at least a dozen proteins involved in mediating mitochondrial fusion and fission, not to mention many more proteins that regulate these proteins. Unfortunately, it is not feasible to analyze all the proteins involved in mitochondrial fusion-fission. Moreover, looking only at protein levels, doesn't necessarily inform about the activity of any protein. Instead, we concentrated in this paper on investigating known links between protein acetylation and mitochondrial dynamics, particularly focusing on the proteins that have known links to acetylation (i.e., DRP1, OPA1, MFNs). We have added a note in the discussion acknowledging that other means of regulation could also be occurring in parallel.
*Figs. 1-5 were focused on mitochondrial morphology, whether KD and NMN changed mitochondrial funciton should be explored, such as to use seahorse to check ECR and OCR. *
Response: Although our question was focused on morphology, we agree that mitochondrial function is important. We have added new data showing that BHB increases basal oxygen consumption in HeLa cells (Sup Fig 2), as well as new data showing that BHB and NMN influence oxygen consumption and glycolysis in our zebrafish model (Fig 7)
Response: Our data show that these doses are indeed sufficient. We did look at some higher doses for NMN, but these were toxic, leading to poor survival and were not studied further.
*Minor concerns 1. Line 26: For 'a growing list of neurological disorders, including autism spectrum disorder (ASD)', please add AD in. *
Response: Line 26 is part of the abstract, which we feel should be focused more on the main message of the paper, which does not involve AD. As addressed above, we have added AD as an example in the introduction.
*Line 57: For 'with side effects such as gastrointestinal disturbances, nausea/vomiting, diarrhea, constipation, and hypertriglyceridemia being reported', rate of frequency shall be provided if any. *
Response: We have modified the statement to indicate the relative percent of patients suffering the various side effects.
*Reviewer #1 (Significance (Required)):
The novelty of the current study was to investigate effects of KD and NAD+ on autism. This investigation was not performed before and thus is the novelty.
Weakness, effects of KD and NAD+/NMN on mitochondrial function were not well-investigated and should be done. Introduction was not well done, many key information in the fields were not provided which may mislead the readers an over-evaluation of the novelty of the current study.*
Response: As outlined above, we have edited the introduction to include additional information requested by the reviewer. Moreover, our focus in this manuscript was to look at the mechanisms underlying changes in mitochondrial morphology, not mitochondrial function per se, though this is clearly important and related. Nonetheless, as discussed above, we have also added new data showing how BHB impacts mitochondrial function.
*My expertise lies in NAD+, mitochondria, and brain health.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The study examined the effect of beta-hydroxybutyrate and nicotinamide nucleotide on mitochondrial morphology and the molecular pathways which mitigate this effect as well as the effect of these treatments on behavior in zebrafish. The study is well done and well written. The only thing I think that could be improved are the bar in the graph some the significant comparisons. It is sometimes difficult to see which groups are being compared.*
Response: We're happy to adjust how the data is displayed in the relevant bar graphs, but it is not clear exactly what changes the reviewer would like. To some degree this will depend on the specific guideline of the final journal where we hope the manuscript will be published. As such, we have not made changes at this point.
***Referees cross-commenting**
The other reviewers do have some fair comments. Multiple doses would be helpful and showing bioenergetic data would complement the morphological measurements. Additionally, behavioral assays showing changes in social behavior in the Zebrafish would provide a stronger link to ASD. *
Response: As discussed above, we have added new information on doses and mitochondrial bioenergetics. With respect to behaviour, we have added thigmotaxis data and reworked the discussion around behaviour and neurodevelopment so that it is less specific to ASD.
*Reviewer #2 (Significance (Required)):
As beta-hydroxybutyrate is an important substrate for the ketogenic diet, this study helps explain the potential mechanisms in which the ketogenic diet may enhance mitochondrial function.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this paper, Uddin and colleagues have investigated components of the ketogenic diet to understand changes in both mitochondrial morphology and protein expression, and zebrafish locomotor behaviour. They investigate whether beta-hydroxybutyrate (BHB) or nicotinamide nucleotide (NMN) application can later human mitochondria in HeLA cell lines, and also recue a locomotion defect in shank3b+/- zebrafish larvae that have previously been proposed as a model for autism. This study is strengthened by showing data from two species; however the link between the HeLA cell line data and larval zebrafish is not strong. The study would be improved by assessing zebrafish mitochondrial changes after drug application, and testing more than one concentration of BH and NMN in the behavioural assay. This is an interesting study, and it is nicely written and presented. I have made some comments to strengthen the study below.
Major comments My expertise is in modelling some aspects of autism in zebrafish. To this end I have focussed on the zebrafish part of this manuscript more fully. I have several comments related to the zebrafish experiments. 1. The changes in mitochondrial morphology, peroxisome number and mitochondrial protein levels were measured in HeLA cells and not comparable data is shown for zebrafish. The same experiments should be repeated using larval zebrafish or a zebrafish cell line. *
Response: We chose to use HeLa cells for the mechanistic studies due to practical reasons. Cell lines offer a controlled and well-established system for investigating cellular processes and molecular mechanisms. Measuring these parameters in tissues is significantly more challenging and requires different reagents (e.g., antibodies) and methodology (electron microscopy) that are not feasible in the current study.
On the other hand, zebrafish larvae were employed for the behavior studies, which cannot be conducted using cell lines. By utilizing zebrafish, we were able to examine the effects of beta-hydroxybutyrate (BHB) and nicotinamide nucleotide (NMN) on locomotor behavior, providing valuable insights into potential therapeutic implications for autism.
While we acknowledge the limitations of not directly measuring mitochondrial morphology, peroxisome number, and mitochondrial protein levels in zebrafish, we believe that our study provides significant contributions to understanding the effects of BHB and NMN in zebrafish behavior. Future studies could certainly consider incorporating zebrafish-specific experiments to complement the findings in HeLa cells.
Response: These doses chosen initially as they were similar the doses that induced mitochondrial elongation in HeLa cells and were tolerated by the fish larvae. As we saw promising effects at these initial doses, we decided to explore them in more detail. While we agree that it would be worth comparing the effects of additional doses, as well as looking at their effects at other timepoints, such work would be a major endeavour and is beyond the scope of our initial investigations, which we feel are worth reporting in their current state.
With respect to the treatment paradigm, fish larvae were treated 10-48 hours post fertilization, as this is a critical neurogenic developmental timepoint that is often used for exposure studies. Fish do not fully hatch until 3-4 days post fertilization, and display only minimal movement before 5 days, which is why we waited until 5 days to look at movement.
Response: We do not observe any gross changes in fish morphology that might explain a decrease in locomotion. Unfortunately, it is not feasible to look at mitochondrial morphology in the fish at this time. However, based on previous published work showing that the ketogenic diet promotes mitochondrial elongation in mouse brains (PMID:32380723), we would expect mitochondrial morphology also to be changed in the fish. Nonetheless, as we have not examined this directly in fish, we are not making this specific claim in this manuscript.
Response: As requested, we have reanalyzed the zebrafish movement data for a more refined analysis. In the revised version (Fig 6), we include analysis of both speed and distance travelled within a defined time. Importantly, these findings still support differences between WT and shank3b+/- fish that are restored by BHB and NMN to varying degrees.
Response: The reviewer makes an important point that the movement behaviour phenotypes that we see do not necessarily represent classic ASD phenotypes (i.e., repetitive behaviour, reduced sociability, and reduced communication). To begin to address this issue, we analyzed thigmotaxis, which can be a measure of anxiety. Notably, we also see differences that are reversed by BHB and NMN. However, we cannot model all ASD behaviours in a fish model, and we are not set up to look at social behaviour, especially in the young fish that we were studying. As such, even though Shank3 is a recognized ASD gene, and the shank3b+/- model we are studying is a validated ASD model (PMID: 29619162), we have re-phrased the manuscript in the context of neurodevelopment generally, rather than with respect to ASD specifically. As such, we ascribe the movement and thigmotaxis phenotypes as neurodevelopmental phenotypes that are improved by BHB and NMN.
*For the statistics, as far as I can tell, all of the data should be analysed by ANOVA or the non-parametric equivalent followed by a post-hoc test. Please check this and add information about normality in. *
Response: As requested, we have clarified our statistical methodology throughout the manuscript.
For the mechanistic data, we used t-tests for direct comparisons between two groups (e.g., vehicle vs. treatment). While multiple conditions such as vehicles, NMN, BHB, or etomoxir were tested, statistical comparisons were only conducted comparisons between the vehicle and each treatment group individually. As we are not also making comparisons between treatments this is not a multiple comparison, and ANOVA is not applicable in this context. We have clarified this rationale in the manuscript to avoid any confusion.
For the zebrafish study, where multiple factors were involved (e.g., treatments across different time points or conditions), we performed a two-way ANOVA followed by Tukey's post-hoc test to identify specific group differences. This approach was appropriate for analyzing these datasets and ensures robust conclusion.
With respect to normality testing, all datasets were assessed for normality using the Shapiro-Wilk test, and no violations of normality were observed. The updated text now includes these details.
*Minor comments
This has bee corrected.
This has bee corrected.
This has bee corrected.
__Response: __A total of 80-150 cells were counted per condition, and the analyses were performed on 3 independent replicates with 2 independent technical replicates for each treatment condition. The quantification of mean mitochondrial branch length in Figure 1B was measured using Image-J and the MiNA plugin. The measurements were taken from three independent replicates using a standard region of interest (ROI) and randomly selected areas from each image.
In Figure 1D, NAD+ levels were measured 24 hours after treatment of vehicle, βHB, NMN, or Eto+βHB in HeLa cells (n=3-6/group). Each sample lysate represents an independent experimental dish from which coverslips were collected for image analysis.
The difference in sample numbers between Figure 1B and 1D arises because image analysis involves individual cells fixed and stained on coverslips, whereas the NAD assay requires the whole lysate from the entire cell culture dish. Therefore, the higher cell count in Figure 1B represents the number of cells analyzed on coverslips, while Figure 1D represents NAD levels from the lysate normalized to the protein concentration.
*Reviewer #3 (Significance (Required)):
I think that this will be interesting to autism researchers and it could lead to more investigation of the ketogenic diet. Some more work is needed, likely in other model organisms, before this research can be translated to human patients. *
__Response: __We agree that the findings of our study could be of interest to autism researchers and have implications for further investigation of the ketogenic diet (KD). It is important to note that further work, including studies in other model organisms, would be beneficial before translating this research to human patients.
Our study aimed to provide mechanistic insights into the effects of the KD on mitochondrial morphology and behavior. We recognize that the translation of research findings to human patients requires rigorous investigation, including preclinical and clinical studies. Our study contributes to the understanding of the underlying mechanisms involved in the KD's effects, laying the groundwork for future research and potential therapeutic avenues.
We appreciate your perspective and emphasize that our intention is to provide valuable insights into the mechanisms underlying the KD's effects rather than suggesting immediate translation to human patients. Further investigation and validation in diverse models and clinical settings will be necessary before considering clinical applications.
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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In this paper, Uddin and colleagues have investigated components of the ketogenic diet to understand changes in both mitochondrial morphology and protein expression, and zebrafish locomotor behaviour. They investigate whether beta-hydroxybutyrate (BHB) or nicotinamide nucleotide (NMN) application can later human mitochondria in HeLA cell lines, and also recue a locomotion defect in shank3b+/- zebrafish larvae that have previously been proposed as a model for autism. This study is strengthened by showing data from two species; however the link between the HeLA cell line data and larval zebrafish is not strong. The study would be improved by assessing zebrafish mitochondrial changes after drug application, and testing more than one concentration of BH and NMN in the behavioural assay.
This is an interesting study, and it is nicely written and presented. I have made some comments to strengthen the study below.
Major comments
My expertise is in modelling some aspects of autism in zebrafish. To this end I have focussed on the zebrafish part of this manuscript more fully. I have several comments related to the zebrafish experiments.
Minor comments
I think that this will be interesting to autism researchers and it could lead to more investigation of the ketogenic diet. Some more work is needed, likely in other model organisms, before this research can be translated to human patients.
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Uddin GM and colleagues presented a research article entitled 'The Ketogenic Diet Metabolite 1 β-Hydroxybutyrate Promotes Mitochondrial Elongation via Deacetylation and Improves Autism-like Behaviour in Zebrafish'. Roles of ketogenic diet (KD) and NAD+ precursors in health promotion and longevity, as well as on the alleviation of a broad range of diseases are evident. However, their roles in autism are not well done, which is the novelty of the current study. Addressing below questions will improve the quality of the paper.
Major concerns
Minor concerns
The novelty of the current study was to investigate effects of KD and NAD+ on autism. This investigation was not performed before and thus is the novelty.
Weakness, effects of KD and NAD+/NMN on mitochondrial function were not well-investigated and should be done. Introduction was not well done, many key information in the fields were not provided which may mislead the readers an over-evaluation of the novelty of the current study.
My expertise lies in NAD+, mitochondria, and brain health.
Type of placebo acupunctureWe ranked the various placebo acupuncture inter-ventions on a 1-5 scale, where 1 represents a placebotreatment that was most likely to produce physiologicaleffects. We ranked needling at acupuncture pointswithout electrical stimulation but indicator lights on as1w3 w6 ; needling at non-acupuncture points with elec-trical stimulation as 2 w5; superficial needling at non-acupuncture points (20-50 mm) avoiding Qi andmanual stimulation as 3w1 w2 w7 w8 w10-w12; non-penetrat-ing needle as 4w9; and laser turned off, held over thesymptomatic points without using any mechanicalpressure as 5.w4A meta-regression of the 12 trials found nostatistically significant relation between the type ofplacebo intervention and the effect of acupuncture(P=0.60). Supplementary subgroup analyses found astatistically significant difference in effect of acupunc-ture between the two trials using non-penetrative
2: Needling non-acupuncture points with electrical stimulation.
3: Superficial needling at non-acupuncture points, avoiding Qi or manual stimulation.
4: Non-penetrating needle.
5: Inactive laser held over points without pressure.
Reviewer #1 (Public review):
Summary:
Planar cell polarity core proteins Frizzled (Fz)/Dishevelled (Dvl) and Van Gogh-like (Vangl)/Prickle (Pk) are localized on opposite sides of the cell and engage in reciprocal repression to modulate cellular polarity within the plane of static epithelium. In this interesting manuscript, the authors explore how the anterior core proteins (Vangl/Pk) inhibit the posterior core protein (Dvl). The authors propose that Pk assists Vangl2 in sequestering both Dvl2 and Ror2, while Ror2 is essential for Dvl to transition from Vangl to Fz in response to non-canonical Wnt signaling. Nevertheless, there are several major and minor points that affect the strength of the author's proposed model (and are listed below).
Strengths:
The strengths of the manuscript are found in the very interesting and new concept along with supportive data for a model of how non-canonical Wnt induces Dvl to transition from Vangl to Fz with an opposing role for PK and Vangl2 to suppress Dvl during convergent extension movements. Ror is key player required for the transition and antagonizes Vangl.
Weaknesses:
The weaknesses are in the clarity and resolution of the data that forms the basis of the model. In addition to general whole embryo morphology that is used as evidence for CE defects, two forms of data are presented, co-expression and IP, as well as a strong reliance on IF of exogenously expressed proteins. Thus, it is critical that both forms of evidence be very strong and clear, and this is where there are deficiencies; 1) For vast majority of experiments general morphology and LWR was used as evidence of effects on convergent extension movements rather than keller explants or actual cell movements in the embryo. 2) the microscopy would benefit from super resolution microscopy since in many cases the differences in protein localization are not very pronounced. 3) the IP and Western analysis data often shows very subtle differences, and some cases not apparent.
Major points.
(1) Assessment of CE movement
The authors conducted an analysis of the subcellular localization of PCP core proteins, including Vangl2, Pk, Fz, and Dvl, within animal cap explants (ectodermal explants). The authors primarily used the length-to-width ratio (LWR) to evaluate CE movement as a basis for their model. However, LWR can be influenced by multiple factors and is not sufficient to directly and clearly represent CE defects. While the author showed that Prickle knockdown suppresses animal cap elongation mediated by Activin treatment, they did not test their model using standard assays such as animal cap elongation or dorsal marginal zone (DMZ) Keller explants. Furthermore, although various imaging analyses were performed in Wnt11-overexpressing animal caps and DMZ explants, the Wnt11-overexpressing animal caps did not undergo CE movement. Given that this study focuses on the molecular mechanisms of Vangl2 and Ror2 regulation of Dvl2 during CE, the model should be validated in more appropriate tissues, such as DMZ explants.
(2) Overexpression conditions
Another concern is that most analyses were performed with overexpression conditions. PCP core proteins (Vangl2, Pk, Dvl, and Fz receptors) are known to display polarized subcellular localization in both the neural epithelium and DMZ explants (Ref: PCP and Septins govern the polarized organization of the actin cytoskeleton during convergent extension, Current Biology, 2024). However, in this study, overexpressed PCP core proteins failed to show polarized localization. Previous studies, such as those from the Wallingford lab, typically used 10-30 pg of RNA for PCP core proteins, whereas this study injected 100-500 pg, which is likely excessive and may have created artificial conditions that confound the imaging results.
(3) Subtle and insufficient effects
Several of the reported results show quite modest changes in imaging and immunoprecipitation analyses, which are not sufficient to strongly support the proposed molecular model. For example, most Dvl2 remained localized with Fz7 even under Vangl2 and Pk overexpression (Fig. 4). Similarly, Wnt11 overexpression only slightly reduced the association between Vangl2 and Dvl2 (Sup. Fig. 8), and the Ror2-related experiments also produced only subtle effects (Fig. 8, Sup. Fig. 15).
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review)
The weaknesses are in the clarity and resolution of the data that forms the basis of the model. In addition to whole embryo morphology that is used as evidence for convergent extension (CE) defects, two forms of data are presented, co-expression and IP, as well as a strong reliance on IF of exogenously expressed proteins. Thus, it is critical that both forms of evidence be very strong and clear, and this is where there are deficiencies; 1) For vast majority of experiments general morphology and LWR was used as evidence of effects on convergent extension movements rather than Keller explants or actual cell movements in the embryo. 2) The study would benefit from high or super resolution microscopy, since in many cases the differences in protein localization are not very pronounced. 3) The IP and Western analysis data often show subtle differences, and not apparent in some cases. 4) It is not clear how many biological repeats were performed or how and whether statistical analyses were performed.
(1) To more objectively assess the convergent extension phenotypes, we developed a Fiji macro to automatically quantify the LWR in various injected Xenopus embryos, as detailed in the Methods section. We acknowledge that a limitation in the current manuscript is how to link our mechanistic model at the molecular level with the actual cellular behavior during convergent extension, and we plan to perform cell biological studies in the future to elucidate the link;
(2) We have repeated some of the imaging experiments in DMZ explants using a Zeiss LSM 900 confocal equipped with Airyscan2 detector that can increase the resolution to ~100 nm. The new data are in Suppl. Fig. 4, 9, 11, 16;
(3) We have repeated all IP and western blots at least three times and provided quantification and statistical analyses;
(4) We have added the information on biological repeats and statistical analyses in all figures and figure legends.
Reviewer #2 (Public Review):
The protein localization experiments in animal cap assays are for the most part convincing, but with the caveat that the authors assume that the proteins are acting within the same cell. As Fzd and Vangl2 are thought to localize to opposite cell ends in many contexts, can the authors be sure that the effects they observe are not due to trans interactions?
In our previous publication, we provided evidence that Vangl is necessary and sufficient to recruit Dvl to the plasma membrane within the same cell (Figure 3 in 10.1093/hmg/ddx095). In a more recent publication ( 10.1038/s41467-025-57658-0 ), we further elucidated a mechanism through which Dvl oligomerization switches its binding from Vangl to Fz, and determined that Dvl binding to Vangl and Fz are differentially mediated by its PDZ and DEP domain, respectively. In the current manuscript, we also performed co-IP experiment under various conditions to demonstrate binding between Dvl and Vangl. We feel that these evidences together provide a strong argument for our model where Vangl2 acts within the same cell to sequester Dvl from Fz.
In regards to the Dvl patches induced by Wnt11 (Fig. 3 and Suppl. Fig. 9), we performed separate injection of EGFP- and mSc-tagged Dvl into adjacent blastomeres, and demonstrated that the Wnt11-induced patches arise from symmetrical accumulation of Dvl at contact of two neighboring cells (Suppl. Fig. 9a-c’). This scenario is different from epithelial PCP where Fz/Dvl and Vangl/Pk are asymmetrically accumulated at the contact between two adjacent cells.
The authors propose a model whereby Vangl2 acts as an adaptor between Dvl and Ror, to first prevent ectopic activation of signaling, and then to relay Dvl to Fzd upon Wnt stimulation. This is based on the observation that Ror2 can be co-IPed with Vangl2 but not Dvl; and secondly that the distribution of Ror2 in membrane patches after Wnt11 stimulation is broader than that of Fzd7/Dvl, while Vangl2 localizes to the edges of these patches. The data for both these points is not wholly convincing. The co-IP of Ror2 and Vangl2 is very weak, and the input of Dvl into the same experiment is very low, so any direct interaction could have been missed. Secondly, the broader distribution of Ror2 in membrane patches is very subtle, and further analysis would be needed to firm up this conclusion.
(1) We repeated the co-IP experiment with Myc-tagged Vangl or Dvl. Using the same anti-Myc antibody and experimental condition (including the expression level of Vangl, Dvl and Ror2), we still found that Ror2 could be pulled down by Vangl but not Dvl (Suppl. Fig. 15b). Whereas this data confirms our previous conclusion, we acknowledge that a negative data does not fully exclude the possibility for direct biding between Ror and Dvl.
(2) We re-analyzed the signal intensity of Dvl and Ror in Wnt11-induced patches. By quantifying the intensity ratio between Ror and Dvl along the patches, we found an increase over two folds at the border of the patches (Fig. 7j, bottom panel). We interpret this data to suggest that Ror is accumulated to a higher level than Dvl at the patch borders.
A final caveat to these experiments is that in the animal cap assays, loss of function and gain of function both cause convergence and extension defects, so any genetic interactions need to be treated with caution i.e. two injected factors enhancing a phenotype does not imply they act in the same direction in a pathway, in particular as there are both cis/trans and positive/negative feedbacks between the PCP proteins.
We agree with the reviewer that a difficulty in studying PCP/ non-canonical signaling is that both loss and gain of function of any its components can cause convergence and extension defects. Genetic interactions, especially synergistic interactions, should be interpreted with caution. But we do want to point out that, in a number of case, we were also able to demonstrate epistasis. For instance, we found that Dvl2 over-expression induced CE defects can be rescued by Pk over-expression (Fig. 1e and f), whereas Vangl/ Pk co-injection induced severe CE defects can be reciprocally rescued by Dvl2 over-expression (Fig. 1g). Likewise, we showed that Fz2/ Dvl2 co-injection induced CE defects can be rescued by wild-type Vangl2 but not Vangl2 RH mutant (Suppl. Fig. 6b), and Ror2 can rescue Vangl2 overexpression induced CE defect (Suppl. Fig. 14). Collectively, these functional interaction data consistently demonstrate an antagonism between Dvl/ Fz/ Ror2 and Vangl2/ Pk, which is correlated with our imaging and biochemical studies.
As you can see from the reviews, the referees generally agree that your paper is a potentially valuable contribution to the field. Your observations are important because of the novel model based on the inhibitory feedback regulation between planar cell polarity (PCP) protein complexes. However, the reviewers also stated that the model is only partly supported by data because of insufficient clarity and missing controls in several experiments supporting the proposed model. The paper would be significantly improved if your conclusions are backed up by additional experimentation. Specifically, the referees wanted to see the reproducibility of the results shown in Figures 3, 4, 8, S3, S7, S12.
We hope that you are able to revise the paper along the lines suggested by the referees to increase the impact of your study on the current understanding of PCP signaling mechanisms.
We thank the reviewers for careful reading of our manuscript and for their constructive critiques and suggestions. We have repeated the animal cap studies in original Figures 3, 4, 8 and S3 with DMZ explants, and the new data are in Supplementary Fig. 9, 11, 16 and 4, respectively. We also repeated the biochemical studies in original Figure S 7and 12, and the new data are in Supplementary Fig. 8 and 15.
Reviewer #1 (Recommendations For The Authors):
Major points:(1) The author conducted an analysis of the subcellular localization of PCP core proteins, including Vangl2, Pk, Fz, and Dvl, within animal cap explants (ectodermal explants). To validate the model proposing that 'non-canonical Wnt induces Dvl to transition from Vangl to Fz, while PK inhibits this transition, and they function synergistically with Vangl to suppress Dvl during Convergent Extension (CE),' it is crucial to assess the subcellular localization of PCP core proteins in dorsal marginal zone (DMZ) cells, which are known to undergo CE. Notably, the overexpression of Wnt11 alone, as employed by the author, does not induce animal cap elongation. Therefore, the use of animal cap explants may not be sufficient to substantiate the model during Convergent Extension (CE). Indeed, previous knowledge indicates that Vangl2 and Pk localize to the anterior region in DMZ explants. However, the results presented in this manuscript appear to differ from this established understanding. Consequently, to provide more robust support for the proposed model, it is advisable to replicate the key experiments (Figures 3, 4, 8, and Figure S3) using DMZ explants.
We repeated the experiments in Figure 3, 4, 8 and Figure S3 with DMZ explant and the new data are in new Supplementary Fig. 9, 11, 16 and 4, respectively.In regards to “previous knowledge indicates that Vangl2 and Pk localize to the anterior region in DMZ explants”, we are aware Vangl/ Pk localization to the anterior cell cortex in neural epithelium from the studies by the Sokol and Wallingford labs, but are not aware of similar reports in DMZ explants. When we examined the localization of small amount of injected EGFP-mPk2 (0.1 ng mRNA) in DMZ explants, we saw a somewhat uniform distribution on the plasma membrane (Suppl. Fig. 4). In addition, in a related recent publication, we examined endogenous XVangl2 protein localization in activin induced animal cap explants that do undergo CE. What we observed was that whereas low level injected Dvl2 and Fz form clusters on the plasma member, endogenous XVangl2 remains uniformly distributed on the plasma membrane (Suppl. Fig. 3S-Z in 10.1038/s41467-025-57658-0 ). These observations may suggest potential differences of PCP protein localization during neural vs. mesodermal convergence and extension.
(2) The author suggests that 'Vangl2 and Pk together synergistically disrupt Fz7-Dvl2 patches.' As shown in Figure 4 (panels J' to I'), it is evident that the co-expression of Pk and Vangl2 increases Fz7 endocytosis. Nevertheless, a significant amount of Fz7 still co-localizes with Dvl2. To strengthen the author's hypothesis, additional clear assay is required such as Fluorescence resonance energy transfer (FRET) assay.
We appreciate this valuable advice. Since none of the tagged Fz/ Dvl/ Vangl proteins we had were suitable for FRET, we made proteins tagged with mClover and mRuby2, which were reported as optimized FRET pairs. But in our hands mRuby2 seems to require very long time (~2 days) to mature and become detectable at room temperature, and is not suitable for our Xenopus experiments. We are in the process of establishing a luciferase based NanoBiT system to detect Fz-Dvl and Dvl-Vangl interactions in live cells and cell lysates, and will use it in future studies to investigate their interaction dynamics.
For the current manuscript, we reason that a substantial reduction of Fz7-Dvl2 clusters with Vangl2/ Pk co-injection would still support our idea that Vangl2 and Pk act synergistically to sequester Dvl from Fz to prevent their clustering in response to non-canonical Wnt ligands.
(3) The IP data is less clear and evident. A couple of examples are: a) Fig 2g where the authors report that the Vangl2 R177H variant reduced Vangl2 interaction with Pk and recruitment of Pk to the plasma membrane, but it appears that the variant interacts slightly better than WT Vangl2 with Pk. In Fig. S7a, the authors state that Pk overexpression can indeed significantly reduce Wnt11-induced dissociation of EGFP-Vangl2 and Flag-Dvl2 in the DMZ. However, there is a minimal impact when compared to the Wnt11 absent control. Based on the results presented in Fig S12a the authors indicate that Wnt11 reduces the association between Vangl2 and Dvl2, which can be discerned, but loss of Ror2 does not change this in any obvious way - but the authors indicate it does. In S12b, the authors have suggested that Ror and Dvl do not form a direct binding interaction. However, the interpretation of Figure S12b is not entirely convincing due to several issues. Notably, the expression levels of each protein appear inconsistent, the bands are not sufficiently clear, and there is the detection of three different tag proteins on a single blot. To strengthen the validity of these findings, it is advisable to repeat this experiment with improved quality.
We repeated all the co-IP and western blot analyses pointed out by the reviewer, and performed quantification and statistical analyses.
Fig 2g had a mistake in the labeling and is replaced with new Figure 2g;
Fig. S7a is replaced by new data in Supplementary Figure 8a and b;
Fig. S12a and 12b are replaced by new data in Supplementary Figure 15a, a’ and b, respectively. In 15a and a’, we noticed a consistent decrease of Dvl2-Vangl2 co-IP in Xror2 morphant. The reason for this is not yet clear and will need further study in the future.
Minor points: (1) In all the whole embryo injection assays examining morphology, no Western analysis is performed to show roughly equivalent and appropriate levels of the various proteins are being expressed. Differences will affect the data.
Although we did not do western analyses to examine the protein levels in various functional interaction assays, we did examine how co-expression of Vangl2, mPk2 or Dvl2 may impact each other’s protein levels in Supplementary Fig. 2, which did not reveal any significant change when co-injected in different combination.
(2) The author's prior publication (Bimodal regulation of Dishevelled function by Vangl2 during morphogenesis, Hum Mol Genet. 2017) presented clear evidence of Vangl2 overexpression inducing Dvl2 membrane localization. However, Figure S4 in the current manuscript did not provide clear evidence of membrane localization. To strengthen the hypothesis that Vangl2-RH mutant also induces Dvl2 membrane localization, further comprehensive imaging analysis is needed.
We re-analyzed the imaging data and replaced old Figure S4 with a new Supplementary Fig. 5.
(3) In Supplementary Figure 9, the authors propose that the overexpression of Vangl2/Pk induces Fz7 endocytosis, as indicated by its co-localization with FM4-64. However, it raises a question: how does the Fz7-GFP protein internalize into the cells without endocytosis, as seen in Figures S9a-c'? To enhance readers' understanding, a discussion addressing this point should be included.
We think that this might be a technical issue. As detailed in the Method section, we only incubated the embryos transiently with FM4-64 for 30 minutes, and the embryos were subsequently washed and dissected in 0.1X MMR without the dye. Therefore, only the Fz7-GFP protein endocytosed during the 30 minute-incubation would be labeled by FM-64, whereas that endocytosed before or after the incubation would not. Alternatively, the very few Fz7-GFP puncta occasionally observed in the absence of Vangl2/Pk overexpression could be vesicles trafficking to the plasma membrane.
(4) Statistical analyses are absent for several results, including those in Figure 2f, Figure S4d, and Figure S7b.
We repeated these experiments and included statistical analyses. The new data are in Figure 2f, Supplementary Fig. 5d and Supplementary Fig. 8b.
(5) This manuscript lacks any results regarding Ck1. Therefore, it is advisable to consider removing the discussion or mention of CK1.
We agree, and tune down the discussion on CK1 and removed CK1 from our model in Fig. 9.
Reviewer #2 (Recommendations For The Authors):
(1) In all the convergence and extension assays, the authors should report n numbers (i.e. number of animals), what statistical test is used, and what the error bars show. Ideally dot-plots would be used instead of bar charts as they give a better insight into the data distribution. It might be useful to give a section on the statistical analyses used in the M&M, including e.g. any power calculations carried out, as now required by many journals.
We have follow the advice to use dot-plots for all the quantification analyses in the manuscript. We include in the figure legends the statistical test used and what the error bars show. The number of embryos analyzed were included in each panel in the figures. We also provided more details in the Methods section on how the LWR quantification was carried out.
(2) I think Figure 2g is wrongly labelled? FLAG bands are in all three lanes in the western blot, but not labelled as such in the schematic.
We corrected the schematic labeling in Figure 2g, and thank the reviewer for catching this mistake.
(3) In Figure S7, the authors show that co-IP of Dvl and Vangl2 is reduced by Wnt11 and the effects of Wnt are blocked by Pk. Does Pk have any effect in the absence of Wnt?
We examined the effect of Pk over-expression on Dvl2-Vangl2 co-IP as advised, and did not see a significant impact in the absence of Wnt11 co-injection. The data is included in the new Supplementary Figure 8a. We interpret the data to suggest that “at least under the condition of our co-IP experiment, Pk may not directly impact the steady-state binding between Vangl and Dvl”.
(4) In Figure 3, the authors show (as published previously) that Wnt11 induces patches of Dvl at the plasma membrane. It would be useful to see Dvl in the absence of Wnt and Vangl2/Dvl in the absence of Wnt.
Dvl is widely known as a cytoplasmic protein and its localization has been published by many labs over the past 20-30 years. In our recent publication (10.1038/s41467-025-57658-0 ), we also re-examined Dvl localization when injected at various dosages. So we did not feel it was necessary to show its localization in the absence of Wnt11 again, but included a reference to our prior publication. In regards to Vangl/Dvl distribution in the absence of Wnt11, the readers can see Suppl. Fig. 5b as an example, in addition to our previous publications referenced in the manuscript.
(5) In the review figures, the difference in Fz7-GFP patch formation in d' and e' (vs e.g. a') is not very clear. Could the images be improved or (better) quantified in some way?
We assume that “review figures” refer to Figure 3 or 4? If so, we felt that Fz7-GFP patch formation was clear in Fig. 3d’, e’ or Fig. 4d’, e’. Nevertheless, we repeated these experiments in DMZ explants as advised by Reviewer 1, and additional examples of Fz7-EGFP patch formation can be seen in the new Suppl. Fig. 9d-f’ and Suppl. Fig. 11d-f’.
(6) In Figure 6d, I'm concerned that the loss of flag-Dvl2 might occur via dephosphorylation in the IP reaction. Also the M&M don't include methodological details about buffers and whether phosphatase inhibitors were used. A compelling control would be anti-FLAG pulldown showing retention of phosphorylation. Also Figure 6f shows a reduced ratio of fast-to-slow migrating bands of Dvl with Vangl2/Pk - unless I have misunderstood, is this ratio the wrong way round?
We added co-IP buffer and protease inhibitor information in Methods.
We agree that the concern about dephosphorylation during IP reaction is valid, and that direct pull down of Dvl to show the phosphorylated form is a compelling control. We therefore note that in Suppl. Fig. 8a and 15b, direct pull down of Flag-Dvl or Myc-Dvl (with anti-Flag or anti-Myc) did show the slower migrating, phosphorylated form. Additional examples in which Vangl only co-IP the faster migrating unphosphorylated Dvl include Suppl. Fig. 15a, and in a related paper we published recently (Fig. 3R and R’ in 10.1038/s41467-025-57658-0 ).
Finally, we did wrongly label Figure 6f in the last submission, and the ratio should have been “slow/fast”. We have made the correction, and appreaicte the reviewer for the meticulousness in perusing our manuscript.
(7) In Figure 7, what does Ror2 look like in the absence of Wnt11?
We included new Figure 7a-c to show that without Wnt11 co-injection, Ror2 is uniformly distributed on the plasma membrane.
(8) Also in Figure 7, Ror2 patches are said to be slightly wider than Dvl2 patches "reminiscent of Vangl2" - I wouldn't describe them as being similar. Vangl2 shows a distinct dip in the center of the Dvl patches, Ror2 does not show a dip, and is only (at best) in a slightly wider patch, and I would want to see further examples to be convinced that the localization domain is reproducibly wider. The merge of many samples in 7d may actually be making the distribution harder to see and if the Xror2 and Dvl2 intensities were normalized I'm not sure how different the curves would appear. (i.e. the Xror2 curve looks like a flattened version of the Dvl2 curve).
We have added an additional panel in the new Figure 7j to compare the intensity ratio of Ror/ Dvl2 along the patches, and this analysis reveals an over two folds increase of the ratio at the border region. This quantification may make a more convincing argument that at the patch border region, Dvl is diminished whereas Ror2 accumulate with Vangl2.
(9) In Figure S12a, the authors suggest Wnt11 induced dissociation of Dvl from Vangl2 (by co-IP), and this is reduced after Ror2 MO. This would be more convincing with replicates and quantitation.
We have repeated this experiment with Vangl2 pull down and added quantification. The data is in the new Suppl. Fig. 15a.
(10) In Figure S12b, the authors suggest Ror2 can co-IP Vangl2 but not Dvl. This is not very convincing, as the Dvl input band is very weak, and the Vangl2 co-IP band is very weak.
We repeated the co-IP experiment with Myc-tagged Vangl or Dvl. Using the same anti-Myc antibody and experimental condition (including the expression level of Vangl, Dvl and Ror2), we still found that Ror2 could be pulled down by Vangl but not Dvl (Suppl. Fig. 15b).
(11) "Prickle" spelled "Prickel" in the abstract (and abbreviated to "PK" not "Pk" at one place in the abstract and several places in text)
We have corrected these typos.
(12) Quite a lot of interesting observations are in supplemental figures. Normally it might be expected that extra data supporting a conclusion would be in supplemental, but here some of the supplemental data feels like it is more than simply additional evidence. For instance supplemental Figures 2 and 3 feel more than just supplemental (and Supplemental Figure 3 if merged with Figure 2 would make it easier for the reader). Moreover, for example, the description of the results in Figure 2 is punctuated by references to supplemental Figures 4 and 5 that contain key data to support the conclusions, which means the reader has to flick backwards and forwards from place to place in the manuscript to follow the argument. It is of course up to the authors, but in some cases putting supplemental data back into the main figures (for which there is no size or number limit) would increase clarity.
These are excellent points; in the resubmitted manuscript we have a total of 24 data figures, and we used 8 as main figures since we felt that they provide the most relevant and conclusive evidence to our model. We will consult the copy editors at eLife on how to arrange the rest as main vs. supporting figures when requesting publication as version of record.
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We can remove this as it's below the CTA (No Sellers Found)
Les exportations ont a la fois été sous estimées en valeur de 3 points de pourcentage et en volume de 4 points de pourcentage, le déflateur des exportations ayant été initialement surestimé de 1,6 pt.
les chiffres en collent pas :1.03*1.016=1.0465 et pas 1.04
Note de synthèse : Réunion des parents d'élèves des filières technologiques (Première et Terminale)
À : L'ensemble du personnel enseignant et administratif du Lycée Louis Vincent De : Olivier Palaise, Proviseur Date : 29 septembre 2025 Objet : Alignement stratégique suite à la réunion des parents des filières technologiques
1. Introduction : Alignement sur notre Vision et nos Exigences Communes
L'objectif de cette note est de garantir que l'ensemble du personnel, enseignant comme administratif, partage une compréhension unifiée et cohérente des messages clés que nous avons communiqués aux parents d'élèves des classes de Première et Terminale technologiques (STI2D et STL).
Cet alignement est fondamental : notre crédibilité et l'efficacité de notre accompagnement reposent sur notre capacité à porter une parole collective forte et univoque.
La réussite de nos élèves dépend directement de cette cohérence entre nos exigences, nos discours et nos actions quotidiennes.
2. Les Filières Technologiques : Fer de Lance de l'Établissement et Cadre d'Exigence
J'ai tenu à réaffirmer auprès des parents ma vision stratégique : nos filières technologiques sont le "fer de lance de l'établissement".
Cette valorisation n'est pas un vain mot ; elle justifie le niveau d'exigence particulièrement élevé que nous appliquons.
Nous offrons des parcours d'exception qui se méritent par un engagement sans faille. Les règles fondamentales de la vie au lycée, rappelées avec fermeté, constituent le socle de cette ambition.
Voici les points sur lesquels nous devons maintenir une vigilance collective et absolue :
L'exemple de l'élève habitant Montini, à qui il a été clairement conseillé de "prendre le bus d'avant", illustre notre refus de la complaisance face aux retards évitables.
C'est une préparation directe aux exigences du monde supérieur et professionnel.
Dans des filières qui préparent à des carrières d'ingénieur, d'architecte ou de technicien supérieur, une tenue et une posture professionnelles sont indispensables. Le premier regard compte, et nous devons les y former.
Pour garantir le respect de cette règle, la sanction dissuasive de "4 heures de col le samedi" a été rappelée.
C'est un outil à notre disposition pour maintenir un environnement de travail serein et concentré.
Cette rigueur n'est pas une fin en soi. Elle est le cadre qui permet à nos élèves de saisir les opportunités exceptionnelles offertes par nos filières et de construire les parcours d'avenir ambitieux que nous leur promettons.
3. Le Parcours vers la Réussite : Calendrier, Enjeux et Stratégie
Nous avons insisté sur le fait que les années de Première et de Terminale constituent une période charnière, un véritable sprint où deux objectifs doivent être menés de front : l'obtention du baccalauréat avec la meilleure mention possible et la préparation stratégique du dossier d'orientation post-bac sur Parcoursup.
3.1. Les Enjeux dès la Classe de Première
Il a été expliqué aux parents que les élèves de Première doivent "courir deux lièvres à la fois" :
1. Préparation au Baccalauréat : Au-delà des épreuves anticipées de français, nous avons souligné la nouveauté de l'épreuve de mathématiques qui s'ajoute au calendrier.
2. Anticipation de l'Orientation : La réflexion sur le projet post-bac n'est plus une option.
Elle doit commencer dès maintenant, car les trois bulletins de Première pèseront d'un poids considérable dans l'examen de leur dossier Parcoursup l'année suivante.
3.2. La Double Échéance de la Classe de Terminale
Pour les élèves de Terminale, l'enjeu est immédiat, car ils sont "de plein fouet dans les deux parties".
Le calendrier Parcoursup est extrêmement serré, et les vœux doivent être formulés et validés dès mi-janvier.
L'importance stratégique du Grand Oral a été particulièrement mise en lumière.
L'exemple de l'élève dont la note est passée de 5/20 à 16/20 après une préparation encadrée de quelques heures est la preuve tangible que le travail et la méthode priment sur le "talent" seul.
Le coefficient (14 pour les Terminales de cette année, qui passera à 12 pour la promotion suivante) en fait une épreuve décisive qu'il est impératif de préparer avec le plus grand sérieux.
3.3. Le Dossier Scolaire : Le Facteur Humain dans la Sélection Parcoursup
Nous avons démystifié Parcoursup : ce n'est pas un algorithme froid.
Derrière l'interface, des commissions composées d'enseignants examinent les dossiers. Certains éléments, que nous devons tous avoir à l'esprit, entraînent un rejet quasi-systématique :
1. Absences régulières : La simple mention "élève régulièrement absent" sur un bulletin place immédiatement le dossier sur une "pile d'à côté".
2. Comportement problématique : De même, une appréciation telle que "comportement inadmissible" disqualifie un candidat avant même l'examen de ses notes.
3. Appréciations des professeurs : L'arbitrage est clair.
Entre un élève à 12 de moyenne "au talent" et un autre à 12 dont l'appréciation souligne les efforts ("fait vraiment des efforts"), le second sera toujours préféré.
Notre rôle, en tant qu'enseignants et membres de l'équipe pédagogique, est donc crucial.
Les appréciations que nous rédigeons ne sont pas de simples commentaires ; ce sont des facteurs décisifs qui peuvent ouvrir ou fermer les portes de l'avenir de nos élèves. La précision et la justesse de nos mots sont une responsabilité majeure.
4. L'Excellence des Filières Technologiques : Pédagogie et Débouchés
Les filières STI2D et STL ont été présentées comme des voies d'excellence, caractérisées par une pédagogie concrète, une forte proportion de travaux pratiques et des perspectives de poursuites d'études ambitieuses, y compris vers les plus grandes écoles.
4.1. Baccalauréat STI2D : Le concret au service de la polyvalence
La philosophie de la filière STI2D est de donner du sens aux sciences par le concret, de permettre aux élèves de "comprendre pourquoi ils font des sciences". Ses points forts résident dans :
Un volume conséquent de 12 heures d'enseignement scientifique dès la Première.
Une approche pédagogique basée sur l'expérimentation et la simulation.
Un profil qui reste "généraliste", assurant un "large éventail sur le choix des formations postbac".
La nouveauté d'une épreuve pratique au baccalauréat, qui valorise enfin leur cœur de formation.
4.2. Baccalauréat STL : La pratique en laboratoire comme tremplin
La spécificité du bac STL repose sur une forte coloration physique-chimie, avec une partie expérimentale "extrêmement importante".
Les élèves passent plus d'un tiers de leur temps en travaux pratiques, en groupes à effectif réduit. Les débouchés sont réels et attractifs :
Le succès de ces filières, validé par des taux de réussite remarquables (plus de 96 % en STI2D et 93 % en STL), est le fruit direct de cette approche pédagogique exigeante.
5. Calendrier des Temps Forts de l'Orientation
Pour accompagner nos élèves dans la construction de leur projet, plusieurs rendez-vous majeurs jalonneront l'année. Il est essentiel que nous les encouragions tous à y participer activement.
Événement Date(s) Clé(s) Public Cible
Un Jour à l'Université (Ujalu) Inscriptions dès le 29 sept. Élèves de Terminale
Forum "Aux réaction" 20, 21 et 22 novembre Première et Terminale (transport non organisé par le lycée cette année)
Forum des Formations (au lycée) 6 février 2026 Première et Terminale Journée Portes Ouvertes (au lycée) 12 février Première et Terminale
Ces événements sont des opportunités précieuses que les élèves doivent saisir pour affiner leurs choix.
6. Conclusion : Notre Responsabilité Collective
Je compte sur chacun de vous pour porter et incarner ce message de rigueur, d'ambition et d'accompagnement.
La réussite de nos élèves des filières technologiques est une fierté pour notre établissement, mais elle est avant tout le résultat d'un effort collectif.
Notre vigilance constante, notre communication cohérente et notre exigence bienveillante sont les clés qui leur permettront de réaliser leur plein potentiel et de transformer leurs ambitions en succès.
Author response:
The following is the authors’ response to the original reviews.
Joint Public Review:
Summary:
This study investigates plasticity effects in brain function and structure from training in navigation and verbal memory.
The authors used a longitudinal design with a total of 75 participants across two sites. Participants were randomised to one of three conditions: verbal memory training, navigation training, or a video control condition. The results show behavioural effects in relevant tasks following the training interventions. The central claim of the paper is that network-based measures of task-based activation are affected by the training interventions, but structural brain metrics (T2w-derived volume and diffusion-weighted imaging microstructure) are not impacted by any of the training protocols tested.
Strengths:
(1) This is a well-designed study which uses two training conditions, an active control, and randomisation, as appropriate. It is also notable that the authors combined data acquisition across two sites to reach the needed sample size and accounted for it in their statistical analyses quite thoroughly. In addition, I commend the authors on using pre-registration of the analysis to enhance the reproducibility of their work.
(2) Some analyses in the paper are exhaustive and compelling in showcasing the presence of longitudinal behavioural effects, functional activation changes, and lack of hippocampal volume changes. The breadth of analysis on hippocampal volume (including hippocampal subfields) is convincing in supporting the claim regarding a lack of volumetric effect in the hippocampus.
Weaknesses:
(1) The rationale for the study and its relationship with previous literature is not fully clear from the paper. In particular, there is a very large literature that has already explored the longitudinal effects of different types of training on functional and structural neuroimaging. However, this literature is barely acknowledged in the Introduction, which focuses on cross-sectional studies. Studies like the one by Draganski et al. 2004 are cited but not discussed, and are clumped together with cross-sectional studies, which is confusing. As a reader, it is difficult to understand whether the study was meant to be confirmatory based on previous literature, or whether it fills a specific gap in the literature on longitudinal neuroimaging effects of training interventions.
We thank the reviewer for these comments and feedback.
We want to clarify that through our pre-registered analysis plan, our approach was confirmatory, rather than exploratory (or rather than post-hoc justified.) This confirmatory approach allowed us to critically evaluate the theoretically novel and important hypotheses which tested what no other study like our longitudinal/intervention study proposed or performed previously. We have now clarified this in the introduction.
“This allowed us to address the following novel theoretical questions: 1) what neural changes, if any, result from an intensive within-participant intervention that improves memory or navigation skills in healthy young adults 2) if such changes occur, what is the degree of neural overlap between the acquisition of these cognitive skills.”
“We pre-registered three novel and specific hypotheses, which are described in more detail here (https://osf.io/etxvj) ”
We have also attempted to better separate cross-section and longitudinal studies. Due to space limitations, we have focused on interventional studies that involved gray matter changes that could relevance to either navigation, episodic memory, or the hypothesized time frame we chose for the training. We also note that some of these relevant studies are discussed in more depth in the discussion.
“Successful cognitive interventions suggest that targeted within-participant cognitive training, even for as little as 1-2 weeks, can result in improvements to specific cognitive functions, including changes in focal gray matter [4,23-27]; but see[28].”
We have also added some additional citations to relevant cognitive intervention work, although we agree that this is an extensive literature, only a subset of which we are able to capture here:
“In some instances, interventions may even generalize to areas not explicitly trained but closely related to the training (termed “near transfer”)[29-33].”
(2.1) The main claim regarding the lack of changes in brain structure seems only partially supported by the analyses provided. The limited whole-brain evidence from structural neuroimaging makes it difficult to confirm whether there is indeed no effect of training. Beyond hippocampal analyses, many whole-brain analyses of both volumetric and diffusion-weighted imaging metrics are only based on coarse ROIs (for example, 34 cortical parcellations for grey matter analyses).
Although vertex-wise analyses in FreeSurfer are reported, it is unclear what metrics were examined (cortical thickness? area? volume?).
We appreciate the reviewer’s thoughtful feedback. We apologize for the lack of clarity in the original manuscript regarding the type of metric used in the vertex-wise analysis. We confirm that these analyses were based on cortical volume, not thickness or area. To clarify this, we have explicitly stated in the revised Methods that the vertex-wise analyses were conducted on cortical volume using FreeSurfer’s mri_glmfit.
In addition, in response to the concern regarding the coarse nature of the ROI-based analyses, we have re-analyzed the volumetric data using the more fine-grained Destrieux atlas, which contains 148 cortical ROIs (74 per hemisphere), instead of the original, coarser 34-region atlas. These more detailed analyses still revealed no significant volume changes from pre- to post-training in any of the three groups. We believe this provides stronger support for the lack of training-induced volumetric changes outside the medial temporal lobe.
Relevant revisions have been made to the Results and Methods sections. Below is the updated content added to the manuscript:
In Results:
“We also analyzed gray matter volume changes outside of the medial temporal lobe using FreeSurfer (see Methods) to determine if any cortical or other relevant brain areas might have been affected by the training. We applied a vertex-wise analysis of cortical volume, again finding no significant differences across the entire cortex (see Methods). This finding was further validated using the Destrieux atlas, which includes 74 cortical parcellations per hemisphere (148 ROIs in total). Paired-sample t-tests revealed that none of the ROIs exhibited significant volume changes from pre- to post-test in any of the three groups (all ps > 0.542, FDR-corrected). These findings suggest that training did not result in any measurable cortical volumetric changes.”
In Methods:
“Whole-brain structural analyses were conducted using FreeSurfer (version 7.4.1; https://surfer.nmr.mgh.harvard.edu). T1-weighted anatomical images were processed using the longitudinal processing pipeline. Vertex-wise analyses of cortical volume were performed using FreeSurfer’s general linear modeling tool, mri_glmfit. Group-level comparisons were corrected for multiple comparisons using mri_glmfit-sim, which implements cluster-wise correction based on Monte Carlo simulations. A vertex-wise threshold of Z > 3.0 (corresponding to p < 0.001, two-sided) was applied to detect both positive and negative effects. Clusters were retained if they survived a cluster-wise corrected p < 0.05.
In addition to vertex-wise analysis, cortical parcellation was performed using the Destrieux atlas (aparc.a2009s), which includes 74 cortical regions per hemisphere, yielding 148 ROIs in total. To account for variability in brain size, each ROI volume was normalized by estimated intracranial volume (ICV) and scaled by a factor of 100. Longitudinal comparisons were conducted using paired-sample t-tests. To correct for multiple comparisons, we applied FDR correction (q < 0.05).”
(2.2) Diffusion-weighted imaging seems to focus on whole-tract atlas ROIs, which can be less accurate/sensitive than tractography-defined ROIs or voxel-wise approaches.
We appreciate the reviewer’s important point regarding diffusion-weighted imaging (DWI) analysis. We focused primarily on atlas-defined tract-level ROIs derived from a standard white matter tract atlas as we did not feel that we had the resolution for more fine-grained analyses with our sequences. While this approach has the advantage of robust anatomical correspondence and improved interpretability, we agree that it may be less sensitive than tractography-defined or voxel-wise methods for detecting more subtle, localized training-related changes. Because of limitations in our DWI sequence, which was optimized to be shorter and identical between different scanners, we are not able to provide more fine-grained analysis of the DWI data.
(3) Quality control of images is only mentioned for FA images in subject space. Given that most analyses are based on atlas ROIs, visual checks following registration are fundamental and should be described in further detail.
Thank you for your thoughtful comment. We agree that visual quality control is critical when using atlas-based ROI analyses. In our study, we implemented comprehensive quality control procedures across all structural and functional imaging analyses.
For hippocampal segmentation using ASHS, we performed manual visual inspections of each participant's subfield segmentation to verify the accuracy of the automated outputs. This is now clearly described in the revised Methods section:
“Each participant's subfield segmentations were manually inspected to ensure the accuracy and reliability of the segmentation protocol.”
For FreeSurfer-based hippocampal and cortical segmentation, we also conducted detailed visual inspections and manual edits following the standard FreeSurfer longitudinal pipeline. We have added the following description to the Methods section to clarify this process:
“Visual quality control was conducted by three trained raters who systematically inspected skull stripping, surface reconstruction, and segmentation accuracy at both the within-subject template and individual timepoints. Manual edits were primarily applied to the within-subject template to correct segmentation errors—particularly in challenging regions such as the hippocampus—since corrections to the template automatically propagate to all timepoints. Raters followed standardized FreeSurfer longitudinal editing guidelines to ensure consistent and reproducible corrections across subjects. Discrepancies were resolved via consensus discussion. This quality control approach enhanced the accuracy and consistency of segmentation across longitudinal scans, thereby improving the reliability of morphometric analyses and atlas-based ROI extractions.”
For functional MRI preprocessing, all registration steps—including transformations from individual functional runs to MNI space—were visually checked for each participant to ensure accurate alignment with the Schaefer atlas. We have clarified this point in the revised Methods section with the following statement:
“Prior to ROI extraction, all registration steps—from individual functional space to MNI space—were visually inspected for each participant to confirm accurate alignment between the functional images and the atlas parcellation.”
These additions now more clearly reflect the robust quality control procedures that were employed throughout our pipeline to ensure the validity of atlas-based analyses.
Recommendations for the authors:
(1) As a reader, I would have appreciated a short section in the methods regarding the preregistration and power analysis. Currently, it is not too straightforward to understand which analyses were included in the preregistration, and at what point in the project the pre-registration was written. Finding all the relevant information from OSF is feasible, but it would be more accessible if a summary of the information were available inside the text.
We thank the reviewer for this valuable suggestion. We agree that providing a concise summary within the manuscript's methods section will significantly improve accessibility for readers.
The full preregistration is now explicitly referenced in the Methods:
“Preregistration and Power Analysis
This study was preregistered on the Open Science Framework (OSF; https://osf.io/etxvj). The preregistration was completed on October 30, 2023, after approximately 80% of data collection had been completed, but prior to any analysis of the primary outcome variables. The preregistration outlines the study hypotheses, design, target sample size, and planned behavioral and neuroimaging analyses, including longitudinal ROI comparisons and statistical correction procedures.
A priori power analysis was conducted using G*Power 3.1 to estimate the required sample size for detecting a Group × Time interaction in a mixed-design ANOVA. Assuming a small-to-medium effect size (f = 0.35), we determined that 24 participants per group would provide 80% power to detect a significant effect at α = 0.05. To allow for potential attrition and data exclusion (e.g., due to excessive motion or incomplete datasets), we targeted recruitment of 30 participants per group across two study sites.
All primary hypotheses, analytic plans, and inference criteria are documented in the preregistration. Exploratory analyses are clearly delineated in both the preregistration and the present manuscript.”
(2) The relevance of the study for "disease" is mentioned in the Abstract but is absent in the Introduction. This may be worth removing?
Thank you for pointing this out. We agree that the reference to "disease" in the Abstract was not well-supported in the Introduction. To maintain consistency and avoid overstatement, we have removed the mention of "disease" from the Abstract in the revised manuscript.
In Abstract:
“Training cognitive skills, such as remembering a list of words or navigating a new city, has important implications for everyday life.”
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
We thank the reviewers for their thoughtful comments and overall very supportive feedback.
Reviewer #1 writes: "The study is very thorough and the experiments contain the appropriate controls. (...) The findings of the study can have relevance for human conditions involving disrupted mitochondrial dynamics, caused for example by mutations in mitofusins." Reviewer #2 writes: "The dataset is rich and the time-resolved approach strong." Reviewer #3 writes: "I admire the philosophy of the research, acknowledging an attempt to control for the many possible confounding influences. (...) This is a powerful and thoughtful study that provides a collection of new mechanistic insights into the link between physical and genetic properties of mitochondria in yeast."
We address all points below. We have not yet updated our text and figures since we expect substantial additions from new experiments. But we have included Figure R1 with some additional analyses of existing data at the bottom of the manuscript.
Reviewer1
1.1 Statistical comparisons are missing throughout the manuscript (with the exception of Fig. 2c). Appropriate statistical tests, along with p-values, should be used and reported where different gorups are compared, for example (but not limited to) Fig. 3d and most panels of Fig. 4.
We initially decided not to add too many extra labels to the already very busy plots, given that the magnitude of change mostly speaks for itself. However, we will try to find meaningful statistical tests together with a sensible graphical representation for all of the figures. For one example see Figure R1A.
1.2. I do not agree with the use of Atp6 protein as a direct read-out of mtDNA content. While Atp6 protein levels will decrease with decreasing mtDNA content, the inverse is not necessarily true: decreased Atp6 protein levels do not necessarily indicate decreased mtDNA levels, because they could alternatively or additionally be caused by decreased transcription and/or translation. Therefore, please do not equate Atp6 protein levels to mtDNA levels, and instead rephrase the text referencing the Atp6 experiments in the Results and Discussion sections to measure "mtDNA expression" or "mt-encoded protein" or similar. For example, on p. 14 line 431 should read "mtDNA expression" rather than "decreased synthesis of mtDNA", and line 440 on the same page "mean mtDNA levels" should be "mtDNA expression" or similar.
All three reviewers agree that using Atp6-NG as a direct proxy for mtDNA requires more validation, or at least rephrasing of the text. We agree that this is the most important point to address. We had previously tried using the mtDNA LacO array (Osman et al. 2015) to directly assess the amount of nucleoids per cell. However, the altered mitochondrial morphology of the Fzo1 depleted cells combined with the LacI-GFP which is still in mitochondria even when mtDNA is gone, increases the noise level to a point that we cannot interpret the signal. However, as this manuscript was in the submission process, the Schmoller lab (co-authors #2 and #7) adapted the HI-NESS system to label mtDNA in live yeast cells(Deng et al. 2025). This system promises much better signal to noise and we expect we can address all concerns regarding the actual count of nucleoids per cell. Should this unexpectedly fail for technical reasons, we will try to calibrate the Atp6-levels with DAPI staining at defined time points and will rephrase the text as the reviewer suggests.
1.3. In Fig. 3, the authors use the fluorescence intensity of a mitochondrially-targeted mCardinal as a read-out of mitochondrial mass. Please provide evidence that this is not affected by MMP, either with relevant references or by control experiments (e.g. comparing it to N-acridine orange or other MMP-independent dyes or methods).
Whether or not the import of any mitochondrial protein is dependent on the MMP depends largely on the signal sequence. The preSu9-signaling sequence was previously characterized as largely independent of the MMP compared to other presequences (Martin, Mahlke, and Pfanner 1991), which is why Vowinckel (Vowinckel et al. 2015) and others (Di Bartolomeo et al. 2020; Perić et al. 2016; Ebert et al. 2025) have previously used this as a neutral reference to the strongly MMP-dependent pre-Cox4 signal to estimate MMP. As one control in our own data, we consider that the population-averaged mitochondrial fluorescent signal Figure S3C stays constant in the first few hours, in agreement with the total averaged mitochondrial proteome (Fig R1E). As additional controls, we plan to compare the signal to an MMP independent dye as the reviewer suggests.
1.4. In Fig. 2e-f, the authors use a promoter reporter with Neongreen to answer whether the reduced levels of the nuclear-encoded mitochondrial proteins Mrps5 and Qcr7 are due to decreased expression or to protein degradation, and find no evidence of degradation of the Neongreen reporter protein. However, subcellular localization might affect the availability of the protein to proteases. Although not absolutely required, it would be relevant to know if the Neongreen fusion protein is found in the same subcellular compartment as Mrps5 and Qcr7 at 0h and 9h after Fzo1 depletion.
Here, it seems we need to explain the set-up and interpretation of the data better. The key point we are trying to make with the promoter-Neongreen construct is that the regulation is not mainly at the level of transcription. We are showing that the reduction in the levels of the actual protein (orange bars) is not (mainly) explained by a reduction in expression, since the promoter is similarly active at 0 and at 9 hours (grey bars). If expression from the promoter were strongly reduced, the Neongreen would be diluted with growth and would also decrease, but this is not the case. The fluorophore itself is just floating around in the cytosol and is not subject to the same post-translational regulation as Mrps5 and Qcr7, so there is no reason to expect degradation.
1.5. Fzo1 depletion leads to a very rapid drop in MMP during the first hour of depletion. In the Discussion, can the authors speculate on the possible mechanism of this rapid MMP drop that occurs well before mtDNA or mt-encoded proteins are decreased in level?
This is indeed an interesting point. We think there are likely three reasons causing this initial drop: Firstly, due to the fragmentation the mixing of mitochondrial content is disturbed and smaller fragments may have suboptimal stoichiometry of components (see also (Khan et al. 2024) who look at this in detail including the Fzo1 deletion); secondly, already fairly early, some mitochondrial fragments may not contain any mtDNA and therefore will be unable to synthesize ETC proteins; thirdly, altered morphological features like changes in the surface-to-volume ratios may play a role. Sadly, mechanistically following up on this is not possible with the tools in our hands and therefore outside of the scope of this manuscript. But we are happy to include these speculations in our discussion.
1.6. In Fig. 2a, the mtDNA copy number of Fzo1-depleted cells is ca 1.3-fold of the control cells at the 0h timepoint. Why might this be? Is it an impact of one of the inducers? If so, we might be looking at the combination of two different processes when measuring copy number: one that is an induction caused by the inducer(s), and the other a consequence of Fzo1 depletion itself.
We believe that this 30% increase is within the noise of the experiment rather than an effect of the induction. Since we normalize to t=0 uninduced, the first black data point does not have error bars, emphasizing this difference. None of the protein data suggests that there is an increase in mtDNA encoded proteins (see e.g. 2B, or Atp6 fluorescence data). In the planned HI-NESS experiment, we will see in our single cell data whether there is an actual increase in mtDNA upon TIR induction. Additionally, we will run a qPCR to carefully determine mtDNA levels of untreated wild-type cells, tetracycline treated wild-type cells and tetracycline induced TIR expressing cells to exclude effects of tetracycline as well as the expression of TIR on mtDNA.
Minor comments:
1.7. p. 3, line 71: "ten thousands of dividing cells.." should be "tens of thousands of dividing cells".
Thank you, will correct.
1.8.-p.4, line 116: please be even more clear with what the "depleted" cells and controls are treated with: are depleted cells treated with both inducers, and controls with neither?
We will make this more clear. Depleted cells are treated with both inducers, the control cells are not. However, in Figure 1A and in S1 we do controls to show that inducing TIR per se or adding aTC per se does not change growth rate or mitochondrial morphology. We will make this more clear.
1.9. -p.5, lines 147-148: the authors write "the rate with which the abundance of Cox2 and Var1 proteins decreases was similar to the rate of mtDNA loss" though the actual rate is not shown. Please calculate and show rates for these processes side by side to make comparison possible, or alternatively rephrase the statement.
Indeed this was not phrased well. We will call it dynamics rather than rates.
1.10. -Fig. 2d: changing the y-axis numbering to match those in panels a and b would facilitate comparisons.
Makes sense, we will change this.
1.11. Fig. 2e: it is recommended to label the western blot panels to indicate what protein is being imaged in each (Neongree,, Mrps5, Qcr7).
We will adapt the labelling to make it more clear.
1.12. -p.9, line 262: I suggest referencing Fig. 4e at the end of the first sentence for clarity.
We will modify the sentence as suggested.
1.13. -In the sections related to Fig. 3a and Fig. 5a as well as the connected supplemental data, the authors discuss both the median and the mean of mitochondrial mass and Atp6 protein, respectively. For purposes of clarity, I suggest decreasing the focus on the mean (that is provided only in the supplemental data) and focusing the text mainly on the median. The two show differing trends and it is very good that both are shown, but the clarity of the text can be improved by focusing more on the median where possible.
We will check the phrasing and simplify.
1.14. -p. 14, line 435: the statement that mt mass is maintained over the first 9h of depletion is only true for the mean mt mass, not for the median. Please make this clear or rephrase.
We will check phrasing, make it more clear and also point out the extended proteomics data (see Fig R1), which corresponds to the mean of the populations
1.15.-p.14, line 452: "mitofusions" should be "mitofusins".
Thanks for catching this.
Reviewer 2:
2.1. While inducible TIR is used to reduce background, the manuscript should rigorously exclude auxin/TIR off-targets (growth, mitochondrial phenotypes, gene expression). Please include full matched controls: (plus minus)auxin, (plus minus)TIR, epitope tag alone, and a degron control on an unrelated mitochondrial membrane protein.
We agree that rigorous controls are crucial for the interpretation of the results. However, we think we have already included most of the controls the reviewer is asking for, but we might have not pointed this out clearly enough. For example, in Fig 1A, we could make it more clear by adding more labels in which samples we added aTC, which is only described in the figure legend.
Here is a list of all the controls:
2.2. The Mitoloc preSu9 vs Cox4 import ratio is only a proxy of mitochondrial membrane potential (ΔΨm) and itself depends on mitochondrial mass, protein expression, matrix ATP, and import saturation. The authors need to calibrate ΔΨm with orthogonal dyes (TMRE/TMRM) and pharmacologic titrations (FCCP/antimycin/oligomycin) to generate a response curve; show that Mitoloc tracks dye-based ΔΨm across the relevant range and corrects for mass/photobleaching. Report single-cell ΔΨm vs mass residuals.
We completely agree that the MitoLoc system is only a rough proxy for the actual membrane potential. That is why we make no quantitative claims on the absolute value or absolute difference between groups of cells. We also make very clear in Fig 3B what we are actually measuring and can emphasize again in the text that this is only a proxy. We agree that it is a good idea to compare MitoLoc values to TMRE staining as the reviewer suggests, we will do these experiments in depleted and control cells at different timepoints. Please note though that also dye staining has its caveats, especially in dynamic live cell experiments. TMRM for example is not compatible with the acidic pH 5 medium that is typically used for yeast and subjecting cells to washing steps and higher pH may change both morphology of mitochondria and the MMP, especially in cells that are already “stressed”. We prefer not to complete elaborate pharmacological titration experiments because firstly, this was extensively done in the original MitoLoc paper by the Ralser lab ((Vowinckel et al. 2015), cited 120 times); secondly, the value of the MMP is not the most critical claim of the manuscript. See also 3.12. Please note that in Figure S4D we had already plotted MMP vs mitochondrial concentration.
2.3. To use Atp6-mNeon as a proxy for mtDNA is an assumption. Interpreting Atp6 intensity as "functional mtDNA" could be confounded by translation, turnover, or assembly. Please (i) report mtDNA copy number time courses (you have qPCR), nucleoid counts (DAPI/PicoGreen or TFAM/Abf2 tagging), and (ii) assess translation (e.g., 35S-labeling or puromycin proxies) and turnover (proteasome/AAA protease inhibition, mitophagy mutants -some data are alluded to- plus mRNA levels for mtDNA-encoded genes). This will support the "reduced synthesis" versus "increased degradation" conclusion.
We agree with all three reviewers that Atp6 is only a proxy for mtDNA (Jakubke et al. 2021; Roussou et al. 2024) and the correlation should be checked more carefully. We will use the very recently established Hi-NESS system to follow nucleoids/ mtDNA during depletion experiments. See detailed reply to 1.2.
(ii) in Figure 2C we inhibit mitochondrial translation and show that in this case control and depleted cells have the same level of Cox2, at least suggesting that degradation is not the key mechanism controlling the levels of mtDNA encoded proteins. We cannot do proteasome inhibitor assays since the nature of the AID-TIR systems requires an active proteasome. In figure S5C we show that the Atp6 depletion is similar in an atg32 deletion. This does not completely exclude a contribution of mitophagy to the observed phenotype, but does confirm that mitophagy is not the primary reason for cells becoming petite.
2.4. The promoter-NeonGreen reporters argue against transcriptional down-regulation of nuclear OXPHOS. Please add mRNA (RT-qPCR/RNA-seq) for representative genes and a pulse-chase or degradation-pathway dependency (e.g., proteasome/mitophagy/autophagy mutants) to firmly assign active degradation. The authors need to normalize proteomics to mitochondrial mass (e.g., citrate synthase/porin) to separate organelle abundance from protein turnover.
While we are happy to perform qPCR experiments for selected genes, a full RNA-seq experiment seems outside the scope of this study. As explained above, a proteasome inhibitor experiment is not possible in this set-up. Bulk mitophagy/autophagy seems unlikely to be the cause of the decrease of the nuclear-encoded OXPHOS proteins, since most other mitochondrial proteins do not decrease on average on population level in the first hours. This data is now plotted as additional figure (see below) and will be included in the supplementary of the revised manuscript (Fig R1E).
2.5. Using preSu9-mCardinal intensity as "mitochondrial concentration" is sensitive to expression, import competence, and morphology/segmentation. The authors should provide validation that this metric tracks 3D volume across fragmentation states (e.g., correlation with mito-GFP volumetrics; detergent-free CS activity; TOMM20/Por1 immunoblot per cell).
We agree that this is an important point and the co-authors discussed this point quite intensively. In figure S3A and B we show (using confocal data) that there is a very strong correlation between the total fluorescence signal and the 3D volume reconstruction. However, the slope of the correlation is different between tubular and fragmented mitochondria (compare panels A and B) and see figure legend. Since we are dealing with diffraction-limited objects it is likely that the 3D reconstruction is sensitive to morphology, especially if mitochondria are “clumping”. We therefore think that the total fluorescence signal is actually a better estimate of mitochondrial mass per cell than the 3D volume reconstruction (especially for our data obtained with a conventional epifluorescence microscope). The mean of the total mitochondrial fluorescence also better matches the population average mitochondrial proteome (Fig R1E). To consolidate this assumption, we will additionally compare our data to a strain with Tom70-Neongreen and to MMP independent dyes.
Notably, since the morphology is similarly altered in mothers and buds this is of minor impact for our main point – the unequal distribution between mother and buds.
2.6. The unequal mother-daughter distribution is compelling, but causality remains inferred. Test whether modulating inheritance machinery (actin cables/Myo2, Num1, Mmr1) or altering fission (Dnm1 inhibition) modifies segregation defects and rescues mtDNA/Atp6 decline. Complementation with Fzo1 re-expression at defined times would help order the phenotype cascade.
We agree that rescue experiments would be very useful. We have some preliminary data for tether experiments, for example with Num1. The general problem is that the fragmented mitochondria clump together. We have not found a method to restore an equal distribution between mother and daughter cells. We will try to optimize the assay, but are not overly confident it will work. Mmr1 deletion aggravates the Fzo1 phenotype, likely also because the distribution becomes even more heterogeneous, but we have not rigorously analyzed this.
We like the idea of the Fzo1 re-expression and will run such experiments. This will be especially powerful in combination with the new HI-NESS mtDNA reporter. We may be able to track exactly when cells reach the point-of-no return and become petite. This will also help connecting our mathematical model more directly to the data.
2.7. The model is useful but should include parameter sensitivity (segregation variance, synthesis slopes, initial nucleoid number) and prospective validation (e.g., predict rescue upon partial restoration of synthesis or inheritance, then test experimentally).
We will refine our model to include the to-be-measured nucleoids/mtDNA values. We will include a parameter sensitivity analysis with the updated model.
Reviewer 3:
3.1. About the use of Atp6 as a good proxy for mtDNA content. This is assumed from l285 onwards, based on a previous publication. As the link is fairly central to part of the paper's arguments, and the system in this study is being perturbed in several different ways, a stronger argument or demonstration that this link remains intact (and unchanged, as it is used in comparisons) would seem important.
We agree, see 1.2.
3.2. About confounding variables and processes. The study does an admirable job of being transparent and attempting to control for the many different influences involved in the physical-genetic link. But some remain less clearly unpacked, including some I think could be quite important. For example, there is a lot of focus on mito concentration -- but given the phenotypes are changing the sizes of cells, do concentration changes come from volume changes, mito changes, or both? In "ruling out" mitophagy -- a potentially important (and intuitive) influence, the argument is not presented as directly as it could be and it's not completely clear that it can in fact be ruled out in this way. There are a couple of other instances which I've put in the smaller points below.
Thank you for acknowledging our efforts to show transparent and well-controlled experiments! We address each of the specific points below.
3.3. full genus name when it first appears
We will add the full name.
3.4. I may be wrong here, but I thought the petite phenotype more classically arises from mtDNA deletion mutations, not loss? The way this is phrased implies that mtDNA loss is [always] the cause. Whether I'm wrong on that point or not, the petite phenotype should be described and referenced.
We can expand the text and cite additional relevant papers. The term “petite” refers to any strain that is respiratory incompetent and leads to small colonies (not necessarily small cells!) (Seel et al. 2023). This can be mutations or gene loss (fragments) on the mtDNA (these are called cytoplasmic petite), or chemically induced loss of mtDNA (e.g. EtBr), or mutations of nuclear genes required for respiration (these are termed nuclear petite; some nuclear petites show loss of mtDNA in addition to the mutation in the nuclear genome) (Contamine and Picard 2000).
3.5. para starting l59 -- should mention for context that mitochondria in (healthy, wildtype) yeast are generally much more fused than in other organisms
ok.
3.6. Fig 1C -- very odd choice of y-axis range! either start at zero or ensure that the data fill as much vertical space of the plot as possible
True, this was probably some formatting relic. We will adapt the axis to fill the full space. Most of our axes start at 0, but that doesn’t make so much sense here, since we consider the solidity in the control as “baseline”.
3.7. "wild-type like more tubular mitochondria" reads rather awkwardly. "more tubular mitochondria (as in the wild-type)"?
Thank you, sounds better.
3.8. l106 -- imaging artefacts? are mitos fragmenting because of photo stress? -- this is mentioned in l577-8 in the Methods, but the data from the growth rate and MMP comparison isn't given -- an SI figure would be helpful here. It would be reassuring to know that mito morphology wasn't changing in response to phototoxicity too.
In the methods we just briefly point out that we have done all our “due diligence” controls to check that we do not generate phototoxicity, something that we highlight in the cited review. We do not explicitly have a figure for this, but figure S1A shows that the solidity of the mitochondrial network in control cells stays the same over 9 hours, even though these cells are exposed to the same cultivation and imaging regime as the depleted cells. We will also add a picture of control cells after 9 h. In S1B we show that control cells containing TIR but no AID tag treated with both chemicals imaged over 9 hours also show the same solidity (~mitochondrial morphology) as untreated control. Also, the doubling times of cells grown in our imaging system (Fig R1B) are very similar to the shake flask (Fig R1A). All in all, we are very confident that our imaging settings did not impact our reported phenotypes.
3.9. para l146 -- so this suggests mtDNA-encoded proteins have a very rapid turnover, O(hours) -- is this known/reasonable?
Reference (Christiano et al. 2014) suggests that respiratory chain proteins are shorter lived than the average yeast protein. However, based on Figure 2C we think the dynamics mostly speak for a dilution by growth.
3.10. section l189 -- it's hard to reason fully about these statistics of mitochondrial concentration given that the petite phenotype is fundamentally affecting overall cell volume. can we have details on the cell size distribution in parallel with these results? to put it another way -- how does mitochondrial *amount* per cell change?
This is a good point. We report mostly on mitochondrial “concentrations” because we think this is what the cell actually cares about (mitochondrial activity in relationship to cytosolic activity). But we will include additional graphs on mitochondrial amount as well as size distributions (Fig R1C, related to Fig 4F). We can already point out that the size distribution of the population does not change much in the first hours. The “petite” phenotype refers to small colonies on growth medium with limited supply of a fermentable carbon source, not to smaller size of single cells.
3.11. l199 the mean in Fig S3C certainly does change -- it increases, clearly relative both to control and to its initial value. rather than sweeping this under the carpet we should look in more detail to understand it (a consequence of the increased skew of the distribution)?
This relates somewhat to the previous point. The increase in average concentration is not due to an increased amount in the population, but due to the fact that it is the small buds that get a very high amount of the mitochondria which “exaggerates” the asymmetric/heterogenous distribution. This will be clarified by the figures we mention in the point above.
3.12. para line 206 -- this doesn't make it clear whether your MMP signal is integrated over all mitochondria in the cell, or normalised by mitochondrial content? this matters quite a lot for the interpretation if the distributions of mitochondrial content are changing. reading on, this is even more important for para line 222. Reading further on, there is an equation on l612 that gives a definition, but it doesn't really clarify (apologies if I'm misunderstanding).
For each cell, we basically calculate the relative mitochondrial enrichment of the MMP sensitive vs the MMP insensitive pre-sequence.
So, MMP= (total intensity of mitochondrial pre-Cox4 Neongreen/ total intensity of mitochondrial pre-Su9 Cardinal) / (total cytosolic pre-Cox4 Neongreen/ total cytosolic pre-Su9 Cardinal).
We calculate this value for each cell, but we do not have the optical resolution to calculate it for individual mitochondrial fragments.
Both constructs are driven by the same strong promoter, so transcription of the fluorophore should never limit the uptake. Also, in Figure 3D we compare control and depleted cells with similar total mitochondrial concentration, so the difference must be due to a different import of the two fluorophores, see also Fig S4D. The calculated “MMP” value is of course only a crude proxy for the actual membrane potential in millivolts and we do not want to make any claims on absolute values or quantitative differences. But essentially what we are interested in is “mitochondrial health/activity” and we think the system is good at reporting this. See also 2.2.
3.13. l230 -- a point of personal interest -- low mito concentrations are connected to low "function" (MMP) and give extended division times -- this is interestingly exactly the model needed to reproduce observations in HeLa cells (https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002416). That model went on to predict several aspects of downstream cellular behaviour -- it would be very interesting to see how compatible that picture (parameterised using HeLa observations) is with yeast!
Thank you for pointing out your interesting paper, which we will include in our discussion. Another recent preprint about fission yeast (Chacko et al. 2025) also fits into this picture. Since you were kind enough to disclose your identity, we would be happy to discuss this further with you in person if we can maybe follow-up on this.
3.14. l239 "less mitochondria" -- a bit tricky but I'd say "fewer mitochondria" or "less mitochondrial content"
Thanks, we will think about how to best rephrase this, probably less mitochondrial content.
3.15. Section l234 So here (and in Fig 4) the focus is on overall distributions of mitochondrial concentration in different cells (mother-to-be, mother, bud; gen 1, gen >1). But we've just seen that one effect of fzo1 is to broader the distribution of mitochondrial concentration across cells. Can't we look in more depth at the implications of this heterogeneity? For example in Fig 4F (which is cool) we look at the distribution of all fzo1 mothers-to-be, mothers, and buds. But this loses information about the provenance. For example, do mothers-to-be with extremely low mito concentrations just push everything to the bud, while mothers-to-be with high mito concentrations distribute things more evenly? It would seem very easy and very interesting to somehow subset the distribution of mothers-to-be by concentration and see how different subsets behave
This is a good point. When analyzing the data, we pretty much plotted everything against everything and then chose the graphs that we think will best guide the reader through the story-line. We can make additional supplementary plots where we show the starting concentrations/amounts of the mother in relationship to the resulting split ratio at the end of the cycle (Fig R1D).
3.16. l285 -- experimental design -- do we know that Atp6 will continue to be a good proxy for functional mtDNA in the face of the perturbations provided by Fzo1 depletion? Especially if there is impact on the expression of mitoribosomes, the relationship between mtDNA and Atp6 may look rather different in the mutant?
This is actually our top-priority experiment now. We will use the HI-NESS system and possibly DAPI staining to make a more direct link to mtDNA/ nucleoid numbers, see 1.2.
3.17. l290 -- ruled out mitophagy. This message could be much clearer. Comparing Fig S5C and Fig 3A side-by-side is a needlessly difficult task -- put Fig 3A into Fig S5. Then we see that when mitophagy is compromised, the distribution of mitochondrial concentration has a lower median and much lower upper quartile than in the mitophagy-equipped Fzo1 mutant? What is going on here? For a paper motivated by disentangling coupled mechanisms, this should be made clearer!
Thanks for pointing this out. We can of course easily include the control in the corresponding figure. Compromising mitophagy is likely to generally affect mitochondrial health and turnover a little bit, independent of what is going on with Fzo1. The second evidence that speaks against large-scale mitophagy is the proteomics data: On population level the dynamics of the respiratory chain proteins are very different from those of other (nuclear encoded) mitochondrial proteins. We will add additional supplementary figures to make this more clear, see Fig R1E. Most mitochondrial proteins in the proteomics experiment stay constant in the first few hours, consistent with the imaging data showing that the mean mitochondrial content of the population does not change initially. This again highlights that it is the unequal distribution which is the problem and not massive degradation of mitochondria.
3.18. With the Atp6 signal, how do we know that fluorescence from different cells is comparable? Buds will be smaller than mother cells for example, potentially leading to less occlusion of the fluorescent signal by other content in the cytoplasm
This is of course a general problem that anyone faces doing quantitative fluorescence microscopy. From the technical side, we have done the best we could by taking a reasonable amount of z-slices and by choosing fluorophores that are in a range with little cellular background fluorescence (e.g. Neongreen is much better than GFP). From a practical standpoint, we are always comparing to the control, which is subject to the same technical limitations as the depleted cells and the cell sizes are very similar. So, even if we are systematically overestimating the Atp6 concentration in the bud by a few %, the difference to the control would still be qualitatively true. We therefore do not think that any of our conclusions are affected by this.
3.19. l343 -- maintenance of mtDNA -- here the point about l285 (is the Atp6-mtDNA relationship the same in the Fzo1 mutant) is particularly important, as we're directly tying findings about the protein product to implications about the mtDNA
We will carefully address this, see above.
3.20. l367 -- on a first read this description of the model feels like lots of choices have been made without being fully justified. Why a log-normal distribution (when the fit to the data looks rather flawed); why the choice of 5 groups for nucleoid number (why not 3? or 8?); the process used for parameter fitting is very unclear (after reading the methods I think some of these values are read directly from the data, but the shapes of the distributions remain unexplained). l705 -- presumably the ratio was drawn from a log-normal distribution and then the corresponding nucleoid numbers were rounded to integers? the ratio itself wasn't rounded? (also l367) How were the log-normal distributions fitted to experiments (Figs. S7A,B)? Just by eye?
We will update our model based on measured nucleoid counts and then explain more stringently the choices we make/ parameters we select.
3.21. l711 by random selection -- just at random? ("selection" could be confusing) Overall, it feels like the model may be too complicated for what it needs to show. Either (a) the model should show qualitatively that unequal inheritance and reduced production leads to rapid loss -- which a much simpler model, probably just involving a couple of lines of algebra, could show. Or (b) the model should quantitatively reproduce the particular numerical observations from the experiments -- it's not totally clear that it does this (do the cell-cycle-based decay timescales in Fig 7 correspond to the hour-based decay timescales in other plots, for example). At the moment the model is at a (b) level of detail but it's only clear that it's reporting the (a) level of results.
If the HI-NESS and Fzo1 re-addition experiments work as explained above, all parameters will have direct experimental data, and we should get much closer to (a).
3.22. A lot of the discussion repeats the results; depending on editorial preferences some of this text could probably be pared back to focus on the literature connections and context.
We will think about streamlining the discussion once some of the additional material alluded to above has been added.
3.23. Data availability -- it looks like much of the data required to reproduce the results is not going to be made available. Images and proteomic data are promised, but the data associated with mitochondrial concentration and other features are not mentioned. For FAIR purposes all the data (including statistics from analysis of the images) should be published.
We maybe didn’t phrase this clearly. All data will be made available. Where technically feasible, this will be directly accessible in a repository, otherwise by request to the corresponding author.
On our OMERO server, we have deposited many TB of raw images as well as all the intermediate steps such as segmentation masks, and the csv files with all the extracted data for each cell (including background corrections etc). Additionally, we can include csvs with the data grouped in a way that we used to generate all the box blots etc. As of now, the OMERO data is unfortunately only available by requesting a personal guest login from our bioinformatics facility, but we were promised that with the next technical update there will be a public link available. The proteomics data and the model are already fully accessible. The raw western blot images with corresponding ponceau staining will be included with the final publication either as additional supplementary material or in whatever format matches the journal requirements.
3.24 l660 -- can an overview of the EM protocol be given, to avoid having to buy the Mayer 2024 article?
The cited paper is open access. But we can also include more details in our method section.
References:
Chacko, L. A., H. Nakaoka, R. Morris, W. Marshall, and V. Ananthanarayanan. 2025. 'Mitochondrial function regulates cell growth kinetics to actively maintain mitochondrial homeostasis', bioRxiv.
Christiano, R., N. Nagaraj, F. Frohlich, and T. C. Walther. 2014. 'Global proteome turnover analyses of the Yeasts S. cerevisiae and S. pombe', Cell Rep, 9: 1959-65.
Contamine, V., and M. Picard. 2000. 'Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast', Microbiol Mol Biol Rev, 64: 281-315.
Deng, Jingti, Lucy Swift, Mashiat Zaman, Fatemeh Shahhosseini, Abhishek Sharma, Daniela Bureik, Francesco Padovani, Alissa Benedikt, Amit Jaiswal, Craig Brideau, Savraj Grewal, Kurt M. Schmoller, Pina Colarusso, and Timothy E. Shutt. 2025. 'A novel genetic fluorescent reporter to visualize mitochondrial nucleoids', bioRxiv: 2023.10.23.563667.
Di Bartolomeo, F., C. Malina, K. Campbell, M. Mormino, J. Fuchs, E. Vorontsov, C. M. Gustafsson, and J. Nielsen. 2020. 'Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift', Proc Natl Acad Sci U S A, 117: 7524-35.
Ebert, A. C., N. L. Hepowit, T. A. Martinez, H. Vollmer, H. L. Singkhek, K. D. Frazier, S. A. Kantejeva, M. R. Patel, and J. A. MacGurn. 2025. 'Sphingolipid metabolism drives mitochondria remodeling during aging and oxidative stress', bioRxiv.
Jakubke, C., R. Roussou, A. Maiser, C. Schug, F. Thoma, R. Bunk, D. Horl, H. Leonhardt, P. Walter, T. Klecker, and C. Osman. 2021. 'Cristae-dependent quality control of the mitochondrial genome', Sci Adv, 7: eabi8886.
Khan, Abdul Haseeb, Xuefang Gu, Rutvik J. Patel, Prabha Chuphal, Matheus P. Viana, Aidan I. Brown, Brian M. Zid, and Tatsuhisa Tsuboi. 2024. 'Mitochondrial protein heterogeneity stems from the stochastic nature of co-translational protein targeting in cell senescence', Nature Communications, 15: 8274.
Martin, J., K. Mahlke, and N. Pfanner. 1991. 'Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences', J Biol Chem, 266: 18051-7.
Osman, C., T. R. Noriega, V. Okreglak, J. C. Fung, and P. Walter. 2015. 'Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion', Proc Natl Acad Sci U S A, 112: E947-56.
Perić, Matea, Peter Bou Dib, Sven Dennerlein, Marina Musa, Marina Rudan, Anita Lovrić, Andrea Nikolić, Ana Šarić, Sandra Sobočanec, Željka Mačak, Nuno Raimundo, and Anita Kriško. 2016. 'Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan', Scientific Reports, 6: 28751.
Roussou, Rodaria, Dirk Metzler, Francesco Padovani, Felix Thoma, Rebecca Schwarz, Boris Shraiman, Kurt M. Schmoller, and Christof Osman. 2024. 'Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level', The EMBO Journal, 43: 5340-59-59.
Seel, A., F. Padovani, M. Mayer, A. Finster, D. Bureik, F. Thoma, C. Osman, T. Klecker, and K. M. Schmoller. 2023. 'Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth', Nat Struct Mol Biol, 30: 1549-60.
Vowinckel, J., J. Hartl, R. Butler, and M. Ralser. 2015. 'MitoLoc: A method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells', Mitochondrion, 24: 77-86.
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This article addresses the connection between perturbed mitochondrial structure and genetics in yeast. When mitochondrial fusion is compromised, what is the chain of causality -- the mechanism -- that leads to mtDNA populations becoming depleted? This is a fascinating question, linking physical cell biology to population genetics. I admire the philosophy of the research, acknowledging and attempt to control for the many possible confounding influences. The manuscript describes the context and the research tightly and digestibly; the figures illustrate the results in a clear and natural way.
For transparency, I am Iain Johnston and I am happy for this review to be treated as public domain. To my eyes my most important shortcoming as a review is my relative lack of familiarity with the yeast fzo1 mutant; while I am familiar with analysis of yeast mito morphology and mtDNA segregation, a reviewer familiar with the nuances of this strain and its culture would be a useful complement.
I have a few more general points and a collection of smaller points below that I believe might help make the story more robust.
General points
Smaller points
l47 full genus name when it first appears
l58 I may be wrong here, but I thought the petite phenotype more classically arises from mtDNA deletion mutations, not loss? The way this is phrased implies that mtDNA loss is [always] the cause. Whether I'm wrong on that point or not, the petite phenotype should be described and referenced.
para starting l59 -- should mention for context that mitochondria in (healthy, wildtype) yeast are generally much more fused than in other organisms
Fig 1C -- very odd choice of y-axis range! either start at zero or ensure that the data fill as much vertical space of the plot as possible
l105 "wild-type like more tubular mitochondria" reads rather awkwardly. "more tubular mitochondria (as in the wild-type)"?
l106 -- imaging artefacts? are mitos fragmenting because of photo stress? -- this is mentioned in l577-8 in the Methods, but the data from the growth rate and MMP comparison isn't given -- an SI figure would be helpful here. It would be reassuring to know that mito morphology wasn't changing in response to phototoxicity too.
para l146 -- so this suggests mtDNA-encoded proteins have a very rapid turnover, O(hours) -- is this known/reasonable?
section l189 -- it's hard to reason fully about these statistics of mitochondrial concentration given that the petite phenotype is fundamentally affecting overall cell volume. can we have details on the cell size distribution in parallel with these results? to put it another way -- how does mitochondrial amount per cell change?
l199 the mean in Fig S3C certainly does change -- it increases, clearly relative both to control and to its initial value. rather than sweeping this under the carpet we should look in more detail to understand it (a consequence of the increased skew of the distribution)?
para line 206 -- this doesn't make it clear whether your MMP signal is integrated over all mitochondria in the cell, or normalised by mitochondrial content? this matters quite a lot for the intepretation if the distributions of mitochondrial content are changing. reading on, this is even more important for para line 222. Reading further on, there is an equation on l612 that gives a definition, but it doesn't really clarify (apologies if I'm misunderstanding).
l230 -- a point of personal interest -- low mito concentrations are connected to low "function" (MMP) and give extended division times -- this is interestingly exactly the model needed to reproduce observations in HeLa cells (https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002416). That model went on to predict several aspects of downstream cellular behaviour -- it would be very interesting to see how compatible that picture (parameterised using HeLa observations) is with yeast!
l239 "less mitochondria" -- a bit tricky but I'd say "fewer mitochondria" or "less mitochondrial content"
Section l234 So here (and in Fig 4) the focus is on overall distributions of mitochondrial concentration in different cells (mother-to-be, mother, bud; gen 1, gen >1). But we've just seen that one effect of fzo1 is to broader the distribution of mitochondrial concentration across cells. Can't we look in more depth at the implications of this heterogeneity? For example in Fig 4F (which is cool) we look at the distribution of all fzo1 mothers-to-be, mothers, and buds. But this loses information about the provenance. For example, do mothers-to-be with extremely low mito concentrations just push everything to the bud, while mothers-to-be with high mito concentrations distribute things more evenly? It would seem very easy and very interesting to somehow subset the distribution of mothers-to-be by concentration and see how different subsets behave
l285 -- experimental design -- do we know that Atp6 will continue to be a good proxy for functional mtDNA in the face of the perturbations provided by Fzo1 depletion? Especially if there is impact on the expression of mitoribosomes, the relationship between mtDNA and Atp6 may look rather different in the mutant?
l290 -- ruled out mitophagy. This message could be much clearer. Comparing Fig S5C and Fig 3A side-by-side is a needlessly difficult task -- put Fig 3A into Fig S5. Then we see that when mitophagy is compromised, the distribution of mitochondrial concentration has a lower median and much lower upper quartile than in the mitophagy-equipped Fzo1 mutant? What is going on here? For a paper motivated by disentagling coupled mechanisms, this should be made clearer!
With the Atp6 signal, how do we know that fluorescence from different cells is comparable? Buds will be smaller than mother cells for example, potentially leading to less occlusion of the fluorescent signal by other content in the cytoplasm
l336 -- similar to the Jajoo et al. mechanism in fission yeast -- but are you talking about feedback control of the mtDNA or the protein (or mRNA) product?
l343 -- maintenance of mtDNA -- here the point about l285 (is the Atp6-mtDNA relationship the same in the Fzo1 mutant) is particularly important, as we're directly tying findings about the protein product to implications about the mtDNA
l367 -- on a first read this description of the model feels like lots of choices have been made without being fully justified. Why a log-normal distribution (when the fit to the data looks rather flawed); why the choice of 5 groups for nucleoid number (why not 3? or 8?); the process used for parameter fitting is very unclear (after reading the methods I think some of these values are read directly from the data, but the shapes of the distributions remain unexplained). l705 -- presumably the ratio was drawn from a log-normal distribution and then the corresponding nucleoid numbers were rounded to integers? the ratio itself wasn't rounded? (also l367) How were the log-normal distributions fitted to experiments (Figs. S7A,B)? Just by eye? l711 by random selection -- just at random? ("selection" could be confusing) Overall, it feels like the model may be too complicated for what it needs to show. Either (a) the model should show qualitatively that unequal inheritance and reduced production leads to rapid loss -- which a much simpler model, probably just involving a couple of lines of algebra, could show. Or (b) the model should quantitatively reproduce the particular numerical observations from the experiments -- it's not totally clear that it does this (do the cell-cycle-based decay timescales in Fig 7 correspond to the hour-based decay timescales in other plots, for example). At the moment the model is at a (b) level of detail but it's only clear that it's reporting the (a) level of results.
A lot of the discussion repeats the results; depending on editorial preferences some of this text could probably be pared back to focus on the literature connections and context.
Data availability -- it looks like much of the data required to reproduce the results is not going to be made available. Images and proteomic data are promised, but the data associated with mitochondrial concentration and other features are not mentioned. For FAIR purposes all the data (including statistics from analysis of the images) should be published.
l660 -- can an overview of the EM protocol be given, to avoid having to buy the Mayer 2024 article?
This is a powerful and thoughtful study that provides a collection of new mechanistic insights into the link between physical and genetic properties of mitochondria in yeast. Cell biologists, geneticists, and the mitochondrial field will find this of potentially deep interest. Because of the mode and dynamics of inheritance in budding yeast, findings here may not be directly transferrable to other eukaryotes, but these insights are still of interest for researchers outside of yeast for their insight into how this well-studied system manages its mitochondrial populations.
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Dengler and colleagues use an AID-based acute depletion of Fzo1 in budding yeast, coupling microfluidics live imaging, single-cell quantification (>30k cells), proteomics, an mtDNA-encoded Atp6 reporter, and simple modeling to argue that fusion loss causes (i) rapid fragmentation and ΔΨm decline, (ii) progressive mtDNA/RC depletion, and (iii) unequal mother-daughter mitochondrial inheritance; together with a failure of compensatory synthesis, these changes drive petite formation. The time-resolved design is valuable, but several readouts are indirect, and some claims (particularly those regarding membrane potential, synthesis "failure," and causality) appear over-interpreted without additional controls.
Major points
The dataset is rich and the time-resolved approach strong, but key conclusions rely on indirect proxies and need orthogonal validation and at least one causal rescue experiment to avoid over-interpretation.
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This manuscript by Dengler et al examines the mechanisms underlying the mtDNA depletion observed in cells where mitochondrial fusion is disrupted by depletion of the fusion factor Fzo1. In Saccharomyces cerevisiae, the authors deplete Fzo1 and use live-cell imaging of thousands of cells to follow the effects and their dynamic following Fzo1 depletion. They find that Fzo1-depleted cells show very rapid mitochondrial fragmentation (within 1h of Fzo1 depletion), and also an immediate drop in mitochondrial membrane potential (MMP). MtDNA is lost by 15h, and along with it the expression of mitochondrially-encoded proteins. Nuclear-encoded mitochondrial proteins are also decreased though somewhat later, and the authors find that this is largely due to their degradation (probably a consequence of lack of mitochondrial import into low-MMP cells). Most importantly, the study identifies two separate mechanisms that together contribute to the loss of mt-encoded proteins in Fzo1-depleted cells: unequal distribution of mitochondria during cell division and the reduction of a fusion-dependent compensatory synthesis of mt-encoded proteins. Unexpectedly, Fzo1-depleted cells end up passing an increased (rather than decreased) amount of mitochondria and mitochondria-encoded proteins to their daughters. Over several generations, and combined with the loss of the compensatory synthesis of more mt-encoded proteins, this leads to the progressive loss of mtDNA and mtDNA-encoded proteins in the population.
The study is very thorough and the experiments contain the appropriate controls. The conclusions are convincing and largely supported by the experimental data that has been appropriately replicated. The data presentation is generally clear although the text could benefit from some streamlining.
However, addressing the following major comments is required:
Minor comments:
Referees cross-commenting
I think that the reviews of the other two reviewers are both insightful and constructive. Especially the rescue experiment suggested by Reviewer 2 could provide strong support for the interpretations of the study. Note that all three reviewers ask for validation of the use of Atp6p as a read-out of mtDNA function, and that all agree the data is powerful and the study of value to the field.
The fact that disruption of mt fusion leads to mtDNA loss has been known for some time, but the mechanism behind this phenomenon has remained unknown to date. This thorough and precise study by Dengler et al uses state-of-the-art single-cell analysis to dissect the mechanisms underlying the mtDNA loss following the disruption of mt fusion, and convincingly reveal that it is caused by two different mechanisms: i) the inequal inheritance of mitochondria between mother and bud, and ii) the loss of a compensatory mechanism that normally maintains homeostatic mt protein levels. In the process, the authors shed light on the dynamics of the events following Fzo1 depletion, revealing dramatically fast mt fragmentation and a loss of MMP, which in turn can be expected to act as a stress signal and influence a number of cellular processes.
The findings of the study can have relevance for human conditions involving disrupted mitochondrial dynamics, caused for example by mutations in mitofusins. The study will be of interest to researchers in mitochondrial biology ranging from dynamics and mtDNA maintenance to mitochondrial medicine.
The field of expertise of this reviewer: mtDNA maintenance. I am not able to properly evaluate the modelling in Fig. 7.
Reviewer #3 (Public Review):
It is rare to find systems in neuroscience where a detailed mechanistic link can be made between the biophysical properties of individual neurons and observable behaviors. In this study, Medina and Margoliash examined how the intrinsic physiological properties of a subclass of neurons in HVC, the main nucleus orchestrating the production of birdsong, might have an effect on the temporal structure of a song. This builds on prior work from this lab demonstrating that intrinsic properties of these neurons are highly consistent within individual animals and more similar between animals with similar songs, by identifying specific acoustic features of the song that covary with intrinsic properties and by setting forth a detailed biophysical network model to explain the relationship.
The main experimental finding is that excitability, hyperpolarization-evoked sag, and rebound depolarization are correlated with song duration and the duration of long harmonic elements. This motivates the hypothesis that rebound depolarization acts as a coincidence detector for the offset of inhibition associated with the previous song element and excitation associated with the start of the next element, with the delay and other characteristics of the window determined primarily by Ih. The idea is then that the temporal sensitivity of coincidence detection, which is common to all HVCx neurons, sets a global tempo that relates to the temporal characteristics of a song. This model is supported by some experimental data showing variation in the temporal integration of rebound spiking and by a Hodgkin-Huxley-based computational model that demonstrates proof of principle, including the emergence of a narrow (~50 ms) post-inhibitory window when excitatory input from other principal neurons can effectively evoke spiking.
Overall, the data are convincing and the model is compelling. The manuscript plays to the strengths of zebra finch song learning and the well-characterized microcircuitry and network dynamics of HVC. Of particular note, the design for the electrophysiology experiments employed both a correlational approach exploiting the natural variation in zebra finch song and a more controlled approach comparing birds that were tutored to produce songs that differed primarily along a single acoustical dimension. The modeling is based on Hodgkin-Huxley ionic conductances that have been pharmacologically validated, and the connections and functional properties of the network are consistent with prior work. This makes for a level of mechanistic detail that will likely be fruitful for future work.
Comments on revised version:
I read through everything and I also feel that my comments have been adequately addressed.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1(Public Review):
The correlation between rebound excitation and song structure (e.g., harmonic stack duration) may depend on outliers, such as birds with harmonic stacks >150ms.
If in wild zebra finch, or even if in domesticated zebra finch including our birds and the birds from the other labs that we evaluated, the distribution of durations of longest harmonic stacks has a long tail, it is not apparent that birds with long duration harmonic stacks are properly considered as outliers. Examining the distribution of motif durations (a less derived statistic) in 33 birds (Fig. 2C) does not support the idea that birds with longer duration songs are outliers. Thus, we view the reviewer question as addressing whether there are different mechanisms operating in birds with long harmonic stacks than for other birds. Unfortunately, the numbers of long-duration harmonic stack birds are too small to give confidence in any statistical analysis of that group. Thus, we limited our re-analysis to the data excluding birds with harmonic stacks >150ms (which is arbitrary), examining how these birds influence our conclusions. We conclude that the influence of the excluded birds on the overall result is modest. The updated results are presented in Supplemental Figure 6, and the Results section has been revised to state:
“We found that while some of the p values increased above 0.05 (p = 0.058 for rebound area vs. longest harmonic stack and p = 0.082 for sag ratio and longest harmonic stack), it remained significant for firing frequency and longest stack (Pearson’s R, p = 0.0017) and for sag ratio and motif duration (p = 0.024). However, when sag ratio was compared against the duration of the motif excluding the longest harmonic stack, there was no relationship (p = 0.85).”
There is a disconnect between the physiological measurements and the HH model presented.
We acknowledge that addressing this limitation would involve additional experimental and modeling assumptions. Rather than overextending our interpretations, we have clarified the limitations of the current study in the Discussion:
“While this HH model provides a plausible framework for linking intrinsic properties to sequence propagation, it does not fully account for the observed relationship between IPs and song structure. A principal limitation constraining the current model is the absence of information for the same neurons combining characterization of both IPs and network activity during singing (or song playback), when HVC<sub>X</sub> express activity related to song features. Addressing this gap would requires additional and challenging experiments and is beyond the scope of this study.”
Although disynaptic inhibition between HVC<sub>X</sub> neurons and between HVC<sub>RA</sub> and HVC<sub>X</sub> neurons is well established, I am not aware of any data indicating direct synaptic connections between HVC<sub>X</sub> neurons.
This is an important theoretical point about the reliance of the intervaldetecting network model on HVC<sub>X</sub> neurons and about how the model would change if many of the HVC<sub>X</sub> were swapped for HVC<sub>RA</sub> neurons. Connections between HVC<sub>RA</sub> neurons to HVC<sub>X</sub> neurons are established, whereas there is relative paucity of evidence for HVC<sub>X</sub> to HVC<sub>X</sub> connectivity. This is based on work from Prather and Mooney, 2005 (among others) which performed paired sharp electrode recordings to characterized connections in HVC. This work found very few HVC<sub>X</sub> - HVC<sub>X</sub> connections. However, if connected HVC<sub>X</sub> neurons are physically more distant from each other than are connected HVC<sub>RA</sub> – HVC<sub>X</sub> neurons, they would more likely be missed in blind paired recordings. Using different approaches, recent results from the Roberts lab (Trusel et al.,eLife, 2025) supports the existence of robust HVC<sub>X</sub> - HVC<sub>X</sub> connections.
Reviewer #2(Public Review):
The interpretation of p-values is rigid, and near-significant results (e.g., p = 0.06) are dismissed without discussion.
We revised the text to reflect a more nuanced and consistent interpretation of p-values and updated the reporting to include exact values. For example, the Results section now states:
"Nonetheless, the longest syllable duration was not significantly correlated with the average sag ratio for each bird (Pearson’s R: R<sup>2</sup> = 0.12, p = 0.065, Supplemental Fig. 2, top left panel), though it is trending toward significance (see Discussion)”
The conclusion that harmonic stacks influence intrinsic properties lacks necessary controls.
We have attempted to further clarify that harmonic stacks were used as a representative feature of temporal song structure rather than a unique determinant of intrinsic properties. The Discussion now states:
“Although harmonic stacks provide a useful test case for studying temporal integration, our findings suggest that IPs are broadly linked to song duration and structure, rather than specific syllable types. This is also consistent with prior results that found all HVC<sub>X</sub> ion currents that were modeled were influenced by song learning[31].”
The relationship between rebound area and experimentally tutored birds was not fully explored.
We expanded the analysis to include rebound area in instrumentally tutored birds, which has now been incorporated into Figure 4C. These additional analyses also robustly support our hypotheses. The Results section has been updated to state:
“We then evaluated the IPs of HVC<sub>X</sub> in the birds from the two groups. HVC<sub>X</sub> neurons from birds who sang unmodified songs (N = 5 birds, 31 neurons), which had shorter harmonic stacks and shorter overall duration, had lower sag ratios (Mann-Whitney: p = 0.025), firing frequency (Mann-Whitney, p = 0.0051) and rebound area (Mann-Whitney: p = 0.0003)”
Reviewer #3 (Public Review):
Limited data supports the claim that intrinsic properties influence temporal integration windows.
While we agree that further data could strengthen this claim, we show that this can happen in principle (Figure 5) but believe that the appropriate experiment to test this requires further experiments in-vivo. We emphasize in the Discussion:
“Our findings suggest that post-inhibitory rebound excitation in HVC<sub>X</sub> could expand temporal integration. Ultimately, experiments combining in vitro with in vivo recordings can directly quantify this effect. We hope our results motivate such experiments.”
Technical Corrections
(1) Fixed typographical errors (e.g., Line 177: corrected "r2 = 4" to "r2 = 0.4").
(2) Revised figure legends for clarity (e.g., Figure 4E now includes tutoring design details).
(3) Updated methods to specify how motifs were defined and measured.
Revised Figures
Figure 4: Updated to include analysis of rebound area in instrumentally tutored birds, reflecting the relationship between experimental tutoring and intrinsic properties.
Supplemental Figure 6: Correlation analysis excluding outliers
Reviewer #1 (Public review):
Summary:
This paper proposes a new model of perceptual habituation and tests it over two experiments with both infants and adults. The model combines a neural network for visual processing with a Bayesian rational model for attention (i.e., looking time) allocation. This Bayesian framework allows the authors to measure elegantly diverse factors that might drive attention, such as expected information gain, current information gain, and surprise. The model is then fitted to infant and adult participants' data over two experiments, which systematically vary the amount of habituation trials (Experiment 1) and the type of dishabituation stimulus (familiarity, pose, number, identity and animacy). Results show that a model based on (expected) information gain performs better than a model based on surprise. Additionally, while novelty preference is observed when exposure to familiar stimuli is elevated, no familiarity preference is observed when exposure to familiar stimuli is low or intermediate, which is in contrast with past work.
Strengths:
There are three key strengths of this work:
(1) It integrates a neural network model with a Bayesian rational learner, thus bridging the gap between two fields that have often been disconnected. This is rarely seen in the cognitive science field, but the advantages are very clear from this paper: It is possible to have computational models that not only process visual information, but also actively explore the environment based on overarching attentional processes.
(2) By varying parametrically the amount of stimulus exposure and by testing the effects of multiple novel stimulus types, this work allowed the authors to put classical theories of habituation to the test on much finer scales than previous research has done.
(3) The Bayesian model allows the authors to test what specific aspects are different in infants and adults, showing that infants display greater values for the noise parameter.
Weaknesses:
This model pertains visual habituation. What drives infants' (dis)engagement of attention more broadly, for example, when learning the probabilistic structures of the environment around them (e.g., language, action prediction) may follow different principles and dynamics.
Reviewer #3 (Public review):
Summary:
This paper by Stribling and colleagues sheds light on a decade-long P. aeruginosa outbreak of the high-risk lineage ST-621 in a US Military hospital. The origins of the outbreak date back to the late 90s and it was mainly caused by two distinct subclones SC1 and SC2. The data of this outbreak showed the emergence of antibiotic resistance to cephalosporin, carbapenems and colistin over time highlighting the emerging risk of extensively resistant infections due to P. aeruginosa and the need for ongoing surveillance.
Strengths:
This study, overall, is well constructed and clearly written. Since detailed information on floor plans of the building and transfers between facilities was available, the authors were able to show that these two subclones emerged in two separate buildings of the hospital. The authors support their conclusions with prospective environmental sampling in 2021 and 2022 and link the role of persistent environmental contamination to sustaining nosocomial transmission. Information on resistance genes in repeat isolates for the same patients allowed the authors to detect the emergence of resistance within patients. The conclusions have broader implications for infection control at other facilities. In particular, the paper highlights the value of real-time surveillance and environmental sampling in slowing nosocomial transmission of P. aeruginosa.
Weaknesses:
My major concern is that the authors used fixed thresholds and definitions to classify the origin of an infection. As such, they were not able to give uncertainty measures around transmission routes nor quantify the relative contribution of persistent environmental contamination vs patient-to-patient transmission. The latter would allow the authors to quantify the impact of certain interventions. In addition, these results represent a specific US military facility and the transmission patterns might be specific to that facility. The study also lacked any data on antibiotic use that could have been used to relate to and discuss the temporal trends of antimicrobial resistance.
Comments on revisions:
The authors have addressed my concerns adequately in the revised manuscript.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
This is a manuscript describing outbreaks of Pseudomonas aeruginosa ST 621 in a facility in the US using genomic data. The authors identified and analysed 254 P. aeruginosa ST 621 isolates collected from a facility from 2011 to 2020. The authors described the relatedness of the isolates across different locations, specimen types (sources), and sampling years. Two concurrently emerged subclones were identified from the 254 isolates. The authors predicted that the most recent common ancestor for the isolates can be dated back to approximately 1999 after the opening of the main building of the facility in 1996. Then the authors grouped the 254 isolates into two categories: 1) patient-to-patient; or 2) environment-to-patient using SNP thresholds and known epidemiological links. Finally, the authors described the changes in resistance gene profiles, virulence genes, cell wall biogenesis, and signaling pathway genes of the isolates over the sampling years.
Strengths:
The major strength of this study is the utilisation of genomic data to comprehensively describe the characteristics of a long-term Pseudomonas aeruginosa ST 621 outbreak in a facility. This fills the data gap of a clone that could be clinically important but easily missed from microbiology data alone.
Weaknesses:
The work would further benefit from a more detailed discussion on the limitations due to the lack of data on patient clinical information, ward movement, and swabs collected from healthcare workers to verify the transmission of Pseudomonas aeruginosa ST 621, including potential healthcare worker to patient transmission, patient-to-patient transmission, patient-to-environment transmission, and environment-to-patient transmission. For instance, the definition given in the manuscript for patient-to-patient transmission could not rule out the possibility of the existence of a shared contaminated environment. Equally, as patients were not routinely swabbed, unobserved carriers of Pseudomonas aeruginosa ST 621 could not be identified and the possibility of misclassifying the environment-to-patient transmissions could not be ruled out. Moreover, reporting of changes in rates of resistance to imipenem and cefepime could be improved by showing the exact p-values (perhaps with three decimal places) rather than dichotomising the value at 0.05. By doing so, readers could interpret the strength of the evidence of changes.
Impact of the work:
First, the work adds to the growing evidence implicating sinks as long-term reservoirs for important MDR pathogens, with direct infection control implications. Moreover, the work could potentially motivate investments in generating and integrating genomic data into routine surveillance. The comprehensive descriptions of the Pseudomonas aeruginosa ST 621 clones outbreak is a great example to demonstrate how genomic data can provide additional information about long-term outbreaks that otherwise could not be detected using microbiology data alone. Moreover, identifying the changes in resistance genes and virulence genes over time would not be possible without genomic data. Finally, this work provided additional evidence for the existence of long-term persistence of Pseudomonas aeruginosa ST 621 clones, which likely occur in other similar settings.
We thank the reviewer for their thorough evaluation of our work, and for the suggested improvements. A main goal of this study was to show that integrating routine wgs in the clinic was a game changer for infection control efforts. We appreciate this aspect was highlighted as a strength by this reviewer. While some of the weaknesses identified are inherent to the data (or lack thereof) available for this study, we have revised the manuscript to include a detailed discussion on limitations (sampling, thresholds of genetic relatedness, definition and categories etc.) that could influence the genomic inferences. We also provided exact p-values for the changes in rates of resistance, as requested. Finally, we have positively answered all the specific recommendations suggested by the reviewer and modified the manuscript accordingly.
Reviewer #2 (Public Review):
Summary:
The authors present a report of a large Pseudomonas aeruginosa hospital outbreak affecting more than 80 patients with first sampling dates in 2011 that stretched over more than 10 years and was only identified through genomic surveillance in 2020. The outbreak strain was assigned to the sequence type 621, an ST that has been associated with carpabapenem resistance across the globe. Ongoing transmission coincided with both increasing resistance without acquisition of carbapenemase genes as well as the convergence of mutations towards a host-adapted lifestyle.
Strengths:
The convincing genomic analyses indicate spread throughout the hospital since the beginning of the century and provide important benchmark findings for future comparison.
The sampling was based on all organisms sent to the Multidrug-resistant Organism Repository and Surveillance Network across the U.S. Military Health System.
Using sequencing data from patient and environmental samples for phylogenetic and transmission analyses as well as determining recurring mutations in outbreak isolates allows for insights into the evolution of potentially harmful pathogens with the ultimate aim of reducing their spread in hospitals.
Weaknesses:
The epidemiological information was limited and the sampling methodology was inconsistent, thus complicating the inference of exact transmission routes. Epidemiological data relevant to this analysis include information on the reason for sampling, patient admission and discharge data, and underlying frequency of sampling and sampling results in relation to patient turnover.
We thank the reviewer for their thoughtful feedback on our manuscript and for highlighting the quality of the genomic analyses. We agree that the lack of patient epi data (e.g. date of admission and discharge) and the inconsistent sampling through the years are limitations of this study. We have revised the manuscript to acknowledge these limitations and discuss how not having this data complicates the inference of exact transmission routes. Finally, we have positively answered all the specific recommendations suggested by the reviewer and modified the manuscript accordingly.
Reviewer #3 (Public Review):
Summary:
This paper by Stribling and colleagues sheds light on a decade-long P. aeruginosa outbreak of the high-risk lineage ST-621 in a US Military hospital. The origins of the outbreak date back to the late 90s and it was mainly caused by two distinct subclones SC1 and SC2. The data of this outbreak showed the emergence of antibiotic resistance to cephalosporin, carbapenems, and colistin over time highlighting the emerging risk of extensively resistant infections due to P. aeruginosa and the need for ongoing surveillance.
Strengths:
This study overall is well constructed and clearly written. Since detailed information on floor plans of the building and transfers between facilities was available, the authors were able to show that these two subclones emerged in two separate buildings of the hospital. The authors support their conclusions with prospective environmental sampling in 2021 and 2022 and link the role of persistent environmental contamination to sustaining nosocomial transmission. Information on resistance genes in repeat isolates for the same patients allowed the authors to detect the emergence of resistance within patients. The conclusions have broader implications for infection control at other facilities. In particular, the paper highlights the value of real-time surveillance and environmental sampling in slowing nosocomial transmission of P. aeruginosa.
Weaknesses:
My major concern is that the authors used fixed thresholds and definitions to classify the origin of an infection. As such, they were not able to give uncertainty measures around transmission routes nor quantify the relative contribution of persistent environmental contamination vs patient-to-patient transmission. The latter would allow the authors to quantify the impact of certain interventions. In addition, these results represent a specific US military facility and the transmission patterns might be specific to that facility. The study also lacked any data on antibiotic use that could have been used to relate to and discuss the temporal trends of antimicrobial resistance.
We thank the reviewer for their evaluation of our work and for highlighting the broad implications of our findings regarding the application of real-time surveillance to suppress nosocomial transmission. We agree with the reviewer that fixed thresholds and definitions are imperfect to classify the origin of an infection. The design of this study (e.g. inconsistent sampling through time) was not conducive to provide a comprehensive/quantitative measurement of transmission routes. Thus, we decided to apply conservative thresholds of genetic relatedness and strict conditions (e.g. time between isolate collection, shared hospital location etc.) to favor specificity as our goal was simply to establish that cases of environmentto-patient transmission did happen. In the absence of a truth set, we have not performed sensitivity analysis, but we are conducting a follow-up study to compare inferences from MCMC models to our original fixed-thresholds predictions. This limitation is now discussed in the revised manuscript. Finally, we have positively answered all the specific recommendations suggested by the reviewer and modified the manuscript accordingly including the addition of Figure S3.
Reviewer #1 (Recommendations For The Authors):
The definitions used on lines 391-396 are necessarily somewhat arbitrary, but it would be helpful to have a little bit more justification for the choices made, particularly for the definition of environmental involving the "3x the number of years they were separated". It seems a little hard to square this with the more relaxed 10 SNP cutoff for a patient-to-patient designation. Are there reasons for thinking SNP differences associated with environmental transmission should be smaller than for patient-to-patient, or is the aim here just to set the bar higher for assuming an environmental source? Because these definitions are quite arbitrary, there could also be some value in exploring the sensitivity of the results to these assumptions.
Thank you. We agree with the reviewers that SNP thresholds, albeit necessarily, are arbitrary and that more discussion/justification was needed to put the genomic inferences in context. We have revised the manuscript to indicate that: 1/ the 10 SNP cutoff for a patient-to-patient designation was set to account for the known evolution rate of P. aeruginosa (inferred by BEAST at 2.987E-7 subs/site/year in this study and similar to previous estimates PMID: 24039595) and the observed within host variability (now displayed in revised Fig. 1E). We note that this SNP distance was not sufficient and that an epi link (patients on the same ward at the same time) needed to be established. 2/ the environment-to-patient definition was indeed set to be most conservative (nearly identical isolates in two patients from the same ward with no known temporal overlap for > 365 days). This was indeed done to favor high specificity as this inference relied solely on clinical isolates (i.e. the identical environmental strain in the patientenvironment-patient chain was not sampled). For these clinical isolates to have acquired no/very little mutation in that much time, no/low replication is expected and, although unsampled, we propose this most likely happened on hospital surfaces.
While the term "core genome" should be familiar to most readers, "shell genome" and "cloud genome" are less widely known, and an explanation of what these terms mean here would be helpful.
Thank you. We have revised the manuscript to define the core, shell, and cloud genomes as genes sets found in ≥ 99%, ≥ 95% and ≥ 15% of isolates, respectively.
In the first paragraph of the discussion, it could be added that in many cases for clinically important Gram negatives short read sequencing alone will fail to detect transmission events as outbreaks can be driven by plasmid spread with only very limited clonal spread (see, for example, https://www.nature.com/articles/s41564-021-00879-y )
Thank you. We agree this is an important/emerging aspect of surveillance. However, the goal of this discussion point was to explain why such a large outbreak was missed prior to implementing WGS (short read) surveillance. We feel that discussing “plasmid outbreaks” (which is not at play here, and relatively rare in P. aeruginosa compared to the Enterobacteriaceae) and the need for long read will distract from the narrative.
line 599 What does "Mock" mean here? Would it be more accurate to say it is a simplified floor plan?
Thank you. “Mock” was changed to “simplified”
IPAC abbreviation is only used once - spelling it out in full would increase readability.
Revised manuscript was edited as suggested.
MHS is only used twice.
Revised manuscript was edited to spell out Military Health System
Line 364: full stop missing.
Revised manuscript was edited as suggested.
Line 401: Bayesian rather than bayesian.
Revised manuscript was edited as suggested.
Reviewer #2 (Recommendations For The Authors):
Thank you for giving me the opportunity to review this interesting manuscript.
The conclusions of this paper are mostly well supported by the data presented, but epidemiological information was limited and the sampling methodology was inconsistent, thus complicating inference of exact transmission routes.
Major issues:
What was the baseline frequency of clinical and/or screening samples of Pseudomonas aeruginosa at the hospital? Neither Figure 1D nor Table S1 allows for differentiating between clinical and screening samples. Most isolates were cultured from clinical materials, and there is no information about the patients' length of stay and their respective sampling dates. Is there any possibility of finding out whether the samples were collected for clinical or screening purposes? Would it be possible to include the patients' admission data to determine whether the strains were imported into the hospital or related to a previous stay, e.g. among known carriers? Also, the issue of sampling dates vs. patient stay on the ward should be addressed, as there may be an overlap in patients' stay on the ward but no overlap in terms of sampling dates or even missing samples (missing links).
We have revised the manuscript to address this important point: i) 16 isolates were from surveillance swabs and are labelled “Surveillance” in Table S1. The remaining 237 were clinical isolates; ii) unfortunately, because the sampling was done under a public health surveillance framework, we do not have access to historical patient data (admission/discharge date, wards, rooms, etc.) and we can not calculate length of stay or better identify patient overlap. These limitations are now acknowledged in the discussion of the revised manuscript.
In order to evaluate the extent of the outbreak, more epidemiological data would be useful What is the size of the hospital, what is the average patient turnover, and what is the average length of stay in ICU and non-ICU? Is there any specialization besides the military label?
We have revised the manuscript to indicate that facility A is 425-bed medical center and is the only Level 1 trauma center in the Military Health System. Unfortunately, the data to calculate length of stay, throughout the years, in ICU and non-ICU, was not available to us. This limitation is now also acknowledged in the discussion.
Perhaps the authors could attempwt to discuss the extent to which large outbreaks like these may be considered as part of unavoidable evolutionary processes within the hospital microbiome as opposed to accumulation and transmission of potentially harmful genes/clones, and differentiate between the putative community spread without any epidemiological links on the one hand, and hospital outbreaks that could be targeted by local infection prevention activities on the other hand.,
We respectfully disagree with the suggestion that this large outbreak “may be considered as part of unavoidable evolutionary processes within the hospital microbiome” and should be opposed to “transmission of potentially harmful genes/clones”. As a matter of fact, our data showed that infection control staff at Facility A responded with multiple interventions, including closing sinks, replacing tubing, and using foaming detergents. This resulted in slowing the spread of the ST621 outbreak with just 3 cases identified in 2022, 0 cases in 2023 and 1 case in 2024. This is now discussed in the revised manuscript.
Page 5, lines 88-92 lines 101-104. It seems as if the outbreak was identified only by the means of genomic surveillance. This raises questions as to the rationale for sampling and sequencing, especially prior to 2020. Considering 11 cases per year between 2011 and 2016, one could assume such an outbreak would have been noticed without sequencing data.
The MRSN was created in 2010, in response to the outbreak of MDR Acinetobacter baumannii in US military personnel returning from Iraq and Afghanistan. Between 2011 and 2017, the MRSN collected MDR isolates (mandate for all MDR ESKAPE but compliance varied between years and facilities) from across the Military Health System and, for select isolates (e.g. high-risk isolates carrying ESBLs or carbapenemases) performed molecular typing by PFGE. In 2017 the MRSN started to perform whole genome sequencing of its entire repository. In 2020, a routine prospective sequencing service was started and first detected the ST621 outbreak. A retrospective analysis of historical isolate genomes (2011-2019) identified additional cases. The first paragraph of the discussion lists possible factors to explain why the ST621 escaped detection by traditional approaches. We believe 11 cases per year is not a strong signal when stratified by month, wards, or both, especially for a clone lacking a carbapenemase and without a remarkable antibiotic susceptibility profile.
Did the infection control personnel suspect transmission? If yes, was the sampling and submission of samples to the MRSN adapted based on the epidemiologic findings?
The ST621 outbreak was unsuspected before the initial genomic detection in 2020. Until that point, MDR isolates only (Magiorakos et al PMID: 21793988) were collected but compliance was variable through time. Quickly thereafter (starting in 2021), complete sampling of all clinical P. aeruginosa (MDR or not) from Facility A was started. The manuscript was revised to clarify those details of the sampling strategy.
Is there any information about how many environmental sites were sampled without evidence of ST621 / screening samples were cultured without evidence of Pseudomonas aeruginosa?
For patient isolates, only 16 isolates were from surveillance swabs. The remaining 237 were clinical isolates. No denominator data was available to calculate P. aeruginosa and ST-621 positivity rate in surveillance swabs throughout the time period. For environmental isolates, a total of 159 swabs were taken from 55 distinct locations in 8 wards/units including the ER. This data is now included in the revised manuscript. However, a complete analysis of these swabs (positivity rate for ESKAPE pathogens, P. aeruginosa, per ward/floor/room, per swab type (sink drain, bed rail etc.) etc.) is beyond the scope of this study and is being performed as a follow up investigation.
Page 5 lines 89 and 39 Figure S1B. Please describe how the allelic distance for the cluster threshold was selected.
As indicated in the legend of Figure S1B, no thresholds were applied. All ST621 isolates ever sequenced by the MRSN were included. All except 3 isolates shared between 023 cgMLST allelic differences. The remaining 3 were distant by 88-89 allelic differences. The text was revised to clarify this point.
Page 5 lines 99-100. Could the authors please provide some distribution measures (e.g. IQR).
Done as requested. The revised manuscript now reads “…of just 38 single nucleotide polymorphisms (SNPs), and an IQR of 19 (Fig. 1A, Table S1).”
Page 5 line 102. Could the authors please provide some distribution measures (e.g. IQR).
Please see above. A chart was created and is now included as Fig. S2.
Page 6 line 107 and page 34 figure 1c. In the text it is stated that isolates were collected in 27 wards, the figure 1C depicts 26 wards and n/a.
Thank you for spotting this inconsistency. This has been fixed in the revised manuscript.
Page 6 lines 117-118. Samples collected in the emergency room would imply samples collected on admission, already addressed previously. Did the authors investigate a potential import into the hospital from community reservoirs or were all these isolates collected among patients who had been previously admitted to the hospital and/or tested positive for the outbreak strain?
We agree that samples collected in the ER imply samples collected on admission. Of the 29 ER isolates only 9 (31%) were primary isolates (first detection in a new patient) which suggests a majority were from returning patients at Facility A. Because the sampling was done under a public health surveillance framework, we do not have access to historical patient data (admission/discharge date, wards, rooms, etc.) to investigate/confirm that these 9 patients had previous visits at Facility A. This point is now discussed in the revised manuscript.
Page 6 line 128. This could also represent increased selective pressure. However, according to Table S1, the 28 isolates collected in 2011 (the number does not match with Figure 1D) were from many different wards, thus indicating earlier spread throughout the hospital.
Yes, we agree. Please note that table S1 lists all isolates for 2011 whereas Figure 1D focuses on primary (first isolate from each patients) only.
Page 7 line 133. Both Figure 2 and the discussion section, page 13 line 296 suggest the year 2005 instead of 2004?
Thank you for catching this typographical error. This was corrected to 2004 in the revised manuscript.
Figure 1E. The figure should also depict intra-patient diversity for comparison.
Thank you for this great suggestion. We have revised Figure 1E accordingly.
Page 7, lines 146-147 Could the authors attempt explaining the upper part of the bimodal peaks?
This is an all-vs-all SNP analysis for all inter-patient isolates. For each isolates all distances to other isolates are reported, not only the smallest. The upper peaks represent comparisons to isolates from a different outbreak subclone (SC1 vs SC2).
Page 7, line 150 This is a very small number considering the extent of the outbreak and suggests a large number of missing links. Or does this rather imply continuous import and evolution over time that does not necessarily represent transmission within the hospital?
We believe all cases were due to transmission happening within the hospital. Based on conservative thresholds (genetic relatedness and epi link, or lack thereof) the precise origin from another patient (n=10) or a contaminated surface (n=12) can be inferred. For the remaining 60 patients, with the available sampling, the conditions we chose are not met and we simply do not conclude whether a direct patient-to-patient or an environmental origin was more likely.
Page 8 line 155. What does the temporal overlap refer to - sampling date versus patient's stay on the ward? Please specify.
The temporal overlap was investigated from sampling dates, as dates of patient admission/discharged were not available.
Page 8, line 157: What does primary/serial isolate mean - first and follow-up samples of ST621 per patient?
Yes. Primary isolate is used to designate the first isolate from a patient. Serial isolates designate follow-up samples of ST621.
Page 8 line 165: Table S3 and Figure 3 only refer to environmental samples from three wards. Ward 20 rooms 2 and 18 as well as ward 1 rooms 1 and 6 were hotspots - is there any information on the specific infection control/disinfection measures? Addressed in discussion page 12, lines 273-275, but no information on what was actually done.
The manuscript was revised to indicate the precise disinfection measures that were taken. A follow-up study is ongoing to assess long-term efficacy and monitor possible retrograde growth from previously contaminated sinks.
Page 8 line 175: Evaluation of change in resistance fraction over time - There may have been a selection bias with an inconsistent number of strains sequenced per year.
Yes, incomplete sampling and possible selection bias are now listed with other limitations of this study in the discussion of the revised manuscript.
Page 9 line 183: The referral to Table S1 is unclear, I could not find the number and the specific isolates selected for long-read sequencing.
Thank you. This has been added to the revised Table S1.
Page 10 lines 217-225 and Figure 4C: Perhaps it is possible to better align what is written in the text and the caption of the figure. The caption does not clarify that only one patient develops colistin resistance (what was the reason to include the other patients?).
Thank you. We have revised the text and the caption of the figure to clarify that only isolates from one patient developed colistin resistance. The isolates from the other patients on Fig. 4C are shown to provide context and accurately map the emergence of the PhoQE77fs mutation.
Page 10, lines 228-229 and Table S5: How is it possible to identify those 64 genes in Table S5?
We have revised Table S5 to facilitate the identification of the 64 genes with ≥ 2 independently acquired mutations (excluding SYN). Specifically, we have added column E labeled “Counts independent mutations per locus (excluding SYN)”. A total of 205 rows (in this table each row is a variant) have a value ≥ 2 and these represent 64 genes (upon deduplication of locus tags).
Page 13, lines 280-281: Where is the information on chronic infection presented? Serial cultures would not necessarily mean chronic infection.
Authors response: Yes, we agree this was not the appropriate characterization and this was revised to ‘long-term’ infections.
Page 14 line 306: Emergence of colistin resistance in a single patient, correct?
Yes. This was further clarified in the text.
Page 14 lines 315-320: This should go to the results section. In particular disinfection, closing, and replacing of tubing should be mentioned in the results section in reference to the results presented in Table S3.
Thank you. We have considered this suggestion and have decided to leave this discussion as the closing paragraph of this publication. A follow-up study is ongoing to assess long-term efficacy of these interventions on the ST-621 bur also other outbreak clones at Facility A.
Methods
Page 15 lines 330-333: Perhaps it is possible to avoid redundancy.
Thank you. We have revised the text accordingly.
Page 15 lines 341: Information on which isolates were subjected to long-read sequencing is missing.
Thank you. This has been added to the revised Table S1.
Page 16 line 345: Was there a particular reason why Newbler was chosen?
No. At the time Newbler was the default assembler built in the MRSN bacterial genome analysis pipeline and QC processes.
Page 16, line 357-358: What was the rationale for selecting this isolate as reference genome?
This isolate was chosen because it was collected early in the outbreak and phylogenetic analysis revealed it had low root to tip divergence.
Page 16 line 361: Why 310 isolates, if only 253 were assigned to the outbreak clone and only a subset of those were collected in facility A?
This was a typographical error that has corrected (it now reads “…set of 253 isolates.”) in the revised manuscript.
Page 17 lines 387-395: What is the reason that intra-patient diversity was not included in the set of criteria for SNP distances?
The observed within host variability (now displayed in revised Fig. 1E) was taken into consideration when setting SNP thresholds for categorizing patient-to-patient transmission or environment-to-patient event. This is now clarified in the revised manuscript.
Page 17 line 392: How was the threshold of <=10 SNPs determined?
The 10 SNP cutoff to infer a patient-to-patient transmission event was set to account for the known evolution rate of P. aeruginosa (inferred by BEAST at 2.987E-7 subs/site/year in this study, and similar to previous estimates PMID: 24039595) and the observed within host variability (now displayed in revised Fig. 1E). We note that this SNP distance was not sufficient and that an epi link (patients on the same ward within the same month) needed to be established.
Page 17 line 395 and Figure 2: What was the assumed average mutation rate per genome per year?
Thank you. The mean substitution rate inferred by BEAST was 2.987E-7 similar to estimate from previous studies on P. aeruginosa outbreaks (e.g. PMID: 24039595).
Reviewer #3 (Recommendations For The Authors):
Please find (line-by-line comments) on each section of the manuscript below:
Introduction
Line 86: I am wondering why the authors state ">28 facilities" instead of the exact number of facilities from which these lineages were recovered.
Thank you. Manuscript was revised to provide the exact number of facilities. It now reads “…recovered from 37 and 28 facilities, respectively.”
Methods
It's not clear to me which criteria were used for collecting these isolates (both prospective and retrospective). I understand that some of the data are described in more detail in Lebreton et al but I did not find the specific criteria for the collection of the isolates and I imagine that these might differ if different facilities. Would it be possible to comment on that and add a short paragraph in the Methods section?
Thank you. This lack of clarity was also raised by other reviewers, and we have revised the manuscript to indicate that: 1/MDR isolates only (Magiorakos et al PMID: 21793988) were collected from 2011-2020 with the same criteria for all facilities although compliance was variable through time and between facilities; and 2/ starting in 2021 all P. aeruginosa isolates, irrespective of their susceptibility profile, were collected from Facility A
The data comes from a US Military hospital. Is this related to the US Veterans Affairs Healthcare system? Is there more detailed information about the demographics of the patient population?
Facility A is part of the Military Health System (MHS) which provides care for active service members and their families. This is distinct from the US Veterans Affairs Healthcare system. Only limited patient data was accessible to us as this study was done as part of our public health surveillance activities. Patient age (avg. 57.2 +/- 21.0) and gender (ratio male/female 1.7) are provided in the revised manuscript.
Line 384ff: The origin of infection was inferred based on the SNP threshold and epidemiological links. However, recombination events can complicate the interpretation of SNP data. Have the authors attempted to account for this?
Thank you. We agree that recombination events can complicate the interpretation of SNP data. We used Gubbins v2.3.1 to filter out recombination from the core SNP alignment, as indicated in the revised manuscript.
The authors' definition of environment-to-patient transmission seems conservative (nearly identical strain and no known temporal overlap for > 365 days). Have the authors changed the threshold, performed sensitivity analyses, and tested how this would affect their results?
Indeed, acknowledging that fixed thresholds have limitations in their ability to accurately predict the origin of infections, we took a conservative approach to favor specificity as our goal was simply to establish that cases of environment-to-patient transmission did happen. In the absence of a truth set, we have not performed sensitivity analysis, but we are conducting a follow-up study to compare inferences from MCMC models to our original predictions. This limitation is now discussed in the revised manuscript.
The authors don't seem to incorporate the role of healthcare workers in the transmission process. Could they comment on this? I am assuming that environment-to-patient transmission could either be directly from the environment to the patient or via a healthcare worker. I think it's fine to make simplifying assumptions here but it would be great if this was explicitly described.
Thank you for this suggestion. We have not sampled the hands of healthcare workers in this study. As a result, the reviewer is correct to say that we made the simplifying assumption that healthcare workers would be possible intermediates in either environment-topatient or patient-to-patient transmissions, as previously described by others (PMID: 8452949). This limitation is now discussed in the revised manuscript.
Page 5, line 100: What does "all vs all" mean? Based on the supplement, I assume it's the pairwise distance and then averaged across all of those. It would improve the readability of the manuscript if the authors could briefly define this term and then maybe refer to Table S1.
Thank you. We have created Fig.S2 and revised the manuscript to state that ST-621 isolates from facility A belonged to the same outbreak clone with a distance (averaged all vs all pairwise comparison) of just 38 single nucleotide polymorphisms (SNPs), and an IQR of 19 (Fig. S2, Table S1).
Figure 1D: It would be interesting to see additional figures in the supplement on the percentage of sequenced isolates per year and whether it varies across the different sources/sites. Is there any information on which isolates were chosen for sequencing?
Lack of clarity in the sampling/sequencing scheme was raised by multiple reviewers and we have provided a thorough response to earlier comments. We also have revised the material and methods section accordingly. Finally, we have created Fig. S3 to show the percentage of sequenced isolates per year across different sources/sites, as suggested by the reviewer. No noticeable patterns were observed.
It seems like only a subset of all clinical isolates were sequenced. Would it be possible that SC2 was present already earlier but not picked up until a certain date?
Although all isolates received by the MRSN were sequenced, compliance varied through time so it is true that not all clinical isolates were sequenced between 2011-2019. As such, we fully agree with this hypothesis and discuss this possibility as BEAST analysis placed the origin of SC2 in 2004 while the first detection of an SC2 isolate was in December 2012. This limitation is now discussed in the revised manuscript.
Could the authors elaborate on whether the isolates resulted from single-colony picks? Is it possible that the different absence of a subclone is due to the fact that they picked only a colony?
Yes, the isolates resulted from single-colony picks except when the presence of different colony morphologies was noted. In the latter, representative isolates for each colony morphologies were processed. We have revised the methods to make that clear.
Figure 2: It is difficult to see which nodes belong to which patient due to the small font size. I wonder if it was possible to color the nodes for each patient, to make it more readable.
We tried coloring the nodes but with > 60 distinct patients/colors we decided it did not improve clarity. We have revised figure 2 to increase the font size.
Page 7-8, lines 154-155: Did the authors check whether there were isolates of the same strain (that were found in the environment) present in other patients elsewhere in the ward?
Yes. In rare cases, we observed virtually genetically identical isolates from two patients collected in different wards. Because we only have access to clinical isolate data (collected from patient X in ward Y) and do not have access to patient data (admission/discharge date, wards, rooms, etc.), we do not know but cannot exclude that patients overlap in a room prior to the sampling of their P. aeruginosa isolates. We designed our fixed thresholds to be conservative. As a result, in this analysis, these cases are labelled as “undetermined”.
Page 8: Do the authors have any information on antibiotic use during this timeframe? From the discussion, it seems like there is no patient-level prescription data. Is there any data on overall trends? How were trends in antibiotic use correlated with trends in antibiotic resistance?
Unfortunately, patient-level prescription data (or any other data not linked to the bacterial specimens) was not accessible to us as this study was done as part of our public health surveillance activities.
To infer the origin of infection, the authors used a static method with fixed thresholds and definitions. This study does not provide any uncertainty with their estimates. Maybe the authors could add a sentence in the discussion section that MCMC methods to infer transmission trees incorporating WGS could provide these estimates. These methods have not been applied to PA a lot but two examples where MCMC methods have been used without WGS (though the definition of environmental contamination may differ between these studies and this study).
Thank you for this great suggestion. We have revised the manuscript to include a discussion on the limitations of fixed thresholds to infer transmission chains/origins, and to discuss existing alternatives including MCMC methods.
Line 322-323: This sentence is a bit vague since not all of these HAI are due to P. aeruginosa. I would suggest citing a number that is specific to PA.
Thank you. While our paper shows a particular example of protracted P. aeruginosa outbreak, the roll-out of routine WGS surveillance in the clinic will help prevent hospital-associated drug-resistant infections for more than this species. We believe that broadening the scope in the last sentence of the manuscript is important and we decline to revise as suggested.
pequeno valor
Créditos de natureza superpreferencial: pagamento da parcela por meio de RPV
Tese fixada - O pagamento de crédito superpreferencial (art. 100, § 2º, da CF/1988) deve ser realizado por meio de precatório, exceto se o valor a ser adimplido encontrar-se dentro do limite estabelecido por lei como pequeno valor.
Resumo - É inconstitucional — por violar o art. 100, §§ 2º e 8º, da Constituição Federal de 1988 — o pagamento parcial de valores de natureza alimentícia pertencente a credores superpreferenciais por meio de requisição de pequeno valor (RPV), se o montante devido ultrapassar o limite legalmente fixado para essa modalidade.
O texto constitucional estabelece que os créditos chamados de superpreferenciais — de natureza alimentícia e de titularidade de idosos, pessoas com deficiência ou portadores de doenças graves — devem ser pagos por meio de precatório, salvo se o montante exigível estiver dentro do limite definido como de pequeno valor. Isso, porque a expedição de RPV é medida excepcional, condicionada à existência de previsão legal que defina as obrigações passíveis de quitação por essa via (1).
Conforme jurisprudência desta Corte (2), o fracionamento de precatórios superpreferenciais para possibilitar o pagamento por meio de RPV, além de representar risco de impacto orçamentário significativo, não encontra amparo na Constituição Federal, uma vez que o pagamento dos créditos contra a Fazenda Pública deve ser realizado de forma integral pelo <u>mesmo rito</u> (RPV ou precatório), <u>sem que se mesclem as modalidades</u>.
Na espécie, o acórdão do Tribunal Regional Federal da 4ª Região manteve decisão que reconheceu a possibilidade de fracionamento do precatório, permitindo o pagamento da parcela superpreferencial (até 180 salários-mínimos) por meio de RPV, reservando-se o excedente para quitação via precatório judicial.
Com base nesses e em outros entendimentos, o Plenário, por unanimidade, ao apreciar o Tema 1.156 da repercussão geral: (i) deu provimento ao recurso extraordinário para reconhecer a violação ao art. 100, §§ 2° e 8° da Constituição Federal de 1988; (ii) determinou que o pagamento dos créditos superpreferenciais seja adimplido por meio de expedição de precatórios; e (iii) fixou a tese anteriormente citada.
(1) CF/1988: “Art. 100. Os pagamentos devidos pelas Fazendas Públicas Federal, Estaduais, Distrital e Municipais, em virtude de sentença judiciária, far-se-ão exclusivamente na ordem cronológica de apresentação dos precatórios e à conta dos créditos respectivos, proibida a designação de casos ou de pessoas nas dotações orçamentárias e nos créditos adicionais abertos para este fim (...) § 2º Os débitos de natureza alimentícia cujos titulares, originários ou por sucessão hereditária, tenham 60 (sessenta) anos de idade, ou sejam portadores de doença grave, ou pessoas com deficiência, assim definidos na forma da lei, serão pagos com preferência sobre todos os demais débitos, até o valor equivalente ao triplo fixado em lei para os fins do disposto no § 3º deste artigo, admitido o fracionamento para essa finalidade, sendo que o restante será pago na ordem cronológica de apresentação do precatório. (...) § 8º É vedada a expedição de precatórios complementares ou suplementares de valor pago, bem como o fracionamento, repartição ou quebra do valor da execução para fins de enquadramento de parcela do total ao que dispõe o § 3º deste artigo.” (2) Precedentes citados: ADI 6.556 MC-Ref; e RE 1.300.190, RE 1.310.690, RE 1.310.475, no RE 1.312.089, RE 1.293.528, RE 1.306.206, RE 1.297.760 e RE 1.304.973 (decisões monocráticas).
Legislação: CF/1988: art. 100, § 2º e 8º.
Precedentes: ADI 6.556 MC-Ref; e RE 1.300.190, RE 1.310.690, RE 1.310.475, no RE 1.312.089, RE 1.293.528, RE 1.306.206, RE 1.297.760 e RE 1.304.973 (decisões monocráticas).
Obs.: Não se admite o pagamento de crédito superpreferencial, que tem limite de até 3 vezes o fixado para RPV, por requisição de pequeno valor. Não há que se falar em pagamento de partes em RPV e de outra parte em precatório. Os pagamentos dos superpreferenciais somente devem ser veiculados por RPV se estritamente dentro do limite delimitado para esta forma excepcional de pagamento. Via de regra, é por meio de precatórios o pagamento de tais verbas.
RPV e autonomia dos estados e municípios
Resumo - Os estados e municípios podem redefinir o valor limite da Requisição de Pequeno Valor (RPV) visando à adequação de suas respectivas capacidades financeiras e especificidades orçamentárias.
É inconstitucional — por violar a competência privativa da União para legislar sobre direito processual (CF/1988, art. 21, I), uma vez que as normas que dispõem sobre RPV têm caráter eminentemente processual (2) — legislação estadual que transfere ao credor a responsabilidade pelo encaminhamento da documentação necessária para solicitação do pagamento do RPV diretamente ao órgão público devedor, bem como determina a suspensão do prazo para pagamento.
Os estados e municípios podem redefinir o valor limite da Requisição de Pequeno Valor (RPV) visando à adequação de suas respectivas capacidades financeiras e especificidades orçamentárias.
Os entes federados, desde que respeitado o princípio da proporcionalidade, gozam de autonomia para estabelecer o montante correspondente às obrigações de pequeno valor e, dessa forma, afastar a aplicação do sistema de precatórios. Eles só não podem estabelecer valor demasiado <u>além</u> ou <u>aquém</u> do razoável, tendo como parâmetro as suas disponibilidades financeiras (1)
É inconstitucional — por violar a competência privativa da União para legislar sobre direito processual (CF/1988, art. 21, I), uma vez que as normas que dispõem sobre RPV têm caráter eminentemente processual (2) — legislação estadual que transfere ao credor a responsabilidade pelo encaminhamento da documentação necessária para solicitação do pagamento do RPV diretamente ao órgão público devedor, bem como determina a suspensão do prazo para pagamento.
Ademais, a lei estadual impugnada não se aplica aos processos judiciais de competência da justiça federal, ainda que no exercício da competência federal delegada, já que para eles prevalece o conteúdo de norma editada pelo Conselho da Justiça Federal (CJF), atualmente a Resolução 458/2017.
Com base nesses entendimentos, o Plenário, por unanimidade, julgou parcialmente procedente a ação para (i) declarar a inconstitucionalidade do caput e do parágrafo único do art. 6º da Lei 14.757/2015 do Estado do Rio Grande do Sul; e (ii) dar interpretação conforme a Constituição aos incisos do mesmo art. 6º, para limitar sua aplicação aos processos judiciais de competência da justiça estadual, de modo que eles não deverão ser aplicados aos processos julgados no exercício da competência federal delegada, os quais devem ser regidos pela Resolução do CJF (3).
(1) Precedentes citados: ADI 2.868 e ADI 4.332.
(2) Precedentes citados: RE 632.550 AgR; RE 293.231 e ADI 5.534.
(3) Lei 14.757/2015 do Estado do Rio Grande do Sul: “Art. 1º Serão consideradas de pequeno valor, para os fins do disposto no § 3.º do art. 100 da Constituição Federal, as obrigações que o Estado do Rio Grande do Sul, suas Autarquias e Fundações devam quitar em decorrência de decisão judicial transitada em julgado cujo valor, devidamente atualizado, não exceda a 10 (dez) salários mínimos. Art. 2º O crédito de pequeno valor não estará sujeito ao regime de precatórios e deverá ser pago, mediante depósito judicial, no prazo de até 60 (sessenta) dias, contados da data em que for protocolada, perante o órgão competente, a requisição expedida pelo juízo da execução. Parágrafo único. Nas requisições de pequeno valor expedidas por meio eletrônico, o prazo será contado da data de expedição. Art. 3º São vedados o fracionamento, a repartição ou a quebra do valor da execução para que o pagamento se faça, em parte, na forma estabelecida no ‘caput’ do art. 2º desta Lei e, em parte, com a expedição de precatório. Art. 4º Se o valor da execução ultrapassar o montante estabelecido no art. 1º desta Lei, o pagamento far-se-á por meio de precatório, sendo facultada à parte exequente a renúncia ao crédito do valor excedente, para que possa optar pelo pagamento do saldo sem o precatório, na forma prevista no art. 2º desta Lei. Parágrafo único. A opção pelo recebimento do crédito na forma prevista nesta Lei implica a renúncia ao restante dos créditos porventura existentes oriundos do mesmo processo judicial. Art. 5º As requisições de pequeno valor cujo trânsito em julgado da decisão tenha ocorrido antes da entrada em vigor desta Lei observarão o limite de 40 (quarenta) salários mínimos. Art. 6º A requisição de pequeno valor expedida em meio físico será encaminhada diretamente pelo credor, ou seu representante, ao ente devedor responsável pelo pagamento da obrigação, e deverá ser instruída com os seguintes documentos e informações: I - indicação do número do processo judicial em que foi expedida a requisição; II - indicação da natureza da obrigação a que se refere o pagamento; III - comprovante de situação cadastral das partes e dos advogados no Cadastro de Pessoa Física - CPF - ou no Cadastro Nacional de Pessoa Jurídica - CNPJ - do Ministério da Fazenda; IV - cópia da memória completa do cálculo definitivo, ainda que objeto de renúncia ao valor estabelecido nesta Lei; V - indicação do período compreendido para efeito de cálculo do imposto de renda e das contribuições aos sistemas de previdência e saúde; e VI - cópia da manifestação da Procuradoria-Geral do Estado de concordância com o valor do débito. Parágrafo único. A requisição de pequeno valor que não preencher os requisitos do ‘caput’ deste artigo não será recebida pela autoridade competente, ficando suspenso o prazo do seu pagamento até a apresentação pelo credor dos documentos ou informações faltantes. Art. 7º Esta Lei entra em vigor na data de sua publicação. Art. 8º Revoga-se a Lei nº 13.756, de 15 de julho de 2011.”
Legislação: Lei 14.757/2015 do Estado do Rio Grande do Sul
Precedentes: ADI 2.868, ADI 4.332, RE 632.550 AgR; RE 293.231 e ADI 5.534
RPV: valor previsto no ADCT e fixação de quantia referencial inferior por ente federado
Tese fixada - (I) As unidades federadas podem fixar os limites das respectivas requisições de pequeno valor em patamares inferiores aos previstos no artigo 87 do ADCT, desde que o façam em consonância com sua capacidade econômica. - (II) A aferição da capacidade econômica, para este fim, deve refletir não somente a receita, mas igualmente os graus de endividamento e de litigiosidade do ente federado.
Resumo - Ao editar norma própria, o ente federado, desde que em consonância com sua <u>capacidade econômica</u> e com o princípio da <u>proporcionalidade</u>, pode estabelecer quantia inferior à prevista no art. 87 do ADCT como teto para o pagamento de seus débitos judiciais por meio de Requisição de Pequeno Valor (RPV).
O patamar provisório fixado no ADCT (1) para o pagamento de RPV não é irredutível, cabendo a cada unidade federativa estipular o valor máximo para essa especial modalidade de pagamento de acordo com sua capacidade econômica, cuja aferição deve considerar, além do quantum das receitas auferidas, os graus de endividamento e de litigiosidade do ente público.
No tocante à atuação do Poder Judiciário, deve ser adotada uma postura de autocontenção quando não houver demonstração concreta da desproporcionalidade na fixação do valor referencial.
Com base nesse entendimento, o Plenário, por unanimidade, reconheceu a existência da repercussão geral da questão constitucional suscitada (Tema 1.231 RG) e, no mérito, também por unanimidade, reafirmou a jurisprudência dominante sobre a matéria (2) para dar provimento ao recurso extraordinário, assentando a constitucionalidade da Lei 10.562/2017 do Município de Fortaleza/CE, que fixa como teto para pagamento das RPVs o equivalente ao maior benefício do Regime Geral de Previdência Social. Não se manifestou o ministro André Mendonça.
(1) ADCT: “Art. 87. Para efeito do que dispõem o § 3º do art. 100 da Constituição Federal e o art. 78 deste Ato das Disposições Constitucionais Transitórias serão considerados de pequeno valor, até que se dê a publicação oficial das respectivas leis definidoras pelos entes da Federação, observado o disposto no § 4º do art. 100 da Constituição Federal, os débitos ou obrigações consignados em precatório judiciário, que tenham valor igual ou inferior a: I – quarenta salários-mínimos, perante a Fazenda dos Estados e do Distrito Federal; II – trinta salários-mínimos, perante a Fazenda dos Municípios. Parágrafo único. Se o valor da execução ultrapassar o estabelecido neste artigo, o pagamento far-se-á, sempre, por meio de precatório, sendo facultada à parte exequente a renúncia ao crédito do valor excedente, para que possa optar pelo pagamento do saldo sem o precatório, da forma prevista no § 3º do art. 100.”
(2) Precedentes citados: ADI 2868; ADI 4332; e ADI 5100.
Legislação: ADCT, art. 87 Lei 10.562/2017-Fortaleza/CE
Precedentes: ADI 2868; ADI 4332; e ADI 5100.
Reviewer #3 (Public review):
Summary:
Cholecystokinin (CCK) is highly expressed in auditory thalamocortical (MGB) neurons and CCK has been found to shape cortical plasticity dynamics. In order to understand how CCK shapes synaptic plasticity in the auditory thalamocortical pathway, they assessed the role of CCK signaling across multiple mechanisms of LTP induction with the auditory thalamocortical (MGB - layer IV Auditory Cortex) circuit in mice. In these physiology experiments that leverage multiple mechanisms of LTP induction and a rigorous manipulation of CCK and CCK-dependent signaling, they establish an essential role of auditory thalamocortical LTP on the co-release of CCK from auditory thalamic neurons. By carefully assessing the development of this plasticity over time and CCK expression, they go on to identify a window of time that CCK is produced throughout early and middle adulthood in auditory thalamocortical neurons to establish a window for plasticity from 3 weeks to 1.5 years in mice, with limited LTP occurring outside of this window. The authors go on to show that CCK signaling and its effect on LTP in the auditory cortex is also capable of modifying frequency discrimination accuracy in an auditory PPI task. In evaluating the impact of CCK on modulating PPI task performance, it also seems that in mice <1.5 years old CCK-dependent effects on cortical plasticity is almost saturated. While exogenous CCK can modestly improve discrimination of only very similar tones, exogenous focal delivery of CCK in older mice can significantly improve learning in a PPI task to bring their discrimination ability in line with those from young adult mice.
Strengths:
(1) The clarity of the results along with the rigor multi-angled approach provide significant support for the claim that CCK is essential for auditory thalamocortical synaptic LTP. This approach uses a combination of electrical, acoustic, and optogenetic pathway stimulation alongside conditional expression approaches, germline knockout, viral RNA downregulation and pharmacological blockade. Through the combination of these experimental configures the authors demonstrate that high-frequency stimulation-induced LTP is reliant on co-release of CCK from glutamatergic MGB terminals projecting to the auditory cortex.
(2) The careful analysis of the CCK, CCKB receptor, and LTP expression is also a strength that puts the finding into the context of mechanistic causes and potential therapies for age-dependent sensory/auditory processing changes. Similarly, not only do these data identify a fundamental biological mechanism, but they also provide support for the idea that exogenous asynchronous stimulation of the CCKBR is capable of restoring an age-dependent loss in plasticity.
(3) Although experiments to simultaneously relate LTP and behavioral change or identify a causal relationship between LTP and frequency discrimination are not made, there is convincing evidence that CCK signaling in the auditory cortex (known to determine synaptic LTP) is important for auditory processing/frequency discrimination. These experiments are key for establishing the relevance of this mechanism.
Weaknesses:
The following are weaknesses or limitations of the study that may also fall outside of the scope of this work, but which could be addressed in the future.
(1) Given the magnitude of the evoked responses, one expects that pyramidal neurons in layer IV are primarily those that undergo CCK-dependent plasticity, but the degree to which PV-interneurons and pyramidal neurons participate in this process differently is unclear.
(2) While these data support an important role for CCK in synaptic LTP in the auditory thalamocortical pathway, perhaps temporal processing of acoustic stimuli is as or more important than frequency discrimination. Given the enhanced responsivity of the system, it is unclear whether this mechanism would improve or reduce the fidelity of temporal processing in this circuit. Understanding this dynamic may also require consideration of cell type as raised in weakness #1.
(3) In Figure 1, an example of increased spontaneous and evoked firing activity of single neurons after HFS is provided. Yet it is surprising that the group data are analyzed only for the fEPSP. It seems that single neuron data would also be useful at this point to provide insight into how CCK and HFS affect temporal processing and spontaneous activity/excitability.
Author response:
The following is the authors’ response to the previous reviews
Reviewer #1 (Public review):
This report addresses a compelling topic. However, I have significant concerns, which necessitate a reassessment of the report's overall value.
Anatomical Specificity and Stimulation Site:
While the authors clarify that the ventral MGB (MGv) was the intended stimulation target, the electrode track (Fig. 1A) and viral spread (Fig. 2E) suggest possible involvement of the dorsal MGB (MGd) and broader area. Given that MGv-AI and MGd-AC pathways have distinct-and sometimes opposing-effects on plasticity, the reported LTP values (with unusually small standard deviations) raise concerns about the specificity of the findings. Additional anatomical verification would help resolve this issue.
We thank the reviewer for highlighting the importance of anatomical specificity in MGv targeting. In the revised manuscript, we have taken several steps to address these issues:
(1) Higher-magnification histology has been added to Figure 1A, clearly identifying the electrode tip localized within the MGv.
(2) Figure 2E has been replaced with a new image showing viral expression largely confined to MGB, with minimal spread to surrounding structures.
(3) In the Discussion, we explicitly acknowledge that although targeting was guided by stereotaxic coordinates and histological confirmation, some viral spread throughout the MGB occurred. We also discuss the possibility that both MGv-A1 and MGd-AC pathways may contribute to the recorded responses, which could influence the observed plasticity, as previously suggested by the reviewer.
These additions and acknowledgments are now incorporated to ensure the reader can interpret the data with full consideration of anatomical targeting limitations.
Results section:
“Higher-magnification histology confirmed accurate MGv targeting (Figure 1A, lower-middle panel)’”
Discussion section:
“Although our experiment targeting the MGv was guided by stereotaxic coordinates and verified post hoc, we acknowledge potential contributions from non-lemniscal medial geniculate nucleus dorsal (MGd) projections. Anatomical and physiological evidence indicates that MGv-AC projections provide rapid, frequency‑specific, tonotopically organized excitation, whereas MGd pathways target higher‑order auditory cortex with broader tuning, less precise tonotopy, longer response latencies, and greater context‑dependence, features that can differentially shape cortical sensory integration and plasticity (Lee and Sherman, 2010; Smith et al., 2012; Ohga et al., 2018; Lee, 2015; Hu, 2003). While the co-recruitment of lemniscal and non-lemniscal inputs may enhance the generality of our CCK-dependent mechanism, the differing response characteristics of these pathways suggest subtle differences in their relative engagement in the observed plasticity. Future pathway-specific manipulations will help clarify their respective contributions”
Lee, C.C., and Sherman, S.M. (2010). Topography and physiology of ascending streams in the auditory tectothalamic pathway. Proceedings of the National Academy of Sciences 107, 372-377. doi:10.1073/pnas.0907873107.
Smith, P.H., Uhlrich, D.J., Manning, K.A., and Banks, M.I. (2012). Thalamocortical projections to rat auditory cortex from the ventral and dorsal divisions of the medial geniculate nucleus. Journal of Comparative Neurology 520, 34-51.
Ohga, S., Tsukano, H., Horie, M., Terashima, H., Nishio, N., Kubota, Y., Takahashi, K., Hishida, R., Takebayashi, H., and Shibuki, K. (2018). Direct Relay Pathways from Lemniscal Auditory Thalamus to Secondary Auditory Field in Mice. Cerebral Cortex 28, 4424-4439. 10.1093/cercor/bhy234.
Lee, C.C. (2015). Exploring functions for the non-lemniscal auditory thalamus. Frontiers in Neural Circuits 9, 69.
Hu, B. (2003). Functional organization of lemniscal and nonlemniscal auditory thalamus. Experimental Brain Research 153, 543-549. 10.1007/s00221-003-1611-5.
Figure legend section:
“Post-hoc histology at higher magnification (lower-middle) shows the electrode tip confined within the MGv. White lines delineate the MGv/MGd border based on cytoarchitectonic landmarks.”
Statistical Rigor and Data Variability:
The remarkably low standard deviations in LTP measurements are unexpected based on established variability in thalamocortical plasticity. The authors' response confirms these values are accurate, but further justification, such as methodological controls or replication-would bolster confidence in these results. Additionally, the comparison of in vivo vs. in vitro LTP variability requires more substantive support.
We appreciate the reviewer's concern regarding the unusually small variability. We would like to clarify that the error bars in our figures represent Standard Error of the Mean (SEM) rather than Standard Deviations (SD). As SEM is derived from the SD while incorporating sample size, it is inherently smaller than SD, which may have led to the impression of unrealistically low variability. This has now been explicitly clarified in the figure legends and Methods.
To illustrate the raw variability, we have added Supplementary Figure S1E showing unaveraged fEPSP slopes compare to SEM, corresponding to Figure S1C. This addition ensures transparency and allows readers to directly assess the quality and consistency of our recordings.
Regarding the comparison between in vivo and in vitro LTP variability:
We agree that clarifying the basis of our in vivo vs. in vitro variability comparison is important. For example, in Chen et al., 2019, using identical LTP induction protocols (Fig. J), the SED of in vitro slice measurements (Fig. K) was substantially larger than that of in vivo recordings (Fig. L).
This difference likely reflects:
(1) In vitro: neighboring data points within a single experiment are highly correlated; variability across experiments is large due to heterogeneous sensitivity to LTP induction (10–200% increasement).
(2) In vivo: lower correlation between neighboring data points, but each is averaged from 12 recordings over 2 min, reducing cross-trial variability; sensitivity to LTP induction is less variable across experiments (5–60% changes).
We hope that these clarifications and additional data address the reviewer’s concerns regarding statistical rigor and data variability.
Methods section:
“The slopes of the evoked fEPSPs were calculated and normalized using a customized MATLAB script, and the group data were plotted as mean ± Standard Error of the Mean (SEM).”
“All data are presented as mean ± SEM. Error bars and shaded areas represent SEM. Here, n represents the number of stimulation-recording sites or and N represents the number of animals in each experiment. At each time point, fEPSPs were averaged across 12 consecutive trials (2 min) to reduce within-experiment fluctuation. Normalized time courses were then used for repeated-measures analyses.”
Figure legend section:
“Data are mean ± SEM; error bars indicate SEM.”
“(E) Unaveraged fEPSP slopes are shown for each time point, with individual data points corresponding to all sites included in Fig. 1C; mean ± SEM overlays are shown in black. Note that all individual data points are displayed in this figure, whereas in Figure S1C, only the averaged values are shown.”
Viral Targeting and Specificity:
The manuscript does not clearly address whether cortical neurons were inadvertently infected by AAV9. Given the potential for off-target effects, explicit confirmation (e.g., microphotograph of stimulation site) would strengthen the study's conclusions.
We appreciate the request for quantitative confirmation of off-target cortical infection. We clarify that our histological verification was conducted by systematic sampling rather than exhaustive quantification. Under the same sampling procedure, we did not detect tdTomato-positive cortical somata after AAV9‑Syn‑ChrimsonR‑tdTomato injections into the MGB, whereas we observed rare EYFP-positive cortical somata after AAV9‑EF1a‑DIO‑ChETA‑EYFP (median < 1 cell per 0.4 × 0.4 mm² section, Supplementary Figure S1E). Although these observations do not constitute a formal statistical estimate, they were consistent across sampled sections and are in line with the low-level trans-synaptic transfer reported for AAV9. We have discussed their potential implications for data interpretation in the Discussion.
We hope these clarifications and the newly presented histological evidence address the reviewer’s concerns and further strengthen the rigor of our study.
Discussion section:
“Another potential limitation of our study is the trans-synaptic transfer property of AAV9 (Figure S1F). To mitigate this risk, we carefully control the injection volume, rate, and viral expression time, while also verifying expression post-hoc. Systematic sampling histological analysis detected no tdTomato-positive cortical somata in the ACx (Figure 2E lower panel), whereas rare EYFP-positive cortical somata were observed after AAV9-EF1a-DIO-ChETA-EYFP injections (median < 1 cell in 0.4 × 0.4 mm2 section, Figure S1F, corresponds to Figure 2A upper-middle panel). These construct‑dependent observations align with occasional low‑level trans‑synaptic transfer reported for AAV9 (Zingg et al., 2017) and indicate that off‑target cortical infection was negligible for ChrimsonR and exceedingly rare for ChETA under our experimental conditions.”
Zingg, B., Chou, X.L., Zhang, Z.G., Mesik, L., Liang, F., Tao, H.W., and Zhang, L.I. (2017). AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors. Neuron 93, 33-47. 10.1016/j.neuron.2016.11.045.
Figure legend:
“Representative histological images demonstrating low-level transsynaptic spread following AAV9-EF1a-DIO-ChETA-EYFP injection into the MGv. Rare EYFP-positive cortical neurons were observed (median < 1 cell per 0.4 × 0.4 mm² section). Scale bar: 100 µm.”
Integration of Prior Literature:
The discussion of existing work is adequate but could be more comprehensive. A deeper engagement with contrasting findings would provide better context for the study's contributions.
We appreciate the reviewer’s suggestion to engage more deeply with contrasting findings. In the revised Introduction and Discussion, we have:
(1) Refocused the historical context toward adult auditory thalamocortical plasticity and explicitly contrasted it with visual and somatosensory cortices, while adult ACx exhibits weaker and more gated NMDAR dependence.
(2) Positioned CCK–CCKBR signaling as a permissive/gating mechanism that can complement or partially compensate for postsynaptic NMDAR signaling, potentially reconciling variability across cortical areas and life stages.
(3) Clarified the potential differential contributions of lemniscal (MGv) and non‑lemniscal (MGd) streams to plasticity expression and variability, acknowledging pathway-specific response properties.
These additions are now integrated in the Introduction (paragraphs 2–3) and Discussion (sections “CCK Dependence of Thalamocortical Neuroplasticity in the ACx” and “Developmental and Age‑Dependent CCK‑Mediated Plasticity”), providing a more comprehensive and balanced context for our findings.
Introduction section:
“However, converging evidence shows that thalamocortical inputs retain a capacity for experience-dependent modification in adulthood. Sensory enrichment or deprivation can gate or reinstate thalamocortical plasticity. In the adult ACx, pairing sounds with neuromodulatory drive can reshape cortical representations. In vivo high-frequency stimulation (HFS) of dorsal lateral geniculate nucleus (LGN) or medial geniculate body (MGB) induces LTP in sensory cortices and has been linked to perceptual learning beyond the critical period. Notably, auditory thalamocortical plasticity appears less dependent on NMDA receptors compared to other cortical regions. The mechanisms underlying thalamocortical plasticity in the mature brain remain poorly understood.
Cholecystokinin (CCK) and its receptor CCK-B receptor (CCKBR) are well positioned to influence thalamocortical transmission: Cck mRNA is abundant in MGB neurons and CCKBR is enriched in layer IV of ACx, the principal thalamorecipient layer.”
Discussion section:
“These findings suggest a potential involvement of CCK in thalamocortical plasticity. Our data extend this framework by identifying CCK–CCKBR signaling as a permissive modulator of adult thalamocortical LTP.”
“We propose that CCKBR activation may trigger intracellular calcium release and AMPAR recruitment in parallel to, or partially compensating for,independently of postsynaptic NMDAR signaling, while the complementarity of CCKBR and NMDARs may contribute to robust thalamocortical plasticity. This complementary arrangement may reconcile differences across developmental stages and cortical areas, and highlights neuropeptidergic signaling as a lever to re-enable adult thalamocortical plasticity.
Notably, exogenous CCK alone failed to induce LTP in the absence of accompanying stimulation (Figure S2A and S2B), emphasizing that CCK function as a modulator rather than a direct initiator of LTP. Activation of the thalamocortical pathway is also essential for LTP induction. Although our experiment targeting the MGv was guided by stereotaxic coordinates and verified post hoc, we acknowledge potential contributions from non-lemniscal medial geniculate nucleus dorsal (MGd) projections. Anatomical and physiological evidence indicates that MGv-AC projections provide rapid, frequency‑specific, tonotopically organized excitation, whereas MGd pathways target higher‑order auditory cortex with broader tuning, less precise tonotopy, longer response latencies, and greater context‑dependence, features that can differentially shape cortical sensory integration and plasticity. While the co-recruitment of lemniscal and non-lemniscal inputs may enhance the generality of our CCK-dependent mechanism, the differing response characteristics of these pathways suggest subtle differences in their relative engagement in the observed plasticity. Future pathway-specific manipulations will help clarify their respective contributions. Another potential limitation of our study is the trans-synaptic transfer property of AAV9 (Figure S1F). To mitigate this, we carefully controlled the injection volume, rate, and viral expression time, and conducted post-hoc histological analyses to minimize off-target effects, thereby reducing the likelihood of trans-synaptic transfer confounding the interpretation of our findings.”
Therapeutic Implications:
The authors' discussion of therapeutic potential is now appropriately cautious and well-reasoned.
Conclusion:
While the study presents intriguing findings, the concerns outlined above must be addressed to fully establish the validity and impact of the results. I appreciate the authors' efforts thus far and hope they can provide additional data or clarification to resolve these issues. With these revisions, the manuscript could make a valuable contribution to the field.
Reviewer #2 (Public review):
Summary:
This work used multiple approaches to show that CCK is critical for long-term potentiation (LTP) in the auditory thalamocortical pathway. They also showed that the CCK mediation of LTP is age-dependent and supports frequency discrimination. This work is important because is opens up a new avenue of investigation of the roles of neuropeptides in sensory plasticity.
Strengths:
The main strength is the multiple approaches used to comprehensively examine the role of CCK in auditory thalamocortical LTP. Thus, the authors do provide a compelling set of data that CCK mediates thalamocortical LTP in an age-dependent manner.
Weaknesses:
There are some details that should be addressed, primarily regarding potential baseline differences in comparison groups. The behavioral assessment is relatively limited, but may be fleshed out in future work.
We appreciate the reviewer’s suggestion regarding potential baseline differences. In our study, all groups underwent harmonized procedures, including identical exposure, timing, and acquisition parameters. Group allocation and data collection were performed under standardized conditions. For electrophysiology, baseline fEPSP measures and stimulation intensities were calibrated per site using consistent input-output procedures, with analyses based on normalized slopes relative to each site’s own baseline. For behavior, animals from the same litter served as both experimental and control groups, matched for handling conditions; startle/PPI data were acquired using identical hardware and timing settings. While no additional post hoc re-processing was performed, we have clarified these controls in the Methods to enhance transparency.
We agree that the behavioral assessment is intentionally focused and does not encompass broader auditory perceptual functions (e.g., temporal processing). We now explicitly state this limitation and propose future studies to examine temporal acuity and cell-type-specific manipulations. These experiments will clarify how CCK-dependent thalamocortical plasticity generalizes to other perceptual domains.
Reviewer #3 (Public review):
Summary:
Cholecystokinin (CCK) is highly expressed in auditory thalamocortical (MGB) neurons and CCK has been found to shape cortical plasticity dynamics. In order to understand how CCK shapes synaptic plasticity in the auditory thalamocortical pathway, they assessed the role of CCK signaling across multiple mechanisms of LTP induction with the auditory thalamocortical (MGB - layer IV Auditory Cortex) circuit in mice. In these physiology experiments that leverage multiple mechanisms of LTP induction and a rigorous manipulation of CCK and CCK-dependent signaling, they establish an essential role of auditory thalamocortical LTP on the co-release of CCK from auditory thalamic neurons. By carefully assessing the development of this plasticity over time and CCK expression, they go on to identify a window of time that CCK is produced throughout early and middle adulthood in auditory thalamocortical neurons to establish a window for plasticity from 3 weeks to 1.5 years in mice, with limited LTP occurring outside of this window. The authors go on to show that CCK signaling and its effect on LTP in the auditory cortex is also capable of modifying frequency discrimination accuracy in an auditory PPI task. In evaluating the impact of CCK on modulating PPI task performance, it also seems that in mice <1.5 years old CCK-dependent effects on cortical plasticity is almost saturated. While exogenous CCK can modestly improve discrimination of only very similar tones, exogenous focal delivery of CCK in older mice can significantly improve learning in a PPI task to bring their discrimination ability in line with those from young adult mice.
Strengths:
(1) The clarity of the results, along with the rigor multi-angled approach, provide significant support for the claim that CCK is essential for auditory thalamocortical synaptic LTP. This approach uses a combination of electrical, acoustic, and optogenetic pathway stimulation alongside conditional expression approaches, germline knockout, viral RNA downregulation and pharmacological blockade. Through the combination of these experimental configures the authors demonstrate that high-frequency stimulation-induced LTP is reliant on co-release of CCK from glutamatergic MGB terminals projecting to the auditory cortex.
(2) The careful analysis of the CCK, CCKB receptor, and LTP expression is also a strength that puts the finding into the context of mechanistic causes and potential therapies for age-dependent sensory/auditory processing changes. Similarly, not only do these data identify a fundamental biological mechanism, but they also provide support for the idea that exogenous asynchronous stimulation of the CCKBR is capable of restoring an age-dependent loss in plasticity.
(3) Although experiments to simultaneously relate LTP and behavioral change or identify a causal relationship between LTP and frequency discrimination are not made, there is still convincing evidence that CCK signaling in the auditory cortex (known to determine synaptic LTP) is important for auditory processing/frequency discrimination. These experiments are key for establishing the relevance of this mechanism.
Weaknesses:
(1) Given the magnitude of the evoked responses, one expects that pyramidal neurons in layer IV are primarily those that undergo CCK-dependent plasticity, but the degree to which PV-interneurons and pyramidal neurons participate in this process differently is unclear.
We agree with the reviewer that the relative contributions of pyramidal neurons and PV-interneurons to CCK-dependent thalamocortical plasticity remain to be determined. Our recordings primarily reflected excitatory postsynaptic activity from layer IV pyramidal neurons, given the fEPSP metrics used. As PV-interneurons are essential in shaping cortical inhibition and temporal precision, they may also be modulated by CCK release from thalamocortical inputs. We have explicitly acknowledged this limitation in the Discussion section of the manuscript and propose that future studies should employ cell-type-specific recording or manipulation approaches to dissect the respective roles of inhibitory and excitatory neuronal populations in CCK-dependent thalamocortical plasticity. We appreciate the reviewer’s suggestion and believe this is a valuable direction for ongoing research.
(2) While these data support an important role for CCK in synaptic LTP in the auditory thalamocortical pathway, perhaps temporal processing of acoustic stimuli is as or more important than frequency discrimination. Given the enhanced responsivity of the system, it is unclear whether this mechanism would improve or reduce the fidelity of temporal processing in this circuit. Understanding this dynamic may also require consideration of cell type as raised in weakness #1.
We acknowledge that the current study primarily examined frequency discrimination and did not directly assess temporal processing. Enhanced network responsivity could have variable effects on temporal precision, depending on the balance between excitation and inhibition. PV-interneurons, in particular, are known to support temporal fidelity in auditory processing (Nocon et al., 2023; Cai et al., 2018). We discussion that future work should investigate how CCK modulation influences temporal coding at both the circuit and single-cell level, and whether such changes align with or diverge from the mechanisms underlying frequency discrimination improvements.
(3) In Figure 1, an example of increased spontaneous and evoked firing activity of single neurons after HFS is provided. Yet it is surprising that the group data are analyzed only for the fEPSP. It seems that single neuron data would also be useful at this point to provide insight into how CCK and HFS affect temporal processing and spontaneous activity/excitability, especially given the example in 1F.
We appreciate the reviewer’s suggestion. While we recorded single-unit activity during HFS protocols, long-term stability over >1.5 hours was less consistent compared to fEPSP measurements, leading to higher variability in spike-based metrics. We therefore used fEPSPs as our primary quantitative measure for robustness. We agree, however, that single-neuron data could yield valuable complementary insights. In future experiments combining stable single-unit recording with synaptic measurements will be conducted to better link cellular excitability and network plasticity.
(4) The circuitry that determines PPI requires multiple brain areas, including the auditory cortex. Given the complicated dynamics of this process, it may be helpful to consider what, if anything, is known specifically about how layer IV synaptic plasticity in the auditory cortex may shape this behavior.
We agree that PPI involves multiple cortical and subcortical nodes. In our paradigm, layer IV neurons receive segregated MGv inputs, high-frequency activation of thalamocortical projections induces robust synaptic plasticity in layer IV. The potentiation at these synapses could amplify the cortical representation of weak prepulses, facilitating their detection and enhancing PPI performance. This interpretation is consistent with prior work showing that local CCK infusion combined with auditory stimuli can augment cortical responses (Li et al., 2014). We have expanded the Discussion to highlight that in aged animals, where baseline PPI performance is often reduced due to degraded auditory inputs (Ouagazzal et al., 2006; Young et al., 2010), restoring thalamocortical plasticity via CCK may partially compensate for sensory gating deficits. We further note that the exact contribution of layer IV to PPI circuitry warrants future investigation using pathway-specific perturbations.
Comments on revisions:
The manuscript is much improved and many of the issues or questions have been addressed. Ideally, evidence for the degree of transsynaptic spread for AAV9-Syn-ChrimsonR-tdTomato would also be provided in some form since in the authors' response in sounds like some was observed, as expected.
We thank the reviewer for this important point and for the opportunity to clarify. As requested, we have carefully examined the possibility of transsynaptic spread in our experiments:
We clarify that our histological verification was conducted by systematic sampling rather than exhaustive quantification. Under the same sampling procedure, we did not detect tdTomato-positive cortical somata after AAV9‑Syn‑ChrimsonR‑tdTomato injections into the MGB, whereas we observed rare EYFP-positive cortical somata after AAV9‑EF1a‑DIO‑ChETA‑EYFP (median < 1 cell per 0.4 × 0.4 mm² section, see Figure 2A and Figure S1F), consistent with occasional low-level transsynaptic spread reported in the literature.
We have updated the Discussion sections to clearly report these findings, and to emphasize the potential for vector- and construct-dependent variability in transsynaptic spread. We also explicitly acknowledge this technical limitation and discuss its implications for data interpretation.
We hope these clarifications and additions address the reviewer’s concern regarding viral specificity and transsynaptic spread.
Discussion section:
“Another potential limitation of our study is the trans-synaptic transfer property of AAV9 (Figure S1F). To mitigate this risk, we carefully control the injection volume, rate, and viral expression time, while also verifying expression post-hoc. Systematic sampling histological analysis detected no tdTomato-positive cortical somata in the ACx (Figure 2E lower panel), whereas rare EYFP-positive cortical somata were observed after AAV9-EF1a-DIO-ChETA-EYFP injections (median < 1 cell in 0.4 × 0.4 mm2 section, Figure S1F, corresponds to Figure 2A upper-middle panel). These construct‑dependent observations align with occasional low‑level trans‑synaptic transfer reported for AAV9 (Zingg et al., 2017) and indicate that off‑target cortical infection was negligible for ChrimsonR and exceedingly rare for ChETA under our experimental conditions.”
Zingg, B., Chou, X.L., Zhang, Z.G., Mesik, L., Liang, F., Tao, H.W., and Zhang, L.I. (2017). AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors. Neuron 93, 33-47. 10.1016/j.neuron.2016.11.045.
Figure legend:
" Representative histological images demonstrating low-level transsynaptic spread following AAV9-EF1a-DIO-ChETA-EYFP injection into the MGv. Rare EYFP-positive cortical neurons were observed (median < 1 cell per 0.4 × 0.4 mm² section). Scale bar: 100 µm."
Reviewer #1 (Recommendations for the authors):
Thank you for your efforts in revising the manuscript. While progress has been made, I have a few remaining concerns that I hope you can address to further strengthen the study.
Focus of the Introduction:
Auditory thalamocortical plasticity is known to be NMDA-dependent, albeit with weaker dependence during early development. Given that this work examines thalamocortical LTP in young adult and aged mice, I recommend refining the Introduction to place greater emphasis on auditory thalamocortical plasticity in the adult brain. The current discussion of somatosensory plasticity during early development, while interesting, seems less directly relevant to the present study. A sharper focus on the auditory system would better frame your research questions.
We thank the reviewer for this constructive suggestion. We have revised the Introduction to emphasize adult auditory thalamocortical plasticity and to streamline content less directly related to our study. Specifically:
(1) We now foreground evidence that thalamocortical inputs retain experience-dependent plasticity beyond the critical period in adult ACx, including neuromodulatory pairing, HFS-induced LTP, and experience-dependent reinstatement.
(2) We explicitly note that adult auditory thalamocortical plasticity is more weakly NMDAR-dependent than in other cortices, thereby motivating our focus on CCK–CCKBR signaling as a permissive mechanism for adult LTP.
(3) We have condensed the discussion of somatosensory plasticity during early development to a brief background and shifted the focus to adult auditory mechanisms and knowledge gaps that directly frame our research questions.
These changes appear in the revised Introduction (paragraphs 2–3), which now provide a sharper rationale for investigating CCK‑dependent thalamocortical LTP in young adult and aged mice.
Introduction section:
“However, converging evidence shows that thalamocortical inputs retain a capacity for experience-dependent modification in adulthood. Sensory enrichment or deprivation can gate or reinstate thalamocortical plasticity. In the adult ACx, pairing sounds with neuromodulatory drive can reshape cortical representations. In vivo high-frequency stimulation (HFS) of dorsal lateral geniculate nucleus (LGN) or medial geniculate body (MGB) induces LTP in sensory cortices and has been linked to perceptual learning beyond the critical period. Notably, auditory thalamocortical plasticity appears less dependent on NMDA receptors compared to other cortical regions. The mechanisms underlying thalamocortical plasticity in the mature brain remain poorly understood.
Cholecystokinin (CCK) and its receptor CCK-B receptor (CCKBR) are well positioned to influence thalamocortical transmission: Cck mRNA is abundant in MGB neurons and CCKBR is enriched in layer IV of ACx, the principal thalamorecipient layer.”
Anatomical Specificity of MGv Targeting:
The mouse MGv is a small and deep structure, and precise targeting is critical given the functional differences between MGv and MGd pathways. In the current figures:
Fig. 1A suggests the electrode track may have approached the MGd.
Fig. 2E indicates some viral spread beyond the MGB.
Since MGv-AI and MGd-AC pathways exhibit distinct (and sometimes opposing) effects on plasticity, I encourage you to provide additional clarification or verification of the stimulated/infected regions. This would greatly enhance the interpretability of your LTP data.
Please see above.
Data Variability and Transparency:
The reported thalamocortical LTP values exhibit remarkably small standard deviations, which is somewhat unexpected given typical experimental variability in such measurements. To address this concern, it would be helpful to include example raw traces of the recorded LTP (e.g., in a supplementary figure). This would allow readers to better evaluate the data quality and consistency.
Please see above.
Reviewer #2 (Recommendations for the authors):
Overall, the authors did an excellent job of responding to our critiques, both in their direct responses and in the modified text. The modified text is also more readable than before. Two issues that the authors should consider addressing;
(1) Unless I missed it, there is no commentary stated about the impact of using aged C57 mice, which lose their hearing, such that the effects seen in the older mice could be related to hearing loss rather than aging alone. Some discussion of this point should be made.
We thank the reviewer for raising this important point. C57BL/6 mice are known to develop age-related hearing loss, which could potentially affect PPI performance in older animals. We note that in our internal screening we observed markedly reduced startle amplitudes and frequent negative PPI values in many mice >20 months, indicating severe auditory impairment. To minimize this confound a priori, we excluded mice older than 20 months and restricted the aged cohort to 17–19 months, which consistently exhibited robust startle responses and reliable PPI. While some degree of presbycusis may still be present in this age range in C57BL/6 mice, the improvement of PPI following CCK administration combined with acoustic exposure indicates that the auditory pathways remained sufficiently functional to support sensorimotor gating. In fact, the presence of partial hearing loss in these aged mice may have allowed us to better detect the beneficial effects of CCK, further highlighting its therapeutic potential for age-related deficits. The greater improvement in PPI observed in older mice —as compared to younger mice, whose PPI in control group is already high—likely reflect the combined effects of age-related hearing loss and CCK deficiency, with CCK-induced restoration of thalamocortical plasticity being the primary focus of our study. We have now added a discussion of this point in the revised manuscript.
Discussion section:
“In aged mice, PPI deficits are commonly observed due to impaired auditory processing. Notably, C57BL/6 mice exhibit age-related hearing loss (Johnson et al., 1997). Both age-associated changes in auditory function and CCK deficiency contribute to impaired sensory gating. The presence of partial hearing loss in aged mice may have facilitated the detection of CCK’s beneficial effects, further highlighting its therapeutic potential for age-related deficits. Our results suggest that enhanced thalamocortical plasticity mediated by CCK might partially compensate for these deficits by amplifying residual auditory signals in aged mice.”
Johnson, K.R., Erway, L.C., Cook, S.A., Willott, J.F., and Zheng, Q.Y. (1997). A major gene affecting age-related hearing loss in C57BL/6J mice. Hearing Research 114, 83-92. https://doi.org/10.1016/S0378-5955(97)00155-X.
(2) Minor point - I do not agree with the use of the term "ventral to bregma" to describe where the craniotomies were placed (e.g., line 599). The direction being described is more typically referred to as "lateral." If the authors prefer to use the term "ventral," perhaps additional clarification can be added.
We thank the reviewer for pointing out this issue and apologize for any confusion. We agree that “ventral to bregma” is not the standard terminology and have revised the Methods section to use “below the temporal ridge”. We have also clarified that the craniotomy for accessing the auditory cortex was performed on the lateral aspect of the skull in rodents, just below the temporal ridge. We hope this revision resolves the ambiguity.
Method section:
“A craniotomy was performed over the temporal bone, as the auditory cortex is located on the lateral surface of the brain (coordinates: 1.5 to 3.0 mm below the temporal ridge and 2.0 to 4.0 mm posterior to bregma for mice; 2.5 to 6.5 mm below the temporal ridge and 3.0 to 5.0 mm posterior to bregma for rats) to access the auditory cortex.”
“Six-week after CCK-sensor virus injection, a craniotomy was performed to access the auditory cortex at the temporal bone (1.5 to 3.0 mm below the temporal ridge and 2.0 to 4.0 mm posterior to bregma), and the dura mater was opened.”
Reviewer #1 (Public review):
Summary
The manuscript by K.H. Lee et al. presents Spyglass, a new open-source framework for building reproducible pipelines in systems neuroscience. The framework integrates the NWB (Neurodata Without Borders) data standard with the DataJoint relational database system to organize and manage analysis workflows. It enables the construction of complete pipelines, from raw data acquisition to final figures. The authors demonstrate their capabilities through examples, including spike sorting, LFP filtering, and sharp-wave ripple (SWR) detection. Additionally, the framework supports interactive visualizations via integration with Figurl, a platform for sharing neuroscience figures online.
Strengths:
Reproducibility in data analysis remains a significant challenge within the neuroscience community, posing a barrier to scientific progress. While many journals now require authors to share their data and code upon publication, this alone does not ensure that the code will execute properly or reproduce the original results. Recognizing this gap, the authors aim to address the community's need for a robust tool to build reproducible pipelines in systems neuroscience.
Weaknesses:
The issues identified here may serve as a foundation for future development efforts.
(1) User-friendliness:
The primary concern is usability. The manuscript does not clearly define the intended user base within a modern systems neuroscience lab. Improving user experience and lowering the barrier to entry would significantly enhance the framework's potential for broad adoption. The authors provide an online example notebook and a local setup notebook. However, the local setup process is overly complex, with many restrictive steps that could discourage new users. A more streamlined and clearly documented onboarding process is essential. Additionally, the lack of Windows support represents a practical limitation, particularly if the goal is widespread adoption across diverse research environments.
(2) Dependency management and long-term sustainability:
The framework depends on numerous external libraries and tools for data processing. This raises concerns about long-term maintainability, especially given the short lifespan of many academic software projects and the instability often associated with Python's backward compatibility. It would be helpful for the authors to clarify how flexible and modular the pipeline is, and whether it can remain functional if upstream dependencies become deprecated or change substantially.
(3) Extensibility for custom pipelines:
A further limitation is the insufficient documentation regarding the creation of custom pipelines. It is unclear how a user could adapt Spyglass to implement their own analysis workflows, especially if these differ from the provided examples (e.g., spike sorting, LFP analysis that are very specific to the hippocampal field). A clearer explanation or example of how to extend the framework for unrelated or novel analyses would greatly improve its utility and encourage community contributions.
(4) Flexibility vs. Standardization:
The authors may benefit from more explicitly defining the intended role of the framework: is Spyglass designed as a flexible, general-purpose tool for developing custom data analysis pipelines, or is its primary goal to provide a standardized framework for freezing and preserving pipelines post-publication to ensure reproducibility? While both goals are valuable, attempting to fully support both may introduce unnecessary complexity and result in a tool that is not well-suited for either purpose. The manuscript briefly touches on this tradeoff in the introduction, and the latter-pipeline preservation-may be the more natural fit for the package. If so, this intended use should be clearly communicated in the documentation to help users understand its scope and strengths.
Impact:
This work represents a significant milestone in advancing reproducible data analysis pipelines in neuroscience. Beyond reproducibility, the integration of cloud-based execution and shareable, interactive figures has the potential to transform how scientific collaboration and data dissemination are conducted. The authors are at the forefront of this shift, contributing valuable tools that push the field toward more transparent and accessible research practices.
Reviewer #2 (Public review):
Summary:
This valuable paper presents Spyglass, a comprehensive software framework designed to address the critical challenges of reproducibility and data sharing in neuroscience. The authors have developed a robust ecosystem built on community standards such as NWB and DataJoint, and demonstrate its utility by applying it to datasets from two independent labs, successfully validating the framework's ability to reproduce and extend published findings. While the framework offers a powerful blueprint for modern, reproducible research, its immediate broad impact may be tempered by the significant upfront investment required for adoption and its current focus on electrophysiological data. Nevertheless, Spyglass stands as an important and practical contribution, providing a well-documented and thoughtfully designed path toward more transparent and collaborative science.
Strengths:
(1) Principled solution to a foundational challenge:
The work offers a concrete and comprehensive framework for reproducibility in neuroscience, moving beyond abstract principles to provide an implemented, end-to-end ecosystem.
(2) Pragmatic and robust architectural design:
Features such as the "cyclic iteration" motif for spike-sorting curation and the "merge" motif for pipeline consolidation demonstrate deep, practical experience with neurophysiological analysis and address real-world challenges.
(3) Cross-laboratory validation:
The successful replication and extension of published hippocampal decoding findings across independent datasets strongly support the framework's utility and underscore its potential for enabling reproducible science.
(4) Accessibility through documentation and demos:
Extensive tutorials and the availability of a public demo environment lower some of the barriers to adoption.
Weaknesses:
(1) High barrier to adoption:
The requirement to convert all data into NWB, maintain a relational database, and train users in structured workflows is a significant hurdle, particularly for smaller labs.
(2) Limited tool integration:
The current pipelines, while useful, still resemble proof-of-principle demonstrations. Closer integration with established analysis libraries such as Pynapple and others could broaden the toolkit and reduce duplication of effort.
(3) Experimental metadata support:
While NWB provides a solid foundation for storing neurophysiology data streams, it still lacks broad and standardized support for experimental metadata, including descriptions of conditions, subject details, and procedures, as well as links across datasets. This limitation constrains one of Spyglass's key promises: enabling reproducible, cross-laboratory science. The authors should clarify how Spyglass plans to address or mitigate this gap - for example, by adopting or contributing to metadata extensions, providing templates for experimental conditions, or integrating with complementary systems that manage metadata across datasets.
(4) Cross-laboratory interoperability:
While demonstrated across two datasets, the manuscript does not fully address how Spyglass will handle the diversity of metadata standards, acquisition systems, and lab-specific practices that remain major obstacles to reproducibility.
(5) Visualization limitations:
Beyond the export system and Figurl, NWB offers relatively few options for interactive data exploration. The ability to explore data flexibly and discover new phenomena remains limited, which constrains one of the potential strengths of standardized pipelines.
Spyglass is well-positioned to become a community framework for reproducible neuroscience workflows, with the potential to set new standards for transparency and data sharing. With expanded modality coverage, tighter integration of existing community tools, stronger solutions for cross-lab interoperability, and richer visualization capabilities, it could have a transformative impact on the field.
Reviewer #1 (Public review):
Summary:
This paper aims to characterise the physiological and computational underpinnings of the accumulation of intermittent glimpses of sensory evidence.
Strengths:
(1) Elegant combination of electroencephalography and computational modelling.
(2) The authors describe results of two separate experiments, with very similar results, in effect providing an internal replication.
(3) Innovative task design, including different gap durations.
Weaknesses:
(1) The authors introduce the CPP as tracking an intermediary (motor-independent) evidence integration process, and the MBL as motor preparation that maintains a sustained representation of the decision variable. It would help if the authors could more directly and quantitatively assess whether their current data are in line with this. That is, do these signals exhibit key features of evidence accumulation (slope proportional to evidence strength, terminating at a common amplitude that reflects the bound)? Additionally, plotting these signals report locked (to the button press) would help here. What do the results mean for the narrative of this paper?
(2) The novelty of this work lies partly in the aim to characterize how the CPP and MBL interact (page 5, line 3-5). However, this analysis seems to be missing. E.g., at the single-trial level, do relatively strong CPP pulses predict faster/larger MBL? The simulations in Figure 5 are interesting, but more could be done with the measured physiology.
(3) The focus on CPP and MBL is hypothesis-driven but also narrow. Since we know only a little about the physiology during this "gaps" task, have the authors considered computing TFRs from different sensor groupings (perhaps in a supplementary figure?).
(4) The idea of a potential bound crossing during P1 is elegant, albeit a little simplistic. I wonder if the authors could more directly show a physiological signature of this. For example, by focusing on the MBL or occipital alpha split by the LL, LH, HL and HH conditions, and showing this pulse- as well as report-locked. Related, a primacy effect can also be achieved by modelling (i) self-excitation of the current one-dimensional accumulator, or (ii) two competing accumulators that produce winner-take-all dynamics. Is it possible to distinguish between these models, either with formal model comparison or with diagnostic physiological signatures?
(5) The way the authors specify the random effects of the structure of their mixed linear models should be specified in more detail. Now, they write: "Where possible, we included all main effects of interest as random effects to control for interindividual variability." This sounds as if they started with a model with a full random effect structure and dropped random components when the model would not converge. This might not be sufficiently principled, as random components could be dropped in many different orders and would affect the results. Do all main results hold when using classical random effects statistics on subject-wise regression coefficients?
Reviewer #2 (Public review):
Summary:
This paper investigates putative networks associated with prediction errors in task-based and resting-state fMRI. It attempts to test the idea that prediction errors minimisation includes abstract cognitive functions, referred to as the global prediction error hypothesis, by establishing a parallel between networks found in task-based fMRI where prediction errors are elicited in a controlled manner and those networks that emerge during "resting state".
Strengths:
Clearly, a lot of work and data went into this paper, including 2 task-based fMRI experiments and the resting state data for the same participants, as well as a third EEG-fMRI dataset. Overall, well written with a couple of exceptions on clarity, as per below, and the methodology appears overall sound, with a couple of exceptions listed below that require further justification. It does a good job of acknowledging its own weakness.
Weaknesses:
(1) The paper does a good job of acknowledging its greatest weakness, the fact that it relies heavily on reverse inference, but cannot quite resolve it. As the authors put it, "finding the same networks during a prediction error task and during rest does not mean that the networks' engagement during rest reflects prediction error processing". Again, the authors acknowledge the speculative nature of their claims in the discussion, but given that this is the key claim and essence of the paper, it is hard to see how the evidence is compelling to support that claim.
(2) Given how uncontrolled cognition is during "resting-state" experiments, the parallel made with prediction errors elicited during a task designed for that effect is a little difficult to make. How often are people really surprised when their brains are "at rest", likely replaying a previously experienced event or planning future actions under their control? It seems to be more likely a very low prediction error scenario, if at all surprising.
(3) The quantitative comparison between networks under task and rest was done on a small subset of the ROIs rather than on the full network - why? Noting how small the correlation between task and rest is (r=0.021) and that's only for part of the networks, the evidence is a little tenuous. Running the analysis for the full networks could strengthen the argument.
(4) Looking at the results in Figure 2C, the four-quadrant description of the networks labelled for low and high PE appears a little simplistic. The authors state that this four-quadrant description omits some ROIs as motivated by prior knowledge. This would benefit from a more comprehensive justification. Which ROIs are excluded, and what is the evidence for exclusion?
(5) The EEG-fMRI analysis claiming 3-6Hz fluctuations for PE is hard to reconcile with the fact that fMRI captures activity that is a lot slower, while some PEs are as fast as 150 ms. The discussion acknowledges this but doesn't seem to resolve it - would benefit from a more comprehensive argument.
Reviewer #3 (Public review):
Bogdan et al. present an intriguing and timely investigation into the intrinsic dynamics of prediction error (PE)-related brain states. The manuscript is grounded in an intuitive and compelling theoretical idea: that the brain alternates between high and low PE states even at rest, potentially reflecting an intrinsic drive toward predictive minimization. The authors employ a creative analytic framework combining different prediction tasks and imaging modalities. They shared open code, which will be valuable for future work.
However, the current manuscript would benefit from further clarification and empirical grounding, especially with regard to its theoretical framing (that PE-like state fluctuations are intrinsic and help us minimize PE), interpretation of results, and broader functional significance. Below, I outline a few major comments and suggestions that I think would strengthen the contribution.
(1) Consistency in Theoretical Framing
The title, abstract, and introduction suggest inconsistent theoretical goals of the study.
The title suggests that the goal is to test whether there are intrinsic fluctuations in high and low PE states at rest. The abstract and introduction suggest that the goal is to test whether the brain intrinsically minimizes PE and whether this minimization recruits global brain networks. My comments here are that a) these are fundamentally different claims, and b) both are challenging to falsify. For one, task-like recurrence of PE states during resting might reflect the wiring and geometry of the functional organization of the brain emerging from neurobiological constraints or developmental processes (e.g., experience), but showing that mirroring exists because of the need to minimize PE requires establishing a robust relationship with behavior or showing a causal effect (e.g., that interrupting intrinsic PE state fluctuations affects prediction).
The global PE hypothesis-"PE minimization is a principle that broadly coordinates brain functions of all sorts, including abstract cognitive functions"-is more suitable for discussion rather than the main claim in the abstract, introduction, and all throughout the paper.
Given the above, I recommend that the authors clarify and align their core theoretical goals across the title, abstract, introduction, and results. If the focus is on identifying fluctuations that resemble task-defined PE states at rest, the language should reflect that more narrowly, and save broader claims about global PE minimization for the discussion. This hypothesis also needs to be contextualized within prior work. I'd like to see if there is similar evidence in the literature using animal models.
(2) Interpretation of PE-Related Fluctuations at Rest and Its Functional Relevance
It would strengthen the paper to clarify what is meant by "intrinsic" state fluctuations. Intrinsic might mean task-independent, trait-like, or spontaneously generated. Which do the authors mean here? Is the key prediction that these fluctuations will persist in the absence of a prediction task?
Regardless of the intrinsic argument, I find it challenging to interpret the results as evidence of PE fluctuations at rest. What the authors show directly is that the degree to which a subset of regions within a PE network discriminates high vs. low PE during task correlates with the magnitude of separation between high and low PE states during rest. While this is an interesting relationship, it does not establish that the resting-state brain spontaneously alternates between high and low PE states, nor that it does so in a functionally meaningful way that is related to behavior. How can we rule out brain dynamics of other processes, such as arousal, that also rise and fall with PE? I understand the authors' intention to address the reverse inference concern by testing whether "a participant's unique connectivity response to PE in the reward-processing task should match their specific patterns of resting-state fluctuation". However, I'm not fully convinced that this analysis establishes the functional role of the identified modules to PE because of the following:
Theoretically, relating the activities of the identified modules directly to behavior would demonstrate a stronger functional role.
a) Across participants: Do individuals who exhibit stronger or more distinct PE-related fluctuations at rest also perform better on tasks that require prediction or inference? This could be assessed using the HCP prediction task, though if individual variability is limited (e.g., due to ceiling effects), I would suggest exploring a dataset with a prediction task that has greater behavioral variance.
Or even more broadly, does this variability in resting state PE state fluctuations predict general cognitive abilities like WM and attention (which the HCP dataset also provides)? I appreciate the inclusion of the win-loss control, and I can see the intention to address specificity. This would test whether PE state fluctuations reflect something about general cognition, but also above and beyond these attentional or WM processes that we know are fluctuating.
b) Within participants: Do momentary increases in PE-network expression during tasks relate to better or faster prediction? In other words, is there evidence that stronger expression of PE-related states is associated with better behavioral outcomes?
(3) Apriori Hypothesis for EEG Frequency Analysis
It's unclear how to interpret the finding that fMRI fluctuations in the defined modules correlate with frontal Delta/Theta power, specifically in the 3-6 Hz range. However, in the EEG literature, this frequency band is most commonly associated with low arousal, drowsiness, and mind wandering in resting, awake adults, not uniquely with prediction error processing. An a priori hypothesis is lacking here: what specific frequency band would we expect to track spontaneous PE signals at rest, and why? Without this, it is difficult to separate a PE-based interpretation from more general arousal or vigilance fluctuations.
(4) Significance Assessment
The significance of the correlation above and all other correlation analyses should be assessed through a permutation test rather than a single parametric t-test against zero. There are a few reasons: a) EEG and fMRI time series are autocorrelated, violating the independence assumption of parametric tests;<br /> b) Standard t-tests can underestimate the true null distribution's variance, because EEG-fMRI correlations often involve shared slow drifts or noise sources, which can yield spurious correlations and inflating false positives unless tested against an appropriate null.
Building a null distribution that preserves the slow drifts, for example, would help us understand how likely it is for the two time series to be correlated when the slow drifts are still present, and how much better the current correlation is, compared to this more conservative null. You can perform this by phase randomizing one of the two time courses N times (e.g., N=1000), which maintains the autocorrelation structure while breaking any true co-occurrence in patterns between the two time series, and compute a non-parametric p-value. I suggest using this approach in all correlation analyses between two time series.
(5) Analysis choices
If I'm understanding correctly, the algorithm used to identify modules does so by assigning nodes to communities, but it does not itself restrict what edges can be formed from these modules. This makes me wonder whether the decision to focus only on connections between adjacent modules, rather than considering the full connectivity, was an analytic choice by the authors. If so, could you clarify the rationale? In particular, what justifies assuming that the gradient of PE states should be captured by edges formed only between nearby modules (as shown in Figure 2E and Figure 4), rather than by the full connectivity matrix? If this restriction is instead a by-product of the algorithm, please explain why this outcome is appropriate for detecting a global signature of PE states in both task and rest.
When assessing the correspondence across task-fMRI and rs-fMRI in section 2.2.2, why was the pattern during task calculated from selecting a pair of bilateral ROIs (resulting in a group of eight ROIs), and the resting state pattern calculated from posterior-anterior/ventral-dorsal fluctuation modules? Doesn't it make more sense to align the two measures? For example, calculating task effects on these same modules during task and rest?
Reviewer #1 (Public review):
Summary:
Overall, this is an interesting paper. The authors have found multiple experimental knobs to perturb a mechanical wave behavior driven by pilli feedback. The authors framed this as nonreciprocal interactions - while I can see how nonreciprocity could play a role - what about mechanical feedback? Phenomenological models are fine, but a lack of mechanistic understanding is a weakness. I think it will be more interesting to frame the model based on potential mechanochemical feedback to understand microscopic mechanisms. Regardless, more can be done to better constrain the model through finding knobs to explain experimental observations (in Figures 3, 4, 5, and 7).
Strengths:
The report of mechanical waves in bacterial collectives. The mechanism has potential application in a multicellular context, such as morphogenesis.
Weaknesses:
My most serious concern is about left-right symmetry breaking. I fail to see how the data in Figure 6 shows LR symmetry breaking. All they show is in-out directionality, which is a boundary condition. LR SM means breaking of mirror symmetry - the pattern cannot be superimposed on its mirror image using only rigid body transformations (translation and rotation) - as far as I am aware, this condition is not satisfied in this pattern-forming system.
Reviewer #3 (Public review):
Summary:
This manuscript presents a novel investigation into unidirectionally propagating waves observed on the surface of Pseudomonas nitroreducens bacterial biofilms. The authors explore how these waves, initially spiral in form, transition into combinations of spiral, target, and planar patterns. The study identifies the periodic extension-retraction cycles of type IV pili as the driving mechanism for wave propagation, which preferentially moves from the colony's edge to its center. Furthermore, the manuscript proposes two theoretical models-a phase-oscillator model and a continuum active solid model-to reproduce these phenomena, and demonstrates how external manipulations (e.g., water droplets, temperature, PEG) can control wave patterns and direction, often correlating with oscillation frequency gradients. The work aims to bridge the fields of active-matter physics and bacterial biophysics by providing both experimental observations and theoretical frameworks for understanding these complex biological wave phenomena.
Strengths:
The experimental discovery of unidirectionally propagating waves on bacterial biofilms is highly intriguing and represents a significant contribution to both microbiology and active-matter physics. The detailed observations of wave pattern transitions (spiral to target to planar) and their response to various environmental perturbations (water, temperature, PEG) provide valuable empirical data. The identification of type IV pili as the driving force offers a concrete biological mechanism. The observed correlation between frequency gradients and wave direction is a compelling finding with potential for broader implications in understanding biological pattern formation. This work has the potential to stimulate further research in the collective behavior of living systems and the physical principles underlying biological organization.
Weaknesses:
The manuscript attempts to link unidirectional wave propagation to non-reciprocal couplings but ultimately shows that the wave direction is determined by the gradient of the oscillation frequency. The couplings in the two theoretical models are both isotropic and thus cannot dictate the wave direction. A clear distinction should be made between non-reciprocity as a source of wave generation and non-uniformity as a controlling factor of wave direction.
The relationship between the phase oscillator model and the active solid model is unclear. Given that U and P are both dynamical variables evolving in three-dimensional space, defining the phase Φ precisely in the phase space spanned by U and P could be challenging. A graphical illustration of the definition of Φ would be beneficial. To ensure reproducibility of the numerical results, the parameter values used in the numerical simulations and an explicit definition of the elastic force in the active solid model should be provided.
The link between the theoretical models and experimental results is weak. For example, the propagation of the kink from the lower to the higher part of the surface (Figure 1e) could be addressed within the framework of the active solid model. The mechanism of transition from spiral to target waves (Figure 3a), b)) requires clarification, identifying which model parameter is crucial for inducing this transition. The wave propagation toward the lower frequency side is numerically demonstrated using the phase oscillator model, but a physical or intuitive explanation for this phenomenon is missing. Also, the wave transitions induced by the addition of water droplets and temperature rise are not linked to specific parameters in the theoretical models.
RELOADING SQL BACKUPS
1. mysql -u root -p < dump_db.sql
<) the dump file into MySQL.CREATE DATABASE + USE statements (because you used --databases when dumping), MySQL will recreate the DB and restore it automatically.2. mysqladmin -u root -p create db1
db1.CREATE DATABASE.3. (Alternative: create inside MySQL CLI)
sql
mysql> CREATE DATABASE IF NOT EXISTS restore_db;
mysql> USE restore_db;
mysql> source dump_1.sql;
restore_db).USE restore_db;).source dump_1.sql → runs the dump file line by line (restores tables + data into this DB).⚡ In short:
CREATE DATABASE + USE → just run it, it will auto-create the DB.BACKUP
1. mysqldump -u root -p msys140_activity > dump_1.sql
msys140_activity).dump_1.sql.CREATE DATABASE or USE statements (because no --databases flag).2. mysqldump -u root -p --all-databases > alldb.sql
alldb.sql.CREATE DATABASE and USE statements for each database.3. mysqldump -u root -p --databases msys140_activity csci40_db > multipledb.sql
msys140_activity and csci40_db).multipledb.sql.CREATE DATABASE and USE statements for each database (because of --databases).4. [mysqldump -u root -p msys140_activity > dump_1.sql
VS
mysqldump -u root -p --databases msys140_activity > dump_2.sql]
--databases: dump_1.sql has only table structures + data, but no CREATE DATABASE or USE.--databases: dump_2.sql includes the CREATE DATABASE and USE statements, so you can recreate the DB automatically when restoring.5. mysqldump -u root -p msys140_activity tblCustomers > dump_tables.sql
tblCustomers) from the msys140_activity database.dump_tables.sql.👉 Shortcut to remember:
--databases → Only schema + data.--databases / --all-databases → Schema + data + CREATE DATABASE + USE statements.SPEED is optimized at different levels: 1. database speed 2. hardware speed 3. connectivity speed
Avoid such easy, empty transitions as “firstly,” “secondly” and “finally.” Your reader should be able to understand they have been moved from one aspect of your argument to another without a tell-tale “secondly” informing them that the first point is over and the second point is about to begin. Again, this is where keeping a close eye to your thesis and your outline is so important. If you know where the essay is going, you can transfer your readers smoothly from the analysis of one aspect of the text to the next with meaningful connections and statements rather than empty transitional phrases. Witness the transition from the final sentence in Paragraph 2 into the first sentence and then the topic sentence of Paragraph 3.
make the essay smoothier without announcing what is coming next with "first second thrid transition words"
Three common ways to structure a paper are chronological order, spatial order, and order of importance. Choose the order that will most effectively fit your purpose and support your main point.
there is 3 ways you can structure your model
And because our (digital) prototypes try to be used/validaded mainly by communities instead of by academic peers, we need to care about the practicalities of such prototypes and their insertion in the communities. In my experience, this practical insertion could happen via two complementary strategies: the encompassing one and embedding one. The encompassing strategy could be exemplified by the Smalltalk variants, like Pharo or GToolkit, with their OS and IDE rolled into one approach. Here, a single computing experience includes "everything" a community artifact could need: object networks acting as "app(s)"3, persistance, data formats, IDEs, graphical stack, debbugers and so on. The practicalities are related with the collapse of incidental complexity when the community has a single metatool to bridge their other tools and workflows. We use what I call "interstitial programming" to bridge socio-technical systems by changing what happens in the gaps/bridges between them, instead of changing them from inside. This was the approach I followed with Grafoscopio, since late 2014 and early 2015 until present day, with pretty good results and fluency, allowing us to make several prototypes and empowering practices convering diverse needs: from self (PDF/web) publishing, to civic tech and political oversight, community learning and memory, amont other themes (chosing needs and topics in resonance with the community is key in having this prototypes as living artifacts in such community). The embedding strategy could be exemplified by Lua and its variants, like YueScript. Here, an already existing tool/experience is extended from inside or by complementing and then replacing an existing tool/practice, and while this contrast the "interstitial" approach mentioned above, still shares the concern of dealing with needs felt in the community in its current workflows and tools. This is the strategy I plan to explore this year, particularly regarding the publishing workflows/formats of several local grassroots communities, and to compare with how I'll be implementing part of such ideas in Grafoscopio (keeping on with the encompassing strategy). While previously I thought in Fengari as my way to implement embeddability to increse agency in the (web) tools, the recent developments on hypermedia systems make me think that I can keep avoiding JavaScript4 and implement the strategy server side by reimagining TiddlyWiki in Lua+YueScript. Cardumem is the working name for such idea, and as explained in that link the intend is to provide a similar gentle learning curve between being a content creator and a functionality creator, that TiddlyWiki give us, while being able to generalize the concepts learnt while using and extending the wiki in its own functional DSL to other computing languages (for more details and links to the TW's community discussion visit the previos link). So, regarding the "Not Invented Here syndrome", the differences with TiddlyWiki are enough to justify why we need to invest all that work in Cardumem, as community and (inter)personal knowledge management is a core concern5 in the Grafoscopio community, to the point that we need to reinvent the wheel, for the contexts where the already existing ones don't work as we expect for our needs. While learning Lua and YueScript, I frequently miss a lot of the code liveness and the interactive documentation of the "Argumentative Driven Development" (ADD? 🤔) that I already enjoy within Grafoscopio over Pharo/GToolkit. So I thought that my first job would be to implement some kind of minimal notebook publishing on Lua, inpired by Clojure's Clerk6 and Julia's Pluto, but quite more static, at least as the begining (see Boostrapping a Lua notebook for more details). But finally a minimal Lua long comment + "markup tag" was good enough to have my documentation in the Lua files to postpone the idea, while exploring the HTML interactive interfaces provided by HTMX. Instead the design has been guided by the needs I have with my students/apprentices in my classes this semester at the university and future workshops in the hackerspace. And it has been a pretty fruitful design space/practice, where UI and functionality emerge organically, with the lessons I need to learn to ptovide the experience I need/want. There is still a long path to walk, but the initial advances are promising. Let's see how I walk the exploration map sketched here in this pendular movement from emcompassing to embedding strategies and from abstraction about the to concrete implementations. I will document my advances in the entries to come.
La tecnología pensada para comunidades debe práctica y no solo teórica, y para lograrlo se pueden usar dos estrategias: la envolvente, que ofrece una herramienta integral como Grafoscopio, o la incrustada, que mejora las herramientas que la gente ya utiliza, como se muestra con Cardumem. La idea es encontrar que entre estas dos formas se alinee para que la tecnología llegue a las necesidades reales de una comunidad y no solo el entorno académico u operativo de la programación.
Your research question should inform the structure and contents of your project and everything you cite should be related to your research question in some way.
This line is the book’s quiet thesis about research writing: the question is not just a starting prompt; it’s the organizing principle. Read “inform” as constrain and shape. If a subsection, paragraph, or citation doesn’t help answer the question you’ve posed, it’s ornamental—cut it. Practically, this means (1) operationalizing your terms (What exactly counts as “psychological well-being”? Which population, time frame, and context?), (2) reverse-outlining your draft to check that every section maps to a sub-task of the question (define, contextualize, test, interpret), and (3) applying a ruthless relevance test to sources: each should either supply evidence, methods, or counter-arguments that bear directly on the claim your question implies. This alignment prevents the two most common failures in student research: the “data dump” (too many unfocused sources) and the “tour” (interesting but aimless background). A strong question automatically yields a coherent structure because it dictates what must be established, measured, compared, or explained—and in what order. Quick check: write your research question atop your draft; under every paragraph, jot the specific part of the question it answers. Anything blank signals a tangent.
Today, kanji remain an essential part of the Japanese writing system. They are an integral part of education and are taught from an early age. Despite the advent of technology and globalization, kanji continue to be valued for their depth of meaning and their ability to express complex concepts concisely.
Subtopic 2 & 3
As kanji’s role evolves, preserving this practice as part of Japan’s cultural heritage remains important. Kanji proficiency offers cognitive benefits, and some believe learning kanji for Japan improves memory retention and understanding. Others argue that adjusting the way kanji is taught may help bridge the gap between tradition and modern convenience.
Subtopic 3
What’s more, having the kanji in a narrative form and accompanied by images gives you plenty of context to decipher the meaning quickly and effectively.
Subtopic 3
One of the major difficulties in getting things done in a large organization is the fact that it is practically impossible to pinpoint responsibility. A Bureau or an Office can't be made responsible because, as I said before, they are inanimate and therefore are incapable of perception or of feeling. Limited tours of duty of two or three years in positions of responsibility accentuate this situation.
limited tours of 2-3 years
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Response to the reviewers
We thank the reviewers for recognizing the importance of study, and how it “addresses a long-standing question in the heterogeneity of cellular responses to stressors”, “makes a conceptual advance by identifying transcription factors as the limiting determinant of IFN-β induction in KSHV-infected cells”, and “serves as a crucial starting point for understanding cellular heterogeneity”. We agree that our findings appeal to a broad audience interested in virology, immunology, cell biology, and gene transcription.
We also thank the reviewers for their insightful suggestions that will greatly strengthen our study. Below we detail how we plan to address their comments experimentally and how we have already edited the text to respond to them.
Referee #1
One experiment that may provide some insight into the selective RelA activation is to quantify viral genomes within the high and low IFN producing cells. Perhaps, the genome as a PAMP, is more abundant in the inducing cells.
We have added a note in the Discussion section (line 417) that we have evidence that the cGAS PAMP in our system is mitochondrial DNA, not viral DNA. Moreover, our results suggest that the variation in PAMP levels are not the source of heterogeneity, as this would cause heterogeneous activation of the cGAS-STING-TBK1-IRF3 axis. Instead, we have discovered that TBK1 and IRF3 are activated even in cells without interferon-β induction.
Referee #2
1) While the study presents intriguing evidence for AP-1 involvement in regulating IFN-β responses, the reliance on total c-Jun levels as a readout is limiting. Because c-Jun activity depends on phosphorylation and promoter binding, additional experiments (i.e., phospho-c-Jun analysis or ChIP at the IFNB1 promoter) would strengthen the link between AP-1 activity and the observed reporter outcomes.
We agree that that a stronger link between AP-1 activity and IFN induction would improve our study, so we have cloned interferon-β reporter constructs that contain mutations in the AP-1 binding sites. We plan to use this reporter, as well as IFN-β reporter constructs that contain mutations in either the AP-1, IRF3, or NF-κB binding sites, to mechanistically test the connection between AP-1 and activation of the IFN promoter. As a control, we will test that the mutations block reporter induction after stimulation with a well characterized agonist of the IFN induction pathway such as poly(I:C). We have previously investigated c-Jun and ATF2 phosphorylation during KSHV reactivation and caspase inhibition. Surprisingly, in preliminary experiments we did not detect phosphorylation of either AP-1 subunit. We will confirm this result and add these data to the manuscript.
2) The data presented demonstrating that Serine 386 phosphorylation does not distinguish first responder cells is strong. Including complementary data on Ser396 phosphorylation would strengthen the conclusion, as this well-established activation marker is readily detectable with available reagents and would help confirm that the potentiation of IRF3 activity is not the driver of the observed heterogeneity.
We will complement the Ser386 results with Ser396 staining.
3) Consider updating the title to more directly reflect the findings (e.g. "Interferon-β induction heterogeneity during KSHV infection is correlated to expression of ATF2 and RelA")
We have updated the title to “Interferon-β induction heterogeneity during KSHV infection is correlated to levels and activation of the transcription factors ATF2 and RelA, and not IRF3”
*4) To ease the interpretation of data, indicate what the black and white circles indicate in the figure legends. *
We have updated the figures to be more intuitive, using + and -.
5) IE ORF50 is used to show no differences between first responders and non-responders, but showing early and late genes across tdTomato positive and negative cells would rule out potential differences in progression through reactivation.
We added a clarification in the Results section (line 195), explaining that we have examined the progression of viral reactivation through single-cell transcriptomics in our previous publication, and that the results indicate that viral gene expression plays a small role in interferon-β heterogeneity. We favor the scRNA-seq dataset for this conclusion, because the tdTomato negative cell population represents a mix of non-reactivating cells, which would not be expected to make IFN, and reactivating cells that fail to induce IFN expression.
6) The data in Figure 5D (quantified in E and F) show a compelling trend. This could be further clarified by plotting a trend line that connects the results of independent experiments, rather than only showing individual data points. Such visualization would make the consistency of the observed trend across experiments more apparent.
We have added lines in the graphs in Figure 5 to ease visualization.
Referee #3
A major worry comes from using lentiviral transduction to insert the reporter promoter into cells without selecting for clones. Lentiviral transduction introduces heterogeneity due to random insertion of their vector. This results in different copy numbers for the reporter construct, leading to heterogeneity in the reporter expression. Additionally, the expression of foreign proteins, particularly in immune cells, can be perceived as danger signals (10.1007/s12015-016-9670-8) and occasionally trigger p65 activation. To control for this, the authors could validate their reporter results by including a non-IFNb promoter (e.g., constitutive) expressing tdTomato and verifying that these cell populations do not also express endogenous IFNb mRNAs.
We did not select clonal cell lines because different cells may have different reactivation propensity. Moreover, we did not want the tdTomato signal to reflect specific regulation of a single genomic region. We have now added an explanation as to why we did not clonally select that cell lines in the Results section (line 157). Our control conditions that do not result in IFN-β induction show that lentiviral insertion is not sufficient to cause IFN induction, as we did not detect IFN-β mRNA in the untreated reporter cells (first bar in Figure 1C). We also clarified in the Results section (line 184) that the selective enrichment of both IFN-β and tdTomato mRNA in the sorted tdTomato+ cells demonstrates that tdTomato is a faithful reporter for rare IFN-β expression, regardless of heterogeneous lentiviral transduction in the population. To further verify that lentiviral transduction does not play a role in introducing heterogeneity in induction of our tdTomato reporter and of IFN-β, we will measure IFN-β levels in BC-3 cells constitutively expressing tdTomato, which we have already created. We may also sort BC-3 cells constitutively expressing tdTomato and check that the tdTomato signal is not predictive of IFN expression in these cells. However, the expectation is that all or most cells will be tdTomato positive, which may make sorting for tdTomato negative cells impossible.
Regarding AP1 and NF-kB activation, the authors could investigate downstream genes such as GADD45B, HSPA1A, and ATF-3 (for AP1), and IL-6, TNFAIP3 (A20) (for both AP1 and NFkB). It would be interesting to determine if these genes are exclusively expressed in tdTomato-expressing cells.
We will quantify the mRNA levels of these genes by performing qPCR on our cDNA from sort experiments. So far, we have detected IL-6 induction but no enrichment of this transcript in the sorted tdTomato+ samples.
While the authors observed no direct correlation between c-Jun alone and IFN-b production, it is conceivable that TPA-induced c-Jun primes the cells that become fully transcriptionally active upon a stimulus like viral reactivation. I propose that the authors attempt to inhibit c-Jun activation during KSHV reactivation (TPA + caspase inhibitor) using inhibitors like SP600125 and subsequently assess whether this blockade reduces the proportion of IFNb+ cells.
We have tried using the suggested inhibitor (SP600125), but found that it inhibits KSHV reactivation, making any result on IFN levels difficult to interpret. Currently, we are testing a dual AP-1 and NF-κB inhibitor (SP100030) and may add these data to the results if we do not encounter similar issues.
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Summary
Kaku and Gaglia's study provides one step further in the ongoing debate surrounding viral versus innate immune heterogeneity. They addressed this question by creating a reporter B cell line (BC-3) that expresses tdTomato under the IFNb promoter. This particular cell line is known to be latently infected with KSHV, and lytic infection can be induced by treatment with the protein kinase C activator TPA.
Through FACS sorting upon KSHV lytic infection, the authors observed a correlation between the promoter reporter activation and endogenous IFNb and IFNl mRNA levels. However, this correlation did not extend to viral replication, indicating that only a fraction of virus-infected cells produce IFN. They identified these cells as "first responders" upon viral replication by treating them with anti-IFNAR, confirming that IFN production is triggered by the cGAS-STING pathway sensing lytic virus infection.
Surprisingly, p-IRF3 activation was not limited to IFN-producing cells, suggesting the involvement of other key transcription factors. Indeed, they found a correlation between NF-kB and AP1 activation and IFN production. The study concludes that the combined action of NF-kB, AP1, and IRF3 is crucial for robust IFN production.
Major comment
The author effectively dissects the necessary components for IFNb activation, despite acknowledging the limitations of their findings. All my potential anecdotal queries, such as the role of other viruses or agonists and the treatment of cells with NF-kB inhibitors, are thoroughly addressed in their discussion.
However, a major worry comes from using lentiviral transduction to insert the reporter promoter into cells without selecting for clones. Lentiviral transduction introduces heterogeneity due to random insertion of their vector. This results in different copy numbers for the reporter construct, leading to heterogeneity in the reporter expression. Additionally, the expression of foreign proteins, particularly in immune cells, can be perceived as danger signals (10.1007/s12015-016-9670-8) and occasionally trigger p65 activation. To control for this, the authors could validate their reporter results by including a non-IFNb promoter (e.g., constitutive) expressing tdTomato and verifying that these cell populations do not also express endogenous IFNb mRNAs.
Minor comments
Regarding AP1 and NF-kB activation, the authors could investigate downstream genes such as GADD45B, HSPA1A, and ATF-3 (for AP1), and IL-6, TNFAIP3 (A20) (for both AP1 and NFkB). It would be interesting to determine if these genes are exclusively expressed in tdTomato-expressing cells.
While the authors observed no direct correlation between c-Jun alone and IFN-b production, it is conceivable that TPA-induced c-Jun primes the cells that become fully transcriptionally active upon a stimulus like viral reactivation. I propose that the authors attempt to inhibit c-Jun activation during KSHV reactivation (TPA + caspase inhibitor) using inhibitors like SP600125 and subsequently assess whether this blockade reduces the proportion of IFNb+ cells.
The study presents a valuable dataset and serves as a crucial starting point for understanding cellular heterogeneity, particularly regarding the known concept of IRF+NFkB in IFNb production. While this mechanism isn't novel (10.1074/jbc.273.5.2714), the authors demonstrated the difference in activation in a cellular level. This finding can be the basis of future research utilizing more physiologically relevant models, such as primary cells or tissues, to identify factors contributing to varying cellular responses.
However, the authors acknowledge that these findings should be interpreted with caution and require further validation through additional studies across different models and viral infections. This research will be particularly relevant to those in basic research seeking to deepen their understanding of the dynamic differences in innate immune responses and viral infections.
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In the manuscript "Interferon-β induction heterogeneity during KSHV infection is correlated to expression and activation of enhanceosome transcription factors other than IRF3", Kaku and Gaglia address a long-standing question in the initiation of host antiviral responses: what drives the heterogeneity in the initiation of IFN responses within a presumably homogenous population of cells. In this study, the authors focus on host factors that contribute to the heterogeneity in IFN induction. They use a KSHV lytic reactivation model (TPA + Caspase inhibitor treated BC-3 cells) and FACS-based reporter assays to enhance the resolution in the detection of molecular drivers of "first responder" cells that make IFN. They find that IRF3 activation alone does not predict IFN expression; rather, the expression of ATF2 and RelA is predictive of IFN-β induction. The authors carefully control for off-target effects of TPA treatment in BJAB cells and paracrine signaling through the inclusion of IFN-neutralizing antibodies. Overall, the manuscript is well-written and easy to follow, and the data compellingly support their conclusion that cell-specific transcription factor activity limits IFN production to cell subsets. Demonstrating coordinated occupancy or functional interplay of these factors would increase confidence in the proposed model and broaden the impact for readers interested in virology, immunology, and transcriptional regulation.
Comments:
Minor Comments:
This study addresses an important and long-stading question in the heterogeneity of cellular responses to stressors, such as viruses. The study is well designed and presented, making it appealling to a broad audience interested in virology, immunology, cell biology, and gene transcription.
At least we only have 4 more hours left until we get the comfort of the darkness again.3
I'm assuming here that readers will know that excessive light makes chicken uncomfortable, thus contributing to poor welfare.
Though the 20th hour of light never gets easier. 3
I'm trying to blend fact and fiction together, as the evidence about light in factory cages is coming from a secondary source. However I only cited with a number, which is linked at the bottom with the corresponding number. I could have made the citation a little more clear while reading.
inside each cell, atoms make up molecules; these in turn make up cell organelles and other cellular inclusions
Cause and effect
Reviewer #1 (Public review):
Summary:
This paper describes a behavioral platform "BuzzWatch" and its application in long-term behavioral monitoring. The study tested the system with different mosquito species and Aedes aegypti colonies and monitored behavioral response to blood feeding, change in photoperiod, and host-cue application at different times of the day.
Strengths:
BuzzWatch is a novel, custom-built behavioral system that can be used to monitor time-of-day-specific and long-term mosquito behaviors. The authors provide detailed documentation of the construction of the assay and custom flight tracking algorithm on a dedicated website, making them accessible to other researchers in the field. The authors performed a wide range of experiments using the BuzzWatch system and discovered differences in midday activity level among Aedes aegypti colonies, and reversible change in the daily activity profile post-blood-feeding.
Weaknesses:
The authors report the population metric "fraction flying" as their main readout of the daily activity profile. It is worth explaining why conventional metrics like travel distance/activity level are not reported. Alternatively, these metrics could be shown, considering the development and implementation of a flight trajectory tracking pipeline in this paper.
The authors defined the sugar-feeding index using occupancy on the sugar feeder. However, the correlation between landing on the sugar feeder and active sugar feeding is not mentioned or tested in this paper. Is sugar feeding always observed when mosquitoes land on the sugar feeder? Do they leave the sugar feeding surface once sugar feeding is complete? One can imagine that texture preference and prolonged occupancy may lead to inaccurate reporting of sugar feeding. While occupancy on the sugar feeder is an informative behavioral readout, its link with sugar feeding activity (consumption) needs to be evaluated. Otherwise, the authors should discuss the caveats that this method presents explicitly to avoid overinterpretation of their results.
Throughout the manuscript, the authors mentioned existing mosquito activity monitoring systems and their drawbacks. However, many of these statements are misleading and sometimes incorrect. The authors claim that beam-break monitors are "limited to counting active versus inactive states". Though these systems provide indirect readouts that may underreport activity, the number of beam-breaks in a time interval is correlated with activity level, as is commonly used and reported in Drosophila and mosquitoes and a number of reports in mosquitoes an updated LAM system with larger behavioral arenas and multiple infrared beams. The authors also mentioned the newer, camera-based alternatives to beam-break monitors, but again referred to these systems as "only detecting activity when a moving insect blocks a light beam"; however, these systems actually use video tracking (e.g., Araujo et al. 2020).
The fold change in behavior presented in Figure 4D is rather confusing. Under the two different photoperiods, it is not clear how an hourly comparison is justified (i.e., comparing the light-on activity in the 20L4D condition with scotophase activity in the 12L12D condition). The same point applies to Figure 4H.
The behavioral changes after changing photoperiod (Figure 4) require a control group (12L12D throughout) to account for age-related effects. This is controlled for the experiment in Figure 3 but not for Figure 4.
Reviewer #2 (Public review):
Summary:
This study establishes a platform for studying mosquito flight activity over the course of several weeks and demonstrates key applications of such a paradigm: the comparison of daily activity profiles across different Aedes aegypti populations and the quantification of responses to physiological and environmental perturbations.
Strengths:
(1) Overall, the authors succeed in setting up a low-cost, scalable tracking system that stably records mosquito flight activity for several weeks and uses it to demonstrate compelling use cases.
(2) The text is organized well, is easy to read, and is understandable for a broad audience.
(3) Instructions for constructing housing and for performing tracking with a dedicated GUI are available on an accompanying website, with open-source (and well-organized) code.
(4) A complementary pair of methods (one testing for activity signals at specific times of the day, and the other capturing broader daily patterns) is used effectively.
Weaknesses:
(1) In the interval-based GLMM results, since each time interval is tested independently, p-values should be corrected for multiple hypotheses (for instance, through controlling the false discovery rate).
(2) The accompanying GUI application needs some modifications to fully work out of the box on a sample video.
Reviewer #3 (Public review):
Summary:
The authors in this paper introduce BuzzWatch, an open-source, low-cost (200-300 Euros) platform for long-term monitoring of mosquito flight and behavior. They use a Raspberry Pi with a Noirv2 Camera set up under laboratory conditions to observe 3 different species of mosquitoes. The system captures a variety of multimodal data, like flight activity, sugar feeding, and host-seeking responses, with the help of external modules like CO2 and fructose-soaked cottons. They also release a GUI in addition to automated tracking and behaviour analysis, which doesn't run on Pi but rather on a personal laptop.
Four main use cases are demonstrated:
(1) Characterizing diel rhythms in various Aedes aegypti populations.
(2) Differentiating behaviors of native African vs. invasive human-adapted subspecies.
(3) Assessing physiological (blood-feeding) and environmental (light regime) perturbations.
(4) Testing time-of-day variation in responses to host-associated cues like CO₂ and heat.
Description (Strengths):
(1) The authors introduce a low-cost, scalable system that uses flight tracking in 2D as an alternative to 3D multi-camera systems.
(2) Due to the low pixel quality required by the system, they can record for weeks at a time, capturing long temporal and behavioral activities.
(3) They also integrate external modules such as lights, CO2, and heat as a way to measure responses to a variety of stimuli.
(4) They also introduce a wiki as a guide for building replication and a help in using the GUI module.
(5) They implement both GLMM hourly and PCA of behavior data.
Limitations - Major Comments:
(1) Most experiments are only done with single replicates per colony. If the setup is claimed to be cheap and replicable, there should be clearer replicates across experiments.
(2) No external validation for the flight tracking algorithm using manual annotation or comparison with field data. The authors focus early on biological proof of principle, but the validity of the tracking algorithm is not presented. How accurate is the algorithm at classifying behaviours (e.g., vs human ground truth)? How reliable is tracking?
(3) Why develop a custom GUI instead of using established packages such as rethomics (https://rethomics.github.io/) that are already available for behavioral analysis?
(4) Why use RGB light strips when perceptual white light for humans is not relevant for mosquitoes? The choice of lighting should be based on the mosquito's visual perception. - https://pmc.ncbi.nlm.nih.gov/articles/PMC12077400/ .
(5) Why use GLMMs instead of GAMs (with explicit periodic components)? With GLMMs, you do not account for temporal structure, which is highly relevant and autocorrelated in behavioral time series data.
(6) What is the proportion of mosquitoes that stay alive throughout the experiments? How do you address dead animals in tracking? No data are available on whether all mosquitoes made it through the monitoring period. No survival data is mentioned in the paper, and in the wiki, it is not clear how it is used or how it affects the analyses - https://theomaire.github.io/buzzwatch/analyze.html#diff-cond .
(7 )The sugar feeding behavior is not manually validated.
(8) Figure 4d is difficult to understand - how did you align time? Why is ZT4 aligning with ZT0? Should you "warp" the time series to compare them (e.g., from dawn to dusk)?
(9) No video recordings are made available for demonstration or validation purposes.
Appraisal
(1) The core conclusions---that BuzzWatch can capture multiscale mosquito behavioral rhythms and quantify the effect of genetic, environmental, and physiological variation - show promise but require stronger validation.
(2) Statistical approaches (GLMM, PCA) are chosen but may not be optimal for temporal data with autocorrelation.
(3) The host-seeking module shows a differential response, which is a potentially valuable feature.
Reviewer #2 (Public review):
Summary:
The manuscript by Kawadkar et al investigates the role of Nup107 in developmental progression via regulation of ecdysone signaling. The authors identify an interesting phenotype of Nup107 whole body RNAi depletion in Drosophila development - developmental arrest at the late larval stage. Nup107-depleted larvae exhibit mis-localization of the Ecdysone receptor (EcR) from the nucleus to the cytoplasm and reduced expression of EcR taret genes in salivary glands, indicative of compromised ecdysone signaling. This mis-localization of EcR in salivary glands was phenocopied when Nup107 was depleted only in the prothoracic gland (PG), suggesting that it is not nuclear transport of EcR but presence of ecdysone (normally secreted from PG) that is affected. Consistently, whole body levels of ecdysone were shown to be reduced in Nup107 KD, particularly at the late third instar stage when a spike in ecdysone normally occurs. Importantly, the authors could rescue the developmental arrest and EcR mis-localization phenotypes of Nup107 KD by adding exogenous ecdysone, supporting the notion that Nup107 depletion disrupts biosynthesis of ecdysone, which arrests normal development. Additionally, they found that rescue of Nup107 KD phenotype can also be achieved by over-expression of the receptor tyrosine kinase torso, which is thought to be the upstream regulator of ecdysone synthesis in the PG. Transcript levels of torso are also shown to be downregulated in the Nup107KD, as are transcript levels of multiple ecdysone biosynthesis genes. Together, these experiments reveal a new role of Nup107 or nuclear pore levels in hormone-driven developmental progression, likely via regulation of levels of torso and torso-stimulated ecdysone biosynthesis.
Strengths:
The developmental phenotypes of an NPC component presented in the manuscript are striking and novel, and the data appears to be of high quality. The rescue experiments are particularly significant, providing strong evidence that Nup107 functions upstream of torso and ecdysone levels in regulation of developmental timing and progression.
Weaknesses:
The underlying mechanism is however not clear, and any insight into how Nup107 may regulate these pathways would greatly strengthen the manuscript. Some suggestions to address this are detailed below.
Major questions:
(1) Determining how specific this phenotype is to Nup107 vs. to reduced NPC levels overall would give some mechanistic insight. Does knocking down other components of the Nup107 subcomplex (the Y-complex) lead to similar phenotypes? Given the published gene regulatory function of Nup107, do other gene regulatory Nups such as Nup98 or Nup153 produce these phenotypes?
(2) In a related issue, does this level of Nup107 KD produce lower NPC levels? It is expected to, but actual quantification of nuclear pores in Nup107-depleted tissues should be added. These and above experiments would help address a key mechanistic question - is this phenotype the result of lower numbers of nuclear pores or specifically of Nup107?
(3) Additional experiments on how Nup107 regulates torso would provide further insight. Does Nup107 regulate transcription of torso or perhaps its mRNA export? Looking at nascent levels of the torso transcript and the localization of its mRNA can help answer this question. Or alternatively, does Nup107 physically bind torso?
(4) The depletion level of Nup107 RNAi specifically in the salivary gland vs. the prothoracic gland should be compared by RT-qPCR or western blotting.
(5) The UAS-torso rescue experiment should also include the control of an additional UAS construct - so Nup107; UAS-control vs Nup107; UAS-torso should be compared in the context of rescue to make sure the Gal4 driver is functioning at similar levels in the rescue experiment.
Minor:
(6) Figures and figure legends can stand to be more explicit and detailed, respectively.
Comments on revisions:
The revised manuscript addresses several outstanding issues, most importantly the question of whether the developmental delay phenotype of Nup107 is exhibited by other Nups.
I recommend that the authors include the data they provide in the rebuttal letter on Nup153 KD not showing the delay phenotype (Figure R1) into the actual manuscript. It's an important mechanistic question raised by multiple reviewers, and would strengthen the authors' conclusions. Ideally, knock downs of other Nups of the Nup107 complex should be investigated, especially given that all those RNAi lines are publicly available.
Figure 6B should also specify whether the torso transcript being measured is mRNA or nascent, as it would help understand whether it's transcription or mRNA stability that is affected by Nup107 KD.
Reviewer #3 (Public review):
These findings suggest that Nup107 is involved in regulating ecdysone signaling during developmental transitions, with depletion of Nup107 disrupting hormone-regulated processes. Moreover, the rescue experiments hint that Nup107 might directly influence EcR signaling and ecdysone biosynthesis, though the precise molecular mechanism remains unclear.
Overall, the manuscript presents compelling data supporting Nup107's role in regulating developmental transitions.
Comments on revisions:
RNAi specificity: The authors now provide a more thorough discussion of off-target effects and justify their reliance on the Nup107KK RNAi line. The explanation regarding the predicted off-target for the GD line and their choice to use the KK line with a known insertion site is appropriate and adequately addresses the original concern.
NPC component specificity: The authors clarify that among the Nup107 complex members tested, only Nup107 knockdown induced developmental arrest. Their inclusion of Nup153 as a control helps to support the specificity of the phenotype, although expanding this analysis beyond a single additional Nup would further strengthen the claim.
Mechanistic clarity: The authors now distinguish between Nup107's upstream role in regulating torso and ecdysone biosynthetic genes versus direct control of EcR translocation. The clarification that EcR nuclear localization is 20E-dependent but Nup107-independent improves interpretive clarity.
The molecular mechanism linking Nup107 to torso regulation remains somewhat speculative. A deeper exploration of whether Nup107 influences transcriptional regulation through chromatin association (as the authors suggest) would strengthen the mechanistic narrative.
Conclusion:
Overall, the authors have addressed the major concerns raised in the initial review, and the revised manuscript presents a more coherent and compelling case for Nup107 as a regulator of developmental timing via the ecdysone signaling axis. While some mechanistic questions remain, the core findings are supported by the data, and the work provides novel insights into NPC function in development.
I wanted to be able to do this for a long time
that would load hypothes.is annotation onto the page
So people can se the annotation even if they do not have the extension instlled
Reviewer #3 (Public review):
Gonzaga-Saavedra et al report an analysis on genomic binding of Polycomb group proteins, and of H2Aub1 and H3K27me3 domain formation in the early Drosophila embryo. Using carefully staged embryos during the nuclear cycles (NC) leading up to the cellular blastoderm stage, the authors provide compelling evidence that H3K27me3 domains at PcG target genes are only established during NC14 and do not exist in NC13. In contrast, H2Aub1 domains already start to appear during NC13. The authors show that E(z), the catalytic subunit of the H3K27 histone methyltransferase PRC2, is readily detected in interphase nuclei during the rapid nuclear divisions in pre-blastoderm embryos. In contrast, the DNA-binding proteins Pho, Cg, and GAF that are known (Pho) or have been postulated (Cg, GAF) to anchor PRC2 and PRC1 to Polycomb Response Elements (PREs) in Polycomb target genes only start to show nuclear localization from NC10 onwards with gradually increasing nuclear concentrations, reaching a maximum during NC14. These data strongly corroborate the simple, straightforward view that targeting of PRC2 and PRC1 to PREs by sequence-specific DNA-binding proteins is a prerequisite for the formation of H3K27me3 and H2Aub1 domains at Polycomb target genes.
The authors then explore the potential role of GAF/Trl in this process. They find that in embryos depleted of GAF/Trl, H3K27me3 domain formation is largely unperturbed.
The authors also depleted the pioneer factor Zelda (Zld) and found that removal of Zld results in a more complex outcome. Zelda appears to counteract the accumulation of H3K27me3 at the Polycomb targets eve and zen, but also appears to be required for effective H3K27me3 domain formation at Polycomb targets such as amos or atonal.
This is a very thorough study that reports data of superior technical quality that are highly relevant for the field. The study by Gonzaga-Saavedra et al extends and strengthens previous work from the labs of Eisen (Li et al, eLife 2014) and Zeitlinger (Chen et al, eLife 2013) to convincingly demonstrate that Polycomb domain formation in the early embryo occurs during ZGA but that such domains do not exist prior to ZGA. This should now finally put to rest earlier claims by the Iovino lab (Zenk et al, Science 2017) that H3K27me3 domains present in the zygote nucleus would be propagated and partially maintained during the rapid nuclear cleavage cycles and serve as seeds for H3K27me3 domain formation during ZGA.
The experiments analyzing H3K27me3 domain formation in embryos depleted of GAF/Trl or Zelda will be of great interest to the field.
Author response:
Reviewer 1:
We appreciate the reviewer’s positive assessment and in revision will expand the Discussion to clarify some of the mechanistic insights of this work, as well as to include expanded treatment of related studies in other model systems.
Reviewer 2:
We are grateful for the reviewer’s thorough and supportive comments. We will carefully revise assertions and conclusions for objectivity. Additional analysis of the Zelda experiments will be performed and experimental data tables will be updated to report these results. For the point about providing “insight into models explaining why H3K27me3 is absent prior to NC14,” we have recently submitted a related preprint that addresses this issue directly (Degen, Gonzaga-Saavedra, and Blythe, bioRxiv 2025). In summary, we find evidence that a maternal PcG imprint is indeed maintained through cleavage divisions, albeit through lower-order methylation states (maximally H3K27me2). We chose not to include these additional results in this manuscript to maintain the focus of this study on ZGA. Our revision of this manuscript will include a section in the Discussion that synthesizes the conclusions of the two studies.
Reviewer 3:
We thank the reviewer for recognizing the strength of our data and conclusions, and we agree that our results help settle conflicting claims in the field. We will emphasize Zelda’s context-dependent effects more clearly in the revised manuscript.
References:
Degen EA, Gonzaga-Saavedra N, Blythe SA. Lower-order methylation states underlie the maintenance and re-establishment of Polycomb modifications in Drosophila embryogenesis. bioRxiv [Preprint]. 2025 Jul 29:2025.07.25.666882. doi: 10.1101/2025.07.25.666882. PMID: 40766521; PMCID: PMC12324246.
"document"?
Test #3!
Reviewer #2 (Public review):
In their study, Avraham-Davidi et al. combined scRNA-seq and spatial mapping studies to profile two preclinical mouse models of colorectal cancer: Apcfl/fl VilincreERT2 (AV) and Apcfl/fl LSL-KrasG12D Trp53fl/fl Rosa26LSL-tdTomato/+ VillinCreERT2 (AKPV). In the first part of the manuscript, the authors describe the analysis of the normal colon and dysplastic lesions induced in these models following tamoxifen injection. They highlight broad variations in immune and stromal cell composition within dysplastic lesions, emphasizing the infiltration of monocytes and granulocytes, the accumulation of IL-17+gdT cells and the presence of a distinct group of endothelial cells. A major focus the study is the remodeling of the epithelial compartment, where most significant changes are observed. Using no-negative matrix factorization, the authors identify molecular programs of epithelial cell functions, emphasizing stemness, Wnt signaling, angiogenesis and inflammation as majors features associated with dysplastic cells. They conclude that findings from scRNA-seq analyses in mouse models are transposable to human CRC. In the second part of the manuscript, the authors aim to provide the spatial contexture for their scRNA-seq findings using Slide-seq and TACCO. They demonstrate that dysplastic lesions are disorganized and contain tumor-specific regions, which contextualize the spatial proximity between specific cell states and gene programs. Finally, they claim that these spatial organizations are conserved in human tumors and associate region-based gene signatures with patient outcome in public datasets. Overall, the data were collected and analyzed using solid and validated methodology to offer a useful resource to the community.
Main comments:
(1) Clarity. The manuscript would benefit from a substantial reorganization to improve clarity and accessibility for a broad readership. The text could be shortened and the number of figure panels reduced to emphasize the novel contributions of this work while minimizing extensive discussions on general and expected findings, such as tissue disorganization in dysplastic lesions. Additionally, figure panels are not consistently introduced in the correct order, and some are not discussed at all (e.g., Fig. S1D; Fig. 3C is introduced before Fig. 3A; several panels in Fig. 4 are not discussed). The annotation of scRNA-seq cell states is insufficiently explained, with no corresponding information about associated genes provided in the figures or tables. Multiple annotations are used to describe cell groups (e.g., TKN01 = γδ T and CD8 T, TKN05 = γδT_IL17+), but these are not jointly accessible in the figures, making the manuscript challenging to follow. It is also not clear what is the respective value of the two mouse models and timepoints of tissue collection in the analysis.
(2) Novelty. While the study is of interest, it does not present major findings that significantly advance the field or motivate new directions and hypotheses. Many conclusions related to tissue composition and patient outcomes, such as the epithelial programs of Wnt signaling, angiogenesis, and stem cells, are well-established and not particularly novel. Greater exploration of the scRNA-seq data beyond cell type composition could enhance the novelty of the findings. For instance, several tumor microenvironment clusters uniquely detected in dysplastic lesions (e.g., Mono2, Mono3, Gran01, Gran02) are identified, but no further investigation is conducted to understand their biological programs, such as applying nNMF as was done for epithelial cells. Additional efforts to explore precise tissue localization and cellular interactions within tissue niches would provide deeper insights and go beyond the limited analyses currently displayed in the manuscript.
(3) Validation. Several statements made by the authors are insufficiently supported by the data presented in the manuscript and should be nuanced in the absence of proper validation. For example: 1.) RNA velocity analyses: The conclusions drawn from these analyses are speculative and need further support. 2.) Annotations of epithelial clusters as dysplastic: These annotations could have been validated through morphological analyses and staining on FFPE slides. 3.) Conservation of mouse epithelial programs in human tumors: The data in Figure S5B does not convincingly demonstrate enrichment of stem cell program 16 in human samples. This should be more explicitly stated in the text, given the emphasis placed on this program by the authors. 4.) Figure S6E: Cluster Epi06 is significantly overrepresented in spatial data compared to scRNA-seq, yet the authors claim that cell type composition is largely recapitulated without further discussion, which reduces confidence in other conclusions drawn.<br /> Furthermore, stronger validation of key dysplastic regions (regions 6, 8, and 11) in mouse and human tissues using antibody-based imaging with markers identified in the analyses would have considerably strengthened the study. Such validation would better contextualize the distribution, composition, and relative abundance of these regions within human tumors, increasing the significance of the findings and aiding the generation of new pathophysiological hypotheses.
Comments on revisions:
The authors have improved the clarity of the manuscript and responded adequately to all my initial comments.<br /> I don't have any other comments. Congratulations to the authors on this work.
Reviewer #1 (Public review):
Summary:
Taber et al report the biochemical characterization of 7 mutations in PHD2 that induce erythrocytosis. Their goal is to provide a mechanism for how these mutations cause the disease. PHD2 hydroxylates HIF1a in the presence of oxygen at two distinct proline residues (P564 and P402) in the "oxygen degradation domain" (ODD). This leads to the ubiquitylation of HIF1a by the VHL E3 ligase and its subsequent degradation. Multiple mutations have been reported in the EGLN1 gene (coding for PHD2), which are associated with pseudohypoxic diseases that include erythrocytosis. Furthermore, 3 mutations in PHD2 also cause pheochromocytoma and paraganglioma (PPGL), a neuroendocrine tumour. These mutations likely cause elevated levels of HIF1a, but their mechanisms are unclear. Here, the authors analyze mutations from 152 case reports and map them on the crystal structure. They then focus on 7 mutations, which they clone in a plasmid and transfect into PHD2-KO to monitor HIF1a transcriptional activity via a luciferase assay. All mutants show impaired activation. Some mutants also impaired stability in pulse chase turnover assays (except A228S, P317R, and F366L). In vitro purified PHD2 mutants display a minor loss in thermal stability and some propensity to aggregate. Using MST technology, they show that P317R is strongly impaired in binding to HIF1a and HIF2a, whereas other mutants are only slightly affected. Using NMR, they show that the PHD2 P317R mutation greatly reduces hydroxylation of P402 (HIF1a NODD), as well as P562 (HIF1a CODD), but to a lesser extent. Finally, BLI shows that the P317R mutation reduces affinity for CODD by 3-fold, but not NODD.
Strengths:
(1) Simple, easy-to-follow manuscript. Generally well-written.
(2) Disease-relevant mutations are studied in PHD2 that provide insights into its mechanism of action.
(3) Good, well-researched background section.
Weaknesses:
(1) Poor use of existing structural data on the complexes of PHD2 with HIF1a peptides and various metals and substrates. A quick survey of the impact of these mutations (as well as analysis by Chowdhury et al, 2016) on the structure and interactions between PHD2 peptides of HIF1a shows that the P317R mutation interferes with peptide binding. By contrast, F366L will affect the hydrophobic core, and A228S is on the surface, and it's not obvious how it would interfere with the stability of the protein.
(2) To determine aggregation and monodispersity of the PHD2 mutants using size-exclusion chromatography (SEC), equal quantities of the protein must be loaded on the column. This is not what was done. As an aside, the colors used for the SEC are very similar and nearly indistinguishable.
(3) The interpretation of some mutants remains incomplete. For A228S, what is the explanation for its reduced activity? It is not substantially less stable than WT and does not seem to affect peptide hydroxylation.
(4) The interpretation of the NMR prolyl hydroxylation is tainted by the high concentrations used here. First of all, there is a likely a typo in the method section; the final concentration of ODD is likely 0.18 mM, and not 0.18 uM (PNAS paper by the same group in 2024 reports using a final concentration of 230 uM). Here, I will assume the concentration is 180 uM. Flashman et al (JBC 2008) showed that the affinity of the NODD site (P402; around 10 uM) for PHD2 is 10-fold weaker than CODD (P564, around 1 uM). This likely explains the much faster kinetics of hydroxylation towards the latter. Now, using the MST data, let's say the P317R mutation reduces the affinity by 40-fold; the affinity becomes 400 uM for NODD (above the protein concentration) and 40 uM for CODD (below the protein concentration). Thus, CODD would still be hydroxylated by the P317R mutant, but not NODD.
(5) The discrepancy between the MST and BLI results does not make sense, especially regarding the P317R mutant. Based on the crystal structures of PHD2 in complex with the ODD peptides, the P317R mutation should have a major impact on the affinity, which is what is reported by MST. This suggests that the MST is more likely to be valid than BLI, and the latter is subject to some kind of artefact. Furthermore, the BLI results are inconsistent with previous results showing that PHD2 has a 10-fold lower affinity for NODD compared to CODD.
(6) Overall, the study provides some insights into mutants inducing erythrocytosis, but the impact is limited. Most insights are provided on the P317R mutant, but this mutant had already been characterized by Chowdhury et al (2016). Some mutants affect the stability of the protein in cells, but then no mechanism is provided for A228S or F366L, which have stabilities similar to WT, yet have impaired HIF1a activation.
Comments on revision:
While the authors have addressed my concerns regarding the SEC experiments and the structural interpretation of most mutants, I remain unconvinced by their interpretation of the P317R mutant and affinity measurements. The BLI and MST data remain inconsistent for P317R binding to CODD, and the authors' response is essentially that the fluorescent labeling of P317R (but not other mutants) uniquely interferes with binding to the NODD/CODD peptides, which does not make a lot of sense. The fluorescent labeling target lysine residues; while there are lysine in PHD2 in proximity to the peptide binding site, labeling these sites would affect binding to all mutants, not only P317R (which does not introduce any new labeling site). Furthermore, the authors did not really address the discrepancy with the observations by Flashman et al (2008) that NODD binds more weakly than CODD, which is inconsistent with their BLI results. Another point that makes me doubt the validity of the BLI results is the poor fit of the sensorgrams and the slow dissociation kinetics, which is inconsistent with the relatively low affinity in the 2-6 uM range.
Reviewer #2 (Public review):
Summary:
Mutations in the prolyl hydroxylase, PHD2, cause erythrocytosis and, in some cases, can result in tumorigenesis. Taber and colleagues test the structural and functional consequences of seven patient-derived missense mutations in PHD2 using cell-based reporter and stability assays, and multiple biophysical assays, and find that most mutations are destabilizing. Interestingly, they discover a PHD2 mutant that can hydroxylate the C-terminal ODD, but not the N-terminal ODD, which suggests the importance of N-terminal ODD for biology. A major strength of the manuscript is the multidisciplinary approach used by the authors to characterize the functional and structural consequences of the mutations. However, the manuscript had several major weaknesses, such as an incomplete description of how the NMR was performed, a justification for using neighboring residues as a surrogate for looking at prolyl hydroxylation directly, or a reference to the clinical case studies describing the phenotypes of patient mutations. Additionally, the experimental descriptions for several experiments are missing descriptions of controls or validation, which limits their strength in supporting the claims of the authors.
Strengths:
(1) This manuscript is well-written and clear.
(2) The authors use multiple assays to look at the effects of several disease-associated mutations, which support the claims.
(3) The identification of P317R as a mutant that loses activity specifically against NODD, which could be a useful tool for further studies in cells.
Weaknesses:
Major:
(1) The source data for the patient mutations (Figure 1) in PHD2 is not referenced, and it's not clear where this data came from or if it's publicly available. There is no section describing this in the methods.
(2) The NMR hydroxylation assay.
A. The description of these experiments is really confusing. The authors have published a recent paper describing a method using 13C-NMR to directly detect proly-hydroxylation over time, and they refer to this manuscript multiple times as the method used for the studies under review. However, it appears the current study is using 15N-HSQC-based experiments to track the CSP of neighboring residues to the target prolines, so not the target prolines themselves. The authors should make this clear in the text, especially on page 9, 5th line, where they describe proline cross-peaks and refer to the 15N-HSQC data in Figure 5B.<br /> B. The authors are using neighboring residues as reporters for proline hydroxylation, without validating this approach. How well do CSPs of A403 and I566 track with proline hydroxylation? Have the authors confirmed this using their 13C-NMR data or mass spec?<br /> C. Peak intensities. In some cases, the peak intensities of the end point residue look weaker than the peak intensities of the starting residue (5B, PHD2 WT I566, 6 ct lines vs. 4 ct lines). Is this because of sample dilution (i.e., should happen globally)? Can the authors comment on this?
(3) Data validating the CRISPR KO HEK293A cells is missing.
(4) The interpretation of the SEC data for the PHD2 mutants is a little problematic. Subtle alterations in the elution profiles may hint at different hydrodynamic radii, but as the samples were not loaded at equal concentrations or volumes, these data seem more anecdotal, rather than definitive. Repeating this multiple times, using matched samples, followed by comparison with standards loaded under identical buffer conditions, would significantly strengthen the conclusions one could make from the data.
Minor:
(1) Justification for picking the seven residues is not clearly articulated. The authors say they picked 7 mutants with "distinct residue changes", but no further rationale is provided.
(2) A major finding of the paper is that a disease-associated mutation, P317R, can differentially affect HIF1 prolyhydroxylation, however, additional follow-up studies have not been performed to test this in cells or to validate the mutant in another method. Is it the position of the proline within the catalytic core, or the identity of the mutation that accounts for the selectivity?
Comments on revision:
The revised manuscript addresses most of my concerns, i.e performing SEC experiments under matched sample concentrations, and incorporating additional data to justify the use of surrogate residues to monitor proline hydroxylation. I appreciate the improvements in the text to clarify the NMR experiments, but I still find their description confusing. Although the authors are using neighboring residues to monitor proline hydroxylation (which they justify convincingly using supplementary data), the language in the text suggests they are (and can?) monitor them directly (i.e. referring to proline cross-peaks in an 15N-HSQC spectrum). The axis labels in Figure 5B also seem to have become mislabeled in this revised version.
Reviewer #3 (Public review):
Summary:
This is an interesting and clinically relevant in vitro study by Taber et al., exploring how mutations in PHD2 contribute to erythrocytosis and/or neuroendocrine tumors. PHD2 regulates HIFα degradation through prolyl-hydroxylation, a key step in the cellular oxygen-sensing pathway.
Using a time-resolved NMR-based assay, the authors systematically analyze seven patient-derived PHD2 mutants and demonstrate that all exhibit structural and/or catalytic defects. Strikingly, the P317R variant retains normal activity toward the C-terminal proline but fails to hydroxylate the N-terminal site. This provides the first direct evidence that N-terminal prolyl-hydroxylation is not dispensable, as previously thought.
The findings offer valuable mechanistic insight into PHD2-driven effects and refine our understanding of HIF regulation in hypoxia-related diseases.
Strengths:
The manuscript has several notable strengths. By applying a novel time-resolved NMR approach, the authors directly assess hydroxylation at both HIF1α ODD sites, offering a clear functional readout. This method allows them to identify the P317R variant as uniquely defective in NODD hydroxylation, despite retaining normal activity toward CODD, thereby challenging the long-held view that the N-terminal proline is biologically dispensable. The work significantly advances our understanding of PHD2 function and its role in oxygen sensing, and might help in the future interpretation and clinical management of associated erythrocytosis.
Weaknesses:
There is a lack of in vivo/ex vivo validation. This is actually required to confirm whether the observed defects in hydroxylation-especially the selective NODD impairment in P317R-are sufficient to drive disease phenotypes such as erythrocytosis.
The reliance on HRE-luciferase reporter assays may not reliably reflect the PHD2 function and highlights a limitation in the assessment of downstream hypoxic signaling.
The study clearly documents the selective defect of the P317R mutant, but the structural basis for this selectivity is not addressed through high-resolution structural analysis (e.g., cryo-EM).
Given the proposed central role of HIF2α in erythrocytosis, direct assessment of HIF2α hydroxylation by the mutants would have strengthened the conclusions.
Comments on revision:
The revised manuscript by Taber et al. addresses the key points raised during the review process in a comprehensive and appropriate manner. While some limitations remain, such as the lack of in vivo validation or direct HIF2α assessment, I agree with the authors that these are beyond the scope of the current in vitro-focused study. The authors' primary goal was to define the structural and functional defects caused by disease-associated PHD2 mutations. In this respect, the evidence they present is largely convincing and methodologically appropriate. Additional clarifications and an expanded discussion of the luciferase assay's limitations and the P317R structural context strengthen the manuscript further.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
Taber et al report the biochemical characterization of 7 mutations in PHD2 that induce erythrocytosis. Their goal is to provide a mechanism for how these mutations cause the disease. PHD2 hydroxylates HIF1a in the presence of oxygen at two distinct proline residues (P564 and P402) in the "oxygen degradation domain" (ODD). This leads to the ubiquitylation of HIF1a by the VHL E3 ligase and its subsequent degradation. Multiple mutations have been reported in the EGLN1 gene (coding for PHD2), which are associated with pseudohypoxic diseases that include erythrocytosis. Furthermore, 3 mutations in PHD2 also cause pheochromocytoma and paraganglioma (PPGL), a neuroendocrine tumour. These mutations likely cause elevated levels of HIF1a, but their mechanisms are unclear. Here, the authors analyze mutations from 152 case reports and map them on the crystal structure. They then focus on 7 mutations, which they clone in a plasmid and transfect into PHD2-KO to monitor HIF1a transcriptional activity via a luciferase assay. All mutants show impaired activation. Some mutants also impaired stability in pulse chase turnover assays (except A228S, P317R, and F366L). In vitro purified PHD2 mutants display a minor loss in thermal stability and some propensity to aggregate. Using MST technology, they show that P317R is strongly impaired in binding to HIF1a and HIF2a, whereas other mutants are only slightly affected. Using NMR, they show that the PHD2 P317R mutation greatly reduces hydroxylation of P402 (HIF1a NODD), as well as P562 (HIF1a CODD), but to a lesser extent. Finally, BLI shows that the P317R mutation reduces affinity for CODD by 3-fold, but not NODD.
Strengths:
(1) Simple, easy-to-follow manuscript. Generally well-written.
(2) Disease-relevant mutations are studied in PHD2 that provide insights into its mechanism of action.
(3) Good, well-researched background section.
Weaknesses:
(1) Poor use of existing structural data on the complexes of PHD2 with HIF1a peptides and various metals and substrates. A quick survey of the impact of these mutations (as well as analysis by Chowdhury et al, 2016) on the structure and interactions between PHD2 peptides of HIF1a shows that the P317R mutation interferes with peptide binding. By contrast, F366L will affect the hydrophobic core, and A228S is on the surface, and it's not obvious how it would interfere with the stability of the protein.
Thank you for the comment. We have further analyzed the mutations on the available PHD2 crystal structures in complex with HIFα to discern how these substitution mutations may impact PHD2 structure and function. This analysis has been added into the discussion.
(2) To determine aggregation and monodispersity of the PHD2 mutants using size-exclusion chromatography (SEC), equal quantities of the protein must be loaded on the column. This is not what was done. As an aside, the colors used for the SEC are very similar and nearly indistinguishable.
Agreed. We have performed an additional experiment as suggested by the reviewer to further assess aggregation and hydrodynamic size. The colors used in the graph were changed for clearer differentiation between samples.
(3) The interpretation of some mutants remains incomplete. For A228S, what is the explanation for its reduced activity? It is not substantially less stable than WT and does not seem to affect peptide hydroxylation.
We agree with the reviewer that the causal mechanism for some of the tested disease-causing mutants remain unclear. The negative findings also raise the notion, perhaps considered controversial, that there may be other substrates of PHD2 that are impacted by certain mutations, which contribute to disease pathogenesis. A brief paragraph discussing this has been included in the discussion.
(4) The interpretation of the NMR prolyl hydroxylation is tainted by the high concentrations used here. First of all, there is a likely a typo in the method section; the final concentration of ODD is likely 0.18 mM, and not 0.18 uM (PNAS paper by the same group in 2024 reports using a final concentration of 230 uM). Here, I will assume the concentration is 180 uM. Flashman et al (JBC 2008) showed that the affinity of the NODD site (P402; around 10 uM) for PHD2 is 10-fold weaker than CODD (P564, around 1 uM). This likely explains the much faster kinetics of hydroxylation towards the latter. Now, using the MST data, let's say the P317R mutation reduces the affinity by 40-fold; the affinity becomes 400 uM for NODD (above the protein concentration) and 40 uM for CODD (below the protein concentration). Thus, CODD would still be hydroxylated by the P317R mutant, but not NODD.
The HIF1α concentration was indeed an oversight, which will be corrected to 0.18 mM. The study by Flashman et al.[1] showing PHD2 having a lower affinity to the NODD than CODD likely contributes to the differential hydroxylation rates via PHD2 WT. We showed here via MST that PHD2 P317R had K[d] of 320 ± 20 uM for HIF1αCODD, which should have led to a severe enzymatic defect, even at the high concentrations used for NMR (180 uM). However, we observed only a subtle reduction in hydroxylation efficiency in comparison to PHD2 WT. Thus, we performed another binding method using BLI that showed a mild binding defect on CODD by PHD2 P317R, consistent with NMR data. The perplexing result is the WT-like binding to the NODD by PHD2 P317R, which appears inconsistent with the severe defect in NODD hydroxylation via PHD2 P317R as measured via NMR. These results suggest that there are supporting residues within the PHD2/NODD interface that help maintain binding to NODD but compromise the efficiency of NODD hydroxylation upon PHD2 P317R mutation.
(5) The discrepancy between the MST and BLI results does not make sense, especially regarding the P317R mutant. Based on the crystal structures of PHD2 in complex with the ODD peptides, the P317R mutation should have a major impact on the affinity, which is what is reported by MST. This suggests that the MST is more likely to be valid than BLI, and the latter is subject to some kind of artefact. Furthermore, the BLI results are inconsistent with previous results showing that PHD2 has a 10-fold lower affinity for NODD compared to CODD.
The reviewer’s structural prediction that P317R mutation should cause a major binding defect, while agreeable with our MST data, is incongruent with our NMR and the data from Chowdhury et al.[2] that showed efficient hydroxylation of CODD via PHD2 P317R. Moreover, we have attempted to model NODD and CODD on apo PHD2 P317R structure and found that the mutation had no major impact on CODD while the mutated residue could clash with NODD, causing a shifting of peptide positioning on the protein. However, these modeling predictions, like any in silico projections, would need experimental validation. As mentioned in our preceding response, we also performed BLI, which showed that PHD2 P317R had a minor binding defect for CODD, consistent with the NMR results and findings by Chowdhury et al[2]. NODD binding was also measured with BLI as purified NODD peptides were not amenable for soluble-based MST assay, which showed similar K[d]’s for PHD2 WT and P317R. Considering the absence of NODD hydroxylation via PHD2 P317R as measured by NMR and modeling on apo PHD2 P317R, we posit that P317R causes deviation of NODD from its original orientation that may not affect binding due to the other interactions from the surrounding elements but unfortunately disallows NODD from turnover. Further study would be required to validate such notion, which we feel is beyond the scope of this manuscript.
(6) Overall, the study provides some insights into mutants inducing erythrocytosis, but the impact is limited. Most insights are provided on the P317R mutant, but this mutant had already been characterized by Chowdhury et al (2016). Some mutants affect the stability of the protein in cells, but then no mechanism is provided for A228S or F366L, which have stabilities similar to WT, yet have impaired HIF1a activation.
We thank the reviewer for raising these and other limitations. We have expanded on the shortcomings of the present study but would like to underscore that the current work using the recently described NMR assay along with other biophysical analyses suggests a previously under-appreciated role of NODD hydroxylation in the normal oxygen-sensing pathway.
Reviewer #2 (Public review):
Summary:
Mutations in the prolyl hydroxylase, PHD2, cause erythrocytosis and, in some cases, can result in tumorigenesis. Taber and colleagues test the structural and functional consequences of seven patientderived missense mutations in PHD2 using cell-based reporter and stability assays, and multiple biophysical assays, and find that most mutations are destabilizing. Interestingly, they discover a PHD2 mutant that can hydroxylate the C-terminal ODD, but not the N-terminal ODD, which suggests the importance of N-terminal ODD for biology. A major strength of the manuscript is the multidisciplinary approach used by the authors to characterize the functional and structural consequences of the mutations. However, the manuscript had several major weaknesses, such as an incomplete description of how the NMR was performed, a justification for using neighboring residues as a surrogate for looking at prolyl hydroxylation directly, or a reference to the clinical case studies describing the phenotypes of patient mutations. Additionally, the experimental descriptions for several experiments are missing descriptions of controls or validation, which limits their strength in supporting the claims of the authors.
Strengths:
(1) This manuscript is well-written and clear.
(2) The authors use multiple assays to look at the effects of several disease-associated mutations, which support the claims.
(3) The identification of P317R as a mutant that loses activity specifically against NODD, which could be a useful tool for further studies in cells.
Weaknesses:
Major:
(1) The source data for the patient mutations (Figure 1) in PHD2 is not referenced, and it's not clear where this data came from or if it's publicly available. There is no section describing this in the methods.
Clinical and patient information on disease-causing PHD2 mutants was compiled from various case reports and summarized in an excel sheet found in the Supplementary Information. The case reports are cited in this excel file. A reference to the supplementary data has been added to the Figure 1 legend and in the introduction.
(2) The NMR hydroxylation assay.
A. The description of these experiments is really confusing. The authors have published a recent paper describing a method using 13C-NMR to directly detect proly-hydroxylation over time, and they refer to this manuscript multiple times as the method used for the studies under review. However, it appears the current study is using 15N-HSQC-based experiments to track the CSP of neighboring residues to the target prolines, so not the target prolines themselves. The authors should make this clear in the text, especially on page 9, 5th line, where they describe proline cross-peaks and refer to the 15N-HSQC data in Figure 5B.
As the reviewer mentioned, the assay that we developed directly measures the target proline residues. This assay is ideal when mutations near the prolines are studied, such as A403, Y565 (He et al[3]). In this previous work, we observed that the shifting of the target proline cross-peaks due to change in electronegativity on the pyrrolidine ring of proline in turn impacted the neighboring residues[3], which meant that the neighboring residues can be used as reporter residues for certain purposes. In this study, we focused on investigating the mutations on PHD2 while leaving the sequence of the HIF-1α unchanged by using solely 15N-HSQC-based experiments without the need for double-labeled samples. Nonetheless, we thank the reviewer for pointing out the confusion in the text and we have corrected and clarified our description of this assay.
B. The authors are using neighboring residues as reporters for proline hydroxylation, without validating this approach. How well do CSPs of A403 and I566 track with proline hydroxylation? Have the authors confirmed this using their 13C-NMR data or mass spec?
For previous studies, we performed intercalated 15N-HSQC and 13C-CON experiments for the kinetic measurements of wild-type HIF-1α and mutants. We observed that the shifting pattern of A403 and I566 in the 15N-HSQC spectra aligned well with the ones of P402 and P564, respectively, in the 13C-CON spectra. Representative data has been added to Supplemental Data.
C. Peak intensities. In some cases, the peak intensities of the end point residue look weaker than the peak intensities of the starting residue (5B, PHD2 WT I566, 6 ct lines vs. 4 ct lines). Is this because of sample dilution (i.e., should happen globally)? Can the authors comment on this?
This is an astute observation by the reviewer. We checked and confirmed that for all kinetic datasets, the peak intensities of the end point residue are always slightly lower than the ones of the starting. This includes the cases for PHD2 A228S and P317R in 5B, although not as obvious as the one of PHD2 WT. We agree with the reviewer that the sample dilution is a factor as a total volume of 16 microliters of reaction components was added to the solution to trigger the reaction after the first spectrum was acquired. It is also likely that rate of prolyl hydroxylation becomes extremely slow with only a low amount of substrate available in the system. Therefore, the reaction would not be 100% complete which was detected by the sensitive NMR experimentation.
(3) Data validating the CRISPR KO HEK293A cells is missing.
We thank the reviewer for noting this oversight. Western blots validating PHD2 KO in HEK293A cells have been added to the Supplementary Data file.
(4) The interpretation of the SEC data for the PHD2 mutants is a little problematic. Subtle alterations in the elution profiles may hint at different hydrodynamic radii, but as the samples were not loaded at equal concentrations or volumes, these data seem more anecdotal, rather than definitive. Repeating this multiple times, using matched samples, followed by comparison with standards loaded under identical buffer conditions, would significantly strengthen the conclusions one could make from the data.
Agreed. We have performed an additional experiment as suggested with equal volume and concentration of each PHD2 construct loaded onto the SEC column for better assessment of aggregation. Notably, our conclusion remained unchanged.
Minor:
(1) Justification for picking the seven residues is not clearly articulated. The authors say they picked 7 mutants with "distinct residue changes", but no further rationale is provided.
Additional justification for the selection of the mutants has been added to the ‘Mutations across the PHD2 enzyme induce erythrocytosis’ section. Briefly, some mutants were chosen based on their frequency in the clinical data and their presence in potential mutational hot spots. Various mutations were noted at W334 and R371, while F366L was identified in multiple individuals. Additionally, 9 cases of PHD2-driven disease were reported to be caused from mutations located between residues 200 to 210 while 13 cases were reported between residues 369-379, so G206C and R371H were chosen to represent potential hot spots. To examine a potential genotype-phenotype relationship, two of the mutants responsible for neuroendocrine tumor development, A228S and H374R, were also selected. Finally, mutations located close or on catalytic core residues (P317R, R371H, and H374R) were chosen to test for suspected defects.
(2) A major finding of the paper is that a disease-associated mutation, P317R, can differentially affect HIF1 prolyhydroxylation, however, additional follow-up studies have not been performed to test this in cells or to validate the mutant in another method. Is it the position of the proline within the catalytic core, or the identity of the mutation that accounts for the selectivity?
This is the very question that we are currently addressing but as a part of a follow-up study. Indeed, one thought is that the preferential defect observed could be the result of the loss of proline, an exceptionally rigid amino acid that makes contact with the backbone twice, or the addition of a specific amino acid, namely arginine, a flexible amino acid with an added charge at this site. Although beyond the scope of this manuscript, we will investigate whether such and other characteristics in this region of PHD2/HIF1α interface contribute to the differential hydroxylation.
Reviewer #3 (Public review):
Summary:
This is an interesting and clinically relevant in vitro study by Taber et al., exploring how mutations in PHD2 contribute to erythrocytosis and/or neuroendocrine tumors. PHD2 regulates HIFα degradation through prolyl-hydroxylation, a key step in the cellular oxygen-sensing pathway.
Using a time-resolved NMR-based assay, the authors systematically analyze seven patient-derived PHD2 mutants and demonstrate that all exhibit structural and/or catalytic defects. Strikingly, the P317R variant retains normal activity toward the C-terminal proline but fails to hydroxylate the N-terminal site. This provides the first direct evidence that N-terminal prolyl-hydroxylation is not dispensable, as previously thought.
The findings offer valuable mechanistic insight into PHD2-driven effects and refine our understanding of HIF regulation in hypoxia-related diseases.
Strengths:
The manuscript has several notable strengths. By applying a novel time-resolved NMR approach, the authors directly assess hydroxylation at both HIF1α ODD sites, offering a clear functional readout. This method allows them to identify the P317R variant as uniquely defective in NODD hydroxylation, despite retaining normal activity toward CODD, thereby challenging the long-held view that the N-terminal proline is biologically dispensable. The work significantly advances our understanding of PHD2 function and its role in oxygen sensing, and might help in the future interpretation and clinical management of associated erythrocytosis.
Weaknesses:
(1) There is a lack of in vivo/ex vivo validation. This is actually required to confirm whether the observed defects in hydroxylation-especially the selective NODD impairment in P317R-are sufficient to drive disease phenotypes such as erythrocytosis.
We thank the reviewer for this comment, and while we agree with this statement, the objective of this study per se was to elucidate the structural and/or functional defect caused by the various diseaseassociated mutations on PHD2. The subsequent study would be to validate whether the identified defects, in particular the selective NODD impairment, would lead to erythrocytosis in vivo. However, we feel that such study would be beyond the scope of this manuscript.
(2) The reliance on HRE-luciferase reporter assays may not reliably reflect the PHD2 function and highlights a limitation in the assessment of downstream hypoxic signaling.
Agreed. All experimental assays and systems have limitations. The HRE-luciferase assay used in the present manuscript also has limitations such as the continuous expression of exogenous PHD2 mutants driven via CMV promoter. Thus, we performed several additional biophysical methodologies to interrogate the disease-causing PHD2 mutants. The limitations of the luciferase assay have been expanded in the revised manuscript.
(3) The study clearly documents the selective defect of the P317R mutant, but the structural basis for this selectivity is not addressed through high-resolution structural analysis (e.g., cryo-EM).
We thank the reviewer for the comment. While solving the structure of PHD2 P317R in complex with HIFα substrate is beyond the scope for this study, a structure of PHD2 P317R in complex with a clinically used inhibitor has been solved (PDB:5LAT). In analyzing this structure and that of PHD2 WT in complex with NODD, Chowdhury et al[2] stated that P317 makes hydrophobic contacts with LXXLAP motif on HIFα and R317 is predicted to interact differently with this motif. While this analysis does not directly elucidate the reason for the preferential NODD defect, it supports the possibility that P317R substitution may be more detrimental for enzymatic activity on NODD than CODD. We have discussed this notion in the revised manuscript.
(4) Given the proposed central role of HIF2α in erythrocytosis, direct assessment of HIF2α hydroxylation by the mutants would have strengthened the conclusions.
We thank the reviewer for this comment, but we feel that such study would be beyond the scope of the present study. We observed that the PHD2 binding patterns to HIF1α and HIF2α were similar, and we have previously assigned >95% of the amino acids in HIF1α ODD for NMR study[3]. Thus, we first focused on the elucidation of possible defects on disease-associated PHD2 mutants using HIF1α as the substrate with the supposition that an identified deregulation on HIF1α could be extended to HIF2α paralog. However, we agree with the reviewer that future studies should examine the impact of PHD2 mutants directly on HIF2α.
References:
(1) Flashman, E. et al. Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases. J Biol Chem 283, 3808-3815 (2008).
(2) Chowdhury, R. et al. Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases. Nat Commun 7, 12673 (2016).
(3) He, W., Gasmi-Seabrook, G.M.C., Ikura, M., Lee, J.E. & Ohh, M. Time-resolved NMR detection of prolyl-hydroxylation in intrinsically disordered region of HIF-1alpha. Proc Natl Acad Sci U S A 121, e2408104121 (2024).
Reviewer #1 (Recommendations for the authors):
(1) To increase the impact and significance of this work, I would recommend determining the mechanism by which A228S and F366L impair PHD2. Are these mutations affecting interactions with proteins other than HIF1a? Furthermore, does the F366L mutation affect the hydroxylation rate? This should be measured. The authors should also perform a more in-depth structural analysis of these mutations and perhaps use AlphaFold to identify how these sites may be involved in other interactions.
We thank the reviewer for the recommendations. A paragraph discussing the quandary of A228S and F366L has been added to the discussion as well as an in-depth structural analysis of each selected mutant. While AlphaFold is excellent at predicting protein structures overall, its capability to predict the effect of single point mutation, such as those in this study, is limited. Therefore, it was not utilized for this paper.
(2) For the aggregation assay, I recommended injecting the same quantity of protein on the SEC. If the aggregation-prone mutants' yields were too low, then reduced amounts of the other mutants should be injected.
Agreed. An additional experiment was performed in which similar concentrations of each mutant protein was loaded onto the SEC column and chromatograms was normalized according to the molecular concentration. Results from this experiment have been added to replace the previously performed aggregation assay. Notably, the data from the revised experiment did not change the outcome or conclusion of the study.
(3) For the NMR kinetics data, the authors should discuss the impact of affinities and concentrations on the reaction rate and incorporate this analysis framework to interpret their data.
Done. As discussed in depth in response to Public Reviewer 1’s fourth comment, we observed only a subtle reduction in hydroxylation efficiency of HIF1aCODD by PHD2 P317R in comparison to PHD2 WT. Upon performing BLI, we found PHD2 P317R displays only a mild binding defect on the CODD and NODD. The WT-like binding to the NODD by PHD2 P317R appears to be inconsistent with the severe defect in NODD hydroxylation via PHD2 P317R as measured via NMR. These results suggest that there are supporting residues within the PHD2/NODD interface that help maintain binding to NODD but compromise the efficiency of NODD hydroxylation upon PHD2 P317R mutation.
Reviewer #2 (Recommendations for the authors):
It is unclear where the source data came from describing the patient mutations, or if it is publicly available. Several minor issues were noted with several of the figures or methods:
(1) Figure 2C. It is not clear what data are being compared for significance. The lines don't seem to clearly distinguish this.
Done. The significance lines have been adjusted in the figure to better convey which data are being compared.
(2) Please incorporate the calculated biophysical constants (KD, TM, etc, average +/- std dev) from the tables into the figures or figure legends that show the data from which they are calculated.
Done. References to the corresponding tables have been added to the appropriate figure legends.
(3) Figure 3C, the data for F366L do not appear normalized in the same way as the other constructs.
CD melt values for F366L were normalized in the same way as other constructs but due to noisier data acquired between 25-37°C, the top value of the sigmoidal curve is slightly higher than the other constructs (F366L: 1.066, WT: 1.007, A228S: 1.000, P317R: 1.015, R371H: 1.005).
(4) For Figure 1B, it would be helpful to highlight the mutants characterized in the current study with a different color/symbol to help show the number of cases.
Done. Dots representing the selected mutants have been highlighted in red in Figure 1B.
(5) A description of the isotopic labeling of PHD2 is missing from the methods.
Due to the nature of the NMR assay, no isotopic labeling was required for PHD2.
Reviewer #3 (Recommendations for the authors):
(1) To further strengthen the manuscript, the authors could consider exploring the relevance of their in vitro findings in a more physiological context.
We thank the reviewer for the suggestion, and we will certainly consider furthering our investigation in a more physiological context for future studies.
(2) If technically feasible, integrating direct analyses of HIF2α regulation by the PHD2 mutants would better reflect the clinical phenotype, given the known importance of HIF2α in erythrocytosis.
We agree that HIF2α is important in the context of erythrocytosis, but through MST we observed no difference in binding pattern between HIF1 and HIF2 and the selected PHD2 mutants. As we had previously assigned >95% of residues for HIF1α ODD for NMR assay, we analyzed HIF1 with the supposition that any defects observed would likely apply to HIF2. However, we agree that future studies on the impact of PHD2 mutants directly on HIF2 would be beneficial to supplement our understanding of pseudohypoxic disease.
(3) Additionally, although perhaps more suitable for future work or discussion, structural modeling or highresolution structural studies of the P317R variant could offer valuable insight into the observed NODD selectivity defect.
We thank the reviewer for the suggestion. While solving the structure of PHD2 P317R in complex with NODD is beyond the scope of this manuscript, a crystal structure of PHD2 P317R in complex with an inhibitor has been solved and insights from this structure have been added to the discussion.
(4) Finally, a brief clarification or discussion of the limitations of the luciferase reporter assay-especially in the context of aggregation-prone mutants-would help readers better interpret the functional data.
We thank the reviewer for the suggestion. The limitations of the luciferase reporter assay in regard to its inability to detect defects with aggregation-prone mutants have been elaborated on in the discussion.
Author response:
The following is the authors’ response to the previous reviews
Reviewer #2 (Public Review):
Summary:
The authors show that a combination of arginine methyltransferase inhibitors synergize with PARP inhibitors to kill ovarian and triple negative cancer cell lines in vitro and in vivo using preclinical mouse models.
Strengths and weaknesses
The experiments are well-performed, convincing and have the appropriate controls (using inhibitors and genetic deletions) and use statistics.
They identify the DNA damage protein ERCC1 to be reduced in expression with PRMT inhibitors. As ERCC1 is known to be synthetic lethal with PARPi, this provides a mechanism for the synergy. They use cell lines only for their study in 2D as well as xenograph models.
We sincerely thank Reviewer #2 for the insightful and constructive feedback, as well as for the kind recognition of the scientific quality of our work: “The experiments are well-performed, convincing and have the appropriate controls (using inhibitors and genetic deletions) and use statistics.” We sincerely thank Reviewer #2 for their thoughtful and constructive comments during both rounds of review, which have significantly improved the quality of our manuscript. In response, we have incorporated new results from additional experiments into the figures (Figures 6M and 6N) and made comprehensive revisions throughout the text, figures, and supplementary materials. Following the reviewer’s valuable suggestions, we also revised the Discussion section. In the “Recommendations for the authors” sections, we have provided detailed point-by-point responses to each comment, which were instrumental in guiding our revisions. We believe these updates have substantially strengthened the manuscript and fully addressed all reviewer concerns.
Reviewer #2 (Recommendations for the authors):
Although the authors have addressed each recommendation from the reviewer, further revision of the manuscript are still necessary, as outlined below.
Add these additional comments in the text to further enhance the comprehension and clarity of the data.
(1) If the authors kept the tumors of various sizes in Figure 7I, it would be important to assess the protein and/or mRNA level of ERCC1 to further support their mechanism.
Question (1): Please add the figures of new experiments (treatment diagram, curves for tumor volume and qRT-PCR data) to Figure 6.
We thank the reviewers for their constructive suggestions. In response to the reviewers’ comments, we have added the treatment diagram and qPCR results to Figure 6. In this experiment, we shortened the treatment duration to seven days to assess early molecular responses to therapy rather than downstream effects. As expected, such short-term treatment did not result in significant differences in tumor growth among groups. The new results are now presented in Figure 6, panels M and N. The corresponding results and figure legends will also be included in the revised version of the manuscript
(2) Figure 2G: please explain why two bands remain for sgPRMT1.
Question (2): In the answer, the authors stated, "Upon knockdown of the major isoforms by CRISPR/Cas9, expression of this minor isoform may have increased as part of a compensatory feedback mechanism, rendering it detectable by immunoblotting." Please put the statement into the discussion section.
We sincerely thank the reviewers for their thoughtful and constructive suggestions. In response to these comments, we have carefully revised the manuscript and incorporated the corresponding information into the Discussion section to provide greater clarity and context for our findings.
(3) (Previously point 5) What is the link with ERCC1 splicing because reduced overall ERCC1 expression is clear?
Question (5): Please add the explanation you provide of links between ERCC1 splicing and PRMTi into the discussion section.
"Furthermore, as shown in Figure 4G, we observed a reduction in the total ERCC1 mRNA reads following PRMTi treatment. This decrease may be attributed, at least in part, to the instability of the alternatively spliced ERCC1 transcripts, which could be more prone to degradation. In combination with the transcriptional downregulation of ERCC1 induced by PRMT inhibition, these alternative splicing events may lead to a further reduction in functional ERCC1 protein levels. This dual impact on ERCC1 expression, through both decreased transcription and the generation of unstable or nonfunctional isoforms, likely contributes to the enhanced cellular sensitivity to PARP inhibitors observed in our study."
We sincerely thank the reviewers for their thoughtful and constructive suggestions. In response to these comments, we have carefully revised the manuscript and incorporated the corresponding information into the Discussion section to provide greater clarity and context for our findings.
(4) (Previously 6) Figure 7J: From the graph, it seems like Olaparib+G715 and G715+G025 have a similar effect on tumor volume (two curves overlap). Please discuss.
Question (6): In the answer, the authors stated, "Our in vitro and in vivo findings, together with previously published data, consistently demonstrate that GSK715 is more potent than both GSK025 and Olaparib. Notably, treatment with GSK715 alone led to significantly greater inhibition of tumor growth compared to either GSK025 or Olaparib administered individually. This higher potency of GSK715 also explains the comparable levels of tumor suppression observed in the combination groups, including GSK715 plus Olaparib and GSK715 plus GSK025. These results suggest that GSK715 is likely the primary driver of efficacy in the two drug combination settings." Please put the statement in the corresponding result section for Figure 6J.
We sincerely thank the reviewers for their thoughtful and constructive suggestions. In response to these comments, we have carefully revised the manuscript and incorporated the corresponding information into the result section for Figure 6J to provide greater clarity and context for our findings.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
In this manuscript entitled "Molecular dynamics of the matrisome across sea anemone life history", Bergheim and colleagues report the prediction, using an established sequence analysis pipeline, of the "matrisome" - that is, the compendium of genes encoding constituents of the extracellular matrix - of the starlet sea anemone Nematostella vectensis. Re-analysis of an existing scRNA-Seq dataset allowed the authors to identify the cell types expressing matrisome components and different developmental stages. Last, the authors apply time-resolved proteomics to provide experimental evidence of the presence of the extracellular matrix proteins at three different stages of the life cycle of the sea anemone (larva, primary polyp, adult) and show that different subsets of matrisome components are present in the ECM at different life stages with, for example, basement membrane components accompanying the transition from larva to primary polyp and elastic fiber components and matricellular proteins accompanying the transition from primary polyp to the adult stage.
Strengths:
The ECM is a structure that has evolved to support the emergence of multicellularity and different transitions that have accompanied the complexification of multicellular organisms. Understanding the molecular makeup of structures that are conserved throughout evolution is thus of paramount importance.
The in-silico predicted matrisome of the sea anemone has the potential to become an essential resource for the scientific community to support big data annotation efforts and understand better the evolution of the matrisome and of ECM proteins, an important endeavor to better understand structure/function relationships. This study is also an excellent example of how integrating datasets generated using different -omic modalities can shed light on various aspects of ECM metabolism, from identifying the cell types of origins of matrisome components using scRNA-Seq to studying ECM dynamics using proteomics.
We greatly appreciate the positive feedback regarding the design of our study and the evolutionary significance of our findings.
Weaknesses:
My concerns pertain to the three following areas of the manuscript:
(1) In-silico definition of the anemone matrisome using sequence analysis:
a) While a similar computational pipeline has been applied to predict the matrisome of several model organisms, the authors fail to provide a comprehensive definition of the anemone matrisome: In the text, the authors state the anemone matrisome is composed of "551 proteins, constituting approximately 3% of its proteome (see page 6, line 14), but Figure 1 lists 829 entries as part of the "curated" matrisome, Supplementary Table S1 lists the same 829 entries and the authors state that "Here, we identified 829 ECM proteins that comprise the matrisome of the sea anemone Nematostella vectensis" (see page 17, line 10). Is the sea anemone matrisome composed of 551 or 829 genes? If we refer to the text, the additional 278 entries should not be considered as part of the matrisome, but what is confusing is that some are listed as glycoproteins and the "new_manual_annotation" proposed by the authors and that refer to the protein domains found in these additional proteins suggest that in fact, some could or should be classified as matrisome proteins. For example, shouldn't the two lectins encoded by NV2.3951 and NV2.3157 be classified as matrisome-affiliated proteins? Based on what has been done for other model organisms, receptors have typically been excluded from the "matrisome" but included as part of the "adhesome" for consistency with previously published matrisome; the reviewer is left wondering whether the components classified as "Other" / "Receptor" should not be excluded from the matrisome and moved to a separate "adhesome" list.
In addition to receptors, the authors identify nearly 70 glycoproteins classified as "Other". Here, does other mean "non-matrisome" or "another matrisome division" that is not core or associated? If the latter, could the authors try to propose a unifying term for these proteins? Unfortunately, since the authors do not provide the reasons for excluding these entries from the bona fide matrisome (list of excluding domains present, localization data), the reader is left wondering how to treat these entries.
Overall, the study would gain in strength if the authors could be more definitive and, if needed, even propose novel additional matrisome annotations to include the components for now listed as "Other" (as was done, for example, for the Drosophila or C. elegans matrisomes).
The reviewer is correct to point out the confusing terminology used throughout our manuscript, where both the total of 829 proteins constituting the curated list of ECM domain proteins and the actual matrisome (excluding "others") were referred to as "matrisomes". In general, we followed the example set by Naba & Hynes in their 2012 paper (Mol Cell Proteomics. 2012 Apr;11(4):M111.014647. doi: 10.1074/mcp.M111.014647), where they define the "matrisome" as encompassing all components of the extracellular matrix ("core matrisome") and those associated with it ("matrisome-associated" proteins). This corresponds to our group of 551 proteins, comprising both core matrisome and matrisomeassociated proteins. The Naba & Hynes paper also contains the inclusive and exclusive domain lists for the matrisome that we applied for our dataset. In the revised manuscript, we have now labelled the group of 829 proteins as "curated ECM domain proteins/genes", which includes all proteins positively selected for containing a bona fide ECM domain. After excluding non-matrisomal proteins such as receptors, we arrive at the 551 proteins that constitute the "Nematostella matrisome". We have maintained this terminology throughout the revised manuscript and have revised Figures 1B and 4B accordingly.
Regarding the category of "other" proteins, which by definition are not part of the matrisome although containing ECM domains, we have taken the reviewer's advice and classified these in more detail. We categorized all receptors as "adhesome" (202 proteins). The remaining group of “other” secreted ECM domain proteins were then further subcategorized. Those exhibiting significant matches in the ToxProt database were subclassified as "putative venoms" (15 proteins). This group also includes the two lectins (NV2.3951 and NV2.3157), which had been originally shifted to the “other” category due to their classification as venoms. We categorized as “adhesive proteins” (28 proteins) factors such as coadhesins that due to their domain architecture resemble bioadhesive proteins described in proteomic studies of other invertebrate species, such as corals or sponges (see also https://doi.org/10.1016/j.jprot.2022.104506). Further sub-categories are stress/injury response proteins (9 proteins) and ion channels (6 proteins). The remaining 17 proteins were categorized as “uncharacterized ECM domain proteins”. These include highly diverse proteins possessing either single ECM domains or novel domain combinations. We decided to retain those in our dataset as candidates for future functional characterization.
b) It is surprising that the authors are not providing the full currently accepted protein names to the entries listed in Supplementary Table S1 and have used instead "new_manual_annotation" that resembles formal protein names. This liberty is misleading. In fact, the "new_manual_annotation" seems biased toward describing the reason the proteins were positively screened for through sequence analysis, but many are misleading because there is, in fact, more known about them, including evidence that they are not ECM proteins. The authors should at least provide the current protein names in addition to their "new_manual_annotations".
c) To truly serve as a resource, the Table should provide links to each gene entry in the Stowers Institute for Medical Research genome database used and some sort of versioning (this could be added to columns A, B, or D). Such enhancements would facilitate the assessment of the rigor of the list beyond the manual QC of just a few entries.
d) Since UniProt is the reference protein knowledge database, providing the UniProt IDs associated with the predicted matrisome entries would also be helpful, giving easy access to information on protein domains, protein structures, orthology information, etc.
e) In conclusion, at present, the study only provides a preliminary draft that should be more rigorously curated and enriched with more comprehensive and authoritative annotations if the authors aspire the list to become the reference anemone matrisome and serve the community.
Table S1 has been updated to include links to the respective Stowers Institute IDs (first two columns), as well as SwissProt IDs and current descriptions from both the Stowers Institute (SI) and Swissprot.
In our manual annotations, we prioritized these over automated ones due to the considerable effort invested in examining each sequence individually. The cnidaria-specific minicollagens and NOWA proteins might serve as an example. According to the SI descriptions, the minicollagens are annotated as “keratin-associated protein, predicted or hypothetical protein, collagen-like protein and pericardin”. We classified these as minicollagens on the basis of overall domain architecture and of signature domains and sequence motifs, such as minicollagen cysteine-rich domains (CRDs) and polyproline stretches (doi: 10.1016/j.tig.2008.07.001). NOWA is a CTLD/CRD-containing protein that is part of nematocyst tubules (doi:10.1016/j.isci.2023.106291). The first two NOWA isoforms, according to Si descriptions, were annotated as aggrecan and brevican core proteins, which is very misleading. We therefore feel that our manual annotations better serve the cnidarian research community in classifying these proteins.
Automated annotations of ECM proteins often rely on similarities between individual domains, neglecting overall domain composition. For example, Swissprot descriptions annotate 31 TSP1 domain-containing proteins in our list as "Hemicentin-1", but closer inspection reveals that only one sequence (NV2.24790) qualifies as Hemicentin-1 due to its characteristic vWFA, Ig-like, TSP1, G2 nidogen, and EGF-like domain architecture. Regarding novel protein annotations, NV2.650 might serve as an example. While SI descriptions annotate this protein as "epidermal growth factor" based on the presence of several EGF-like domains, our analysis reveals two integrin alpha N-terminal domains that classify this sequence as integrin-related. We have therefore assigned a description (Secreted integrin-N-related protein) that references this defining domain and avoids misclassification within the EGF family.
In cases where the automated annotation (including those in Genbank) matched our own findings, we adopted the existing description, as seen with netrin-1 (NV2.7734). We acknowledge that our manual annotations are not flawless and will be refined by future research. Nonetheless, we offer them as an approximation to a more accurate definition of the identified protein list.
(2) Proteomic analysis of the composition of the mesoglea during the sea anemone life cycle:
a) The product of 287 of the 829 genes proposed to encode matrisome components was detected by proteomics. What about the other ~550 matrisome genes? When and where are they expressed? The wording employed by the authors (see line 11, page 13) implies that only these 287 components are "validated" matrisome components. Is that to say that the other ~550 predicted genes do not encode components of the ECM? This should be discussed.
Obviously, our wording was not sufficiently accurate here. In the revised Fig. 1B we indicated that 210 of the 551 matrisome (core and associated) proteins were confirmed by mass spectrometry. In total, 287 proteins were identified by mass spectrometry, meaning that 77 of those are non-matrisomal proteins belonging to the “adhesome” (47) and “other” (30) groups. The fact that the remaining 542 proteins of the matrisome predicted by our in silico analysis could not be identified has two major reasons: (1) Our study was focussed on the molecular dynamics of the mesoglea. Therefore, only mesogleas were isolated for the mass spectrometry analysis and nematocysts were mostly excluded by extensive washing steps. As nematocysts contribute significantly to the predicted matrisome, this group of proteins is underrepresented in the mass spectrometry analysis. (2) A significant fraction of the predicted ECM proteins constitutes soluble factors and transmembrane receptors. These might not be necessarily part of the mesoglea isolates. In addition, the isolation and solubilization method we applied might have technical limitations. Although we used harsh conditions for solubilizing the mesoglea samples (90°C and high DTT concentrations), we cannot exclude that we missed proteins which resisted solubilization and thus trypsinization. We confirmed that all genes predicted by the in silico analysis have transcriptomic profiles as demonstrated in supplementary table S4. We have clarified these points in the revised results part (p.6) and also revised the statement in line 16, page 13.
b) Can the authors comment on how they have treated zero TMT values or proteins for which a TMT ratio could not be calculated because unique to one life stage, for example?
We did not include these proteins in the analysis of the respective statistical comparison. This involved only very few proteins (about 10).
c) Could the authors provide a plot showing the distribution of protein abundances for each matrisome category in the main figure 4? In mammals, the bulk of the ECM is composed of collagens, followed by fibrillar ECM glycoproteins, the other matrisome components being more minor. Is a similar distribution observed in the sea anemone mesoglea?
We have included such a plot showing protein abundances across life stages and protein categories (Fig. 4A). Collagens and basement membrane proteoglycans (perlecan) are the most abundant protein categories in the core matrisome while secreted factors dominate in the matrisome-associated group.
d) Prior proteomic studies on the ECM of vertebrate organisms have shown the importance of allowing certain post-translational modifications during database search to ensure maximizing peptide-to-spectrum matching. Such PTMs include the hydroxylation of lysines and prolines that are collagen-specific PTMs. Multiple reports have shown that omitting these PTMs while analyzing LC-MS/MS data would lead to underestimating the abundance of collagens and the misidentification of certain collagens. The authors may want to reanalyze their dataset and include these PTMs as part of their search criteria to ensure capturing all collagen-derived peptides.
Thank you for this suggestion. We have re-analyzed our dataset including lysine and proline hydroxylation as PTM. While we obtained in total 70 more proteins using this approach, this additional group did not contain any large collagen or minicollagen we had not detected before. We only obtained two additional collagen-like proteins with very short triple helical domains (V2t013973001.1, NV2t024002001.1), one being a fragment. We don’t feel this justifies implementing a re-analysis of the proteome in our study.
e) The authors should ensure that reviewers are provided with access to the private PRIDE repository so the data deposited can also be evaluated. They should also ensure that sufficient meta-data is provided using the SRDF format to allow the re-use of their LCMS/MS datasets.
We apologize for not providing the reviewer access in our initial submission and have asked the editorial office to forward the PRIDE repository link to all reviewers immediately after receiving the reviews. We did upload a metadata.csv file with the proteomics dataset. This file contains an annotation of all TMT labels to the samples and conditions and replicates used in the manuscript. It contains similar information as an SRDF format file. In addition, the search output files on protein and psm level have been provided. So, from our point of view, we provided all necessary information to reproduce the analysis.
(3) Supplementary tables:
The supplementary tables are very difficult to navigate. They would become more accessible to readers and non-specialists if they were accompanied by brief legends or "README" tabs and if the headers were more detailed (see, for example, Table S2, what does "ctrl.ratio_Larvae_rep2" exactly refer to? Or Table S6 whose column headers using extensive abbreviations are quite obscure). Similarly, what do columns K to BX in Supplementary Table S1 correspond to? Without more substantial explanations, readers have no way of assessing these data points.
We have revised the tables and removed any redundant data columns. We also included detailed explanations of the used abbreviations, both in the headers and in a separate README file. Some of the information was apparently lost during the conversion to pdf files. We will therefore upload the original .xls files when submitting the revised manuscript.
Reviewer #2 (Public review):
This work set out to identify all extracellular matrix proteins and associated factors present within the starlet sea anemone Nematostella vectensis at different life stages. Combining existing genomic and transcriptomic datasets, alongside new mass spectometry data, the authors provide a comprehensive description of the Nematostella matrisome. In addition, immunohistochemistry and electron microscopy were used to image whole mount and decellularized mesoglea from all life stages. This served to validate the de-cellularization methods used for proteomic analyses, but also resulted in a very nice description of mesoglea structure at different life stages. A previously published developmental cell type atlas was used to identify the cell type specificity of the matrisome, indicating that the core matrisome is predominantly expressed in the gastrodermis, as well as cnidocytes. The analyses performed were rigorous and the results were clear, supporting the conclusions made by the authors.
Thank you. We greatly appreciate the positive assessment of our study.
Reviewer #3 (Public review):
Summary:
This manuscript by Bergheim et al investigates the molecular and developmental dynamics of the matrisome, a set of gene products that comprise the extracellular matrix, in the sea anemone Nematostella vectensis using transcriptomic and proteomic approaches. Previous work has examined the matrisome of the hydra, a medusozoan, but this is the first study to characterize the matrisome in an anthozoan. The major finding of this work is a description of the components of the matrisome in Nematostella, which turns out to be more complex than that previously observed in hydra. The authors also describe the remodeling of the extracellular matrix that occurs in the transition from larva to primary polyp, and from primary polyp to adult. The authors interpret these data to support previously proposed (Steinmetz et al. 2017) homology between the cnidarian endoderm with the bilaterian mesoderm.
Strengths:
The data described in this work are robust, combining both transcriptome and proteomic interrogation of key stages in the life history of Nematostella, and are of value to the community.
Thank you for your positive assessment of our dataset.
Weaknesses:
The authors offer numerous evolutionary interpretations of their results that I believe are unfounded. The main problem with extending these results, together with previous results from hydra, into an evolutionary synthesis that aims to reconstruct the matrisome of the ancestral cnidarian is that we are considering data from only two species. I agree with the authors' depiction of hydra as "derived" relative to other medusozoans and see it as potentially misleading to consider the hydra matrisome as an exemplar for the medusozoan matrisome. Given the organismal and morphological diversity of the phylum, a more thorough comparative study that compares matrisome components across a selection of anthozoan and medusozoan species using formal comparative methods to examine hypotheses is required.
Specifically, I question the author's interpretation of the evolutionary events depicted in this statement:
"The observation that in Hydra both germ layers contribute to the synthesis of core matrisome proteins (Epp et al. 1986; Zhang et al. 2007) might be related to a secondary loss of the anthozoan-specific mesenteries, which represent extensions of the mesoglea into the body cavity sandwiched by two endodermal layers."
Anthozoans and medusozoans are evolutionary sisters. Therefore, the secondary loss of "anthozoan-like mesenteries" in hydrozoans is at least as likely as the gain of this character state in anthozoans. By extension, there is no reason to prefer the hypothesis that the state observed in Nematostella, where gastroderm is responsible for the synthesis of the core matrisome components, is the ancestral state of the phylum. Moreover, the fossil evidence provided in support of this hypothesis (Ou et al. 2022) is not relevant here because the material described in that work is of a crown group anthozoan, which diversified well after the origin of Anthozoa. The phylogenetic structure of Cnidaria has been extensively studied using phylogenomic approaches and is generally well supported (Kayal et al. 2018; DeBiasse et al. 2024). Based on these analyses, anthozoans are not on a "basal" branch, as the authors suggest. The structure of cnidarian phylogeny bifurcates with Anthozoa forming one clade and Medusozoa forming the other. From the data reported by Bergheim and coworkers, it is not possible to infer the evolutionary events that gave rise to the different matrisome states observed in Nematostella (an anthozoan) and hydra (a medusozoan). Furthermore, I take the observation in Fig 5 that anthozoan matrisomes generally exhibit a higher complexity than other cnidarian species to be more supportive of a lineage-specific expansion of matrisome components in the Anthozoa, rather than those components being representative of an ancestral state for Cnidaria. Whatever the implication, I take strong issue with the statement that "the acquisition of complex life cycles in medusozoa, that are distinguished by the pelagic medusa stage, led to a secondary reduction in the matrisome repertoire." There is no causal link in any of the data or analyses reported by Bergheim and co-workers to support this statement and, as stated above, while we are dealing with limited data, insufficient to address this question, it seems more likely to me that the matrisome expanded in anthozoans, contrasting with the authors' conclusions. While the discussion raises many interesting evolutionary hypotheses related to the origin of the cnidarian matrisome, which is of vital interest if we are to understand the origin of the bilaterian matrisome, a more thorough comparative analysis, inclusive of a much greater cnidarian species diversity, is required if we are to evaluate these hypotheses.
DeBiasse MB, Buckenmeyer A, Macrander J, Babonis LS, Bentlage B, Cartwright P, Prada C, Reitzel AM, Stampar SN, Collins A, et al. 2024. A Cnidarian Phylogenomic Tree Fitted With Hundreds of 18S Leaves. Bulletin of the Society of Systematic Biologists [Internet] 3. Available from: https://ssbbulletin.org/index.php/bssb/article/view/9267
Epp L, Smid I, Tardent P. 1986. Synthesis of the mesoglea by ectoderm and endoderm in reassembled hydra. J Morphol [Internet] 189:271-279. Available from: https://pubmed.ncbi.nlm.nih.gov/29954165/
Kayal E, Bentlage B, Sabrina Pankey M, Ohdera AH, Medina M, Plachetzki DC, Collins AG, Ryan JF. 2018. Phylogenomics provides a robust topology of the major cnidarian lineages and insights on the origins of key organismal traits. BMC Evol Biol [Internet] 18:1-18. Available from: https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-018-1142-0
Ou Q, Shu D, Zhang Z, Han J, Van Iten H, Cheng M, Sun J, Yao X, Wang R, Mayer G. 2022. Dawn of complex animal food webs: A new predatory anthozoan (Cnidaria) from Cambrian. The Innovation 3:100195
Steinmetz PRH, Aman A, Kraus JEM, Technau U. 2017. Gut-like ectodermal tissue in a sea anemone challenges germ layer homology. Nature Ecology & Evolution 2017 1:10 [Internet] 1:1535-1542. Available from: https://www.nature.com/articles/s41559-017-0285-5
Zhang X, Boot-Handford RP, Huxley-Jones J, Forse LN, Mould AP, Robertson DL, Li L, Athiyal M, Sarras MP. 2007. The collagens of hydra provide insight into the evolution of metazoan extracellular matrices. J Biol Chem [Internet] 282:6792-6802. Available from: https://pubmed.ncbi.nlm.nih.gov/17204477/
We agree with the reviewer that only the analysis of several additional anthozoan and medusozoan representatives will yield a valid basis for a reconstruction of the ancestral cnidarian matrisome and allow statements about ancestral or novel features within the phylum. We have therefore revised our statements in the discussion part of the manuscript by implementing the cited literature and also findings from medusozoan genome analysis (e.g. Gold et al., 2018) demonstrating that changes in gene content are as common in the anthozoans as in medusozoans, which questioned the previously stated “basal” state of Nematostella or of anthozoans in general.
Reviewer #1 (Recommendations for the authors):
(1) In Figure 2A, an "o" is missing in the labeling of the "developing cnidcytes" population.
Thank you, we have corrected the typo.
(2) It would be helpful to have the different life stages indicated as headers of the heat maps presented in Figure 4.
We have included symbolic representations for the different life stages on top of the heat maps in addition to the respective labels at the bottom.
Reviewer #2 (Recommendations for the authors):
Important changes:
(1) Figure 2B The x-axis tissue names should be changed to something more easily readable/understandable - some are clear, but others are not. Perhaps abbreviations could be expanded in the legend.
We have expanded the legend in Fig. 2B to render it more easily readable. We have also rotated the maps in A to have them aligned with the ones in Fig.3B.
(2) Figure 3B This figure would be improved by the inclusion of cluster names, to understand better the mapping.
We have added relevant cluster names to Fig. 3B and as stated above aligned the orientation of the maps in Fig. 2B and Fig. 3B.
(3) Figure 3C As with 2B, I find the y-axis cnidocyte cell state names to be unclear at times. Perhaps abbreviations could be expanded in the legend.
All abbreviations were expanded in Fig.3C axis labels.
(4) Many of the supplementary tables are not well exported or easily readable as is (gene names are truncated, headers truncated, etc), which means that they may not be easily usable by researchers in the field interested in following up on this work in other contexts. Indeed, to be more usable, please consider sharing these supplementary data as .csv files, for example, instead of as .pdfs.
We are sorry for this inconvenience, which was obviously caused by the conversion to pdf files. We will upload the original csv files when submitting the revised manuscript.
Smaller nitpicky comments:
(5) Page 2 line 4 & page 3 line 7: Please consider a term other than "pre-bilaterian". The drawing/ordering of a phylogeny of extant species is not meaningful in terms of more or less ancestral. e.g. if the tips are flipped in the drawing of the tree, can we say that bilaterians are pre-cnidarians? What does that mean?
We have used that term on the basis that cnidarians existed before the appearance of bilaterians according to the fossil record and molecular phylogenies (McFadden et al., 2021; Adoutte et al., 2000;Cavalier-Smith et al., 1996; Collins, 1998; Kim et al., 1999; Medina et al., 2001; Wainright et al., 1993). To acknowledge remaining uncertainties in the timing of origin of animals, we will use the term “early-diverging metazoans” instead, which is widely accepted in the cnidarian community.
(6) Page 3 line 9 I was confused by the use of "gastrula-shaped body" to describe cnidarians, which are on the whole very morphologically diverse and don't all resemble gastrulae (that can also be quite diverse).
This term is sometimes used to refer to the diploblastic cnidarian body plan (outer ectoderm, inner endoderm) with a mouth that corresponds to the blastopore. To avoid misunderstandings, we changed it in the revised manuscript to “Cnidarians, the sister group to bilaterians, are characterized by a simple body plan with a central body cavity and a mouth opening surrounded by tentacles.”
Reviewer #3 (Recommendations for the authors):
(1) In general, I felt there was a lot of discussion about protein structure and diversity that is difficult to follow without a figure. I think some of the information in Supplementary Figures S5, S9, and S11 should be in the main figures.
Following the reviewer’s suggestion, we have integrated Fig. S5 (collagens) into the main Fig. 2 and Fig. S9 (polydoms) into Fig. 4. As metalloproteases are not extensively discussed in the manuscript (and also due to the large size of the figure) we have kept Fig. S11 as a supplementary figure.
(2) Page 3, Line 7: The use of the term "pre-bilaterian" is inappropriate. Cnidarians and bilaterians are evolutionary sisters. Therefore, each lineage derives from the same split and is the same age. The cnidarian lineage is not older than the bilaterian lineage.
Following a similar request by reviewer 2 we have replaced this term by “early diverging metazoans”.
(3) Page 5, Line 10. How were in silico matrisomes from early-branching metazoan species predicted?
We applied the same bioinformatic pipeline as for the Nematostella matrisome. We clarified this in the respective methods part.
(4) Page 16, Line 8: This should be Thus.
Obviously, the wording of this sentence was ambiguous. We changed it to ”In contrast, the adult mesoglea is significantly enriched in elastic fiber components, such as fibrillins and fibulin. This compositional shift likely adds to the visco-elastic properties (Gosline 1971a, b) of the growing body column (Fig. 4B,D, supplementary table S7).”
Reviewer #3 (Public review):
Summary:
In this article, Toshniwal et al. investigate the role of pyruvate metabolism in controlling cell growth. They find that elevated expression of the mitochondrial pyruvate carrier (MPC) leads to decreased cell size in the Drosophila fat body, a transformed human hepatocyte cell line (HepG2), and primary rat hepatocytes. Using genetic approaches and metabolic assays, the authors find that elevated pyruvate import into cells with forced expression of MPC increases the cellular NADH/NAD+ ratio, which drives the production of oxaloacetate via pyruvate carboxylase. Genetic, pharmacological, and metabolic approaches suggest that oxaloacetate is used to support gluconeogenesis rather than amino acid synthesis in cells over-expressing MPC. The reduction in cellular amino acids impairs protein synthesis, leading to impaired cell growth.
Strengths:
This study shows that the metabolic program of a cell, and especially its NADH/NAD+ ratio, can play a dominant role in regulating cell growth.
The combination of complementary approaches, ranging from Drosophila genetics to metabolic flux measurements in mammalian cells, strengthens the findings of the paper and shows a conservation of MPC effects across evolution.
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We thank the editor and the reviewers for their positive and constructive comments. Below is our point-by-point responses.
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from simple steatosis, steatohepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma. In the current study, the authors aimed to determine the early molecular signatures differentiating patients with MASLD associated fibrosis from those patients with early MASLD but no symptoms. The authors recruited 109 obese individuals before bariatric surgery. They separated the cohorts as no MASLD (without histological abnormalities) and MASLD. The liver samples were then subjected to transcriptomic and metabolomic analysis. The serum samples were subjected to metabolomic analysis. The authors identified dysregulated lipid metabolism, including glyceride lipids, in the liver samples of MASLD patients compared to the no MASLD ones. Circulating metabolomic changes in lipid profiles slightly correlated with MASLD, possibly due to the no MASLD samples derived from obese patients. Several genes involved in lipid droplet formation were also found elevated in MASLD patients. Besides, elevated levels of amino acids, which are possibly related to collagen synthesis, were observed in MASLD patients. Several antioxidant metabolites were increased in MASLD patients. Furthermore, dysregulated genes involved in mitochondrial function and autophagy were identified in MASLD patients, likely linking oxidative stress to MASLD progression. The authors then determined the representative gene signatures in the development of fibrosis by comparing this cohort with the other two published cohorts. Top enriched pathways in fibrotic patients included GTPase signaling and innate immune responses, suggesting the involvement of GTPase in MASLD progression to fibrosis. The authors then challenged human patient derived 3D spheroid system with a dual PPARa/d agonist and found that this treatment restored the expression levels of GTPase-related genes in MASLD 3D spheroids. In conclusion, the authors suggested the involvement of upregulated GTPase-related genes during fibrosis initiation. Overall, the current study might provide some resources regarding transcriptomic and metabolomic data derived from obese patients with and without MASLD. However, several concerns should be carefully addressed.
The authors recruited 109 patients but only performed transcriptomic and metabolomic analysis in 94 liver samples. Why did the authors exclude other samples?
We thank the reviewer for their question and we understand the confusion. The discrepancy in sample size between liver and plasma cohorts is due to the fact that, for certain cases, we were unable to get sufficient liver tissue slices (“Exclusion criteria included: age The authors mentioned clinical data in Table 1 but did not present the table in this manuscript.
Table 1 (key patient characteristics) was included in the main document after the Methods section, and Table S1 (additional patient characteristics) was provided as a supplemental file in our original submission.
The generated metabolomic data could be a very useful resource to the MASLD community. However, it is very confusing how the data was generated in those supplemental tables. There is no clear labeling of human clinical information in those tables. Also, what do those values mean in columns 47-154? This reviewer assumed that they are the raw data of metabolomic analysis in plasma samples. However, without clear clinical information in these patients, it is impossible that any scientist can use the data to reproduce the authors' findings.
We appreciate this suggestion. To ensure accessibility of the data resources, we created a GitHub repository for both data and code, available at https://github.com/SLINGhub/MASLD_dual_omics____.
The GitHub repository includes clinical data for all 109 participants with patient characteristics and histological gradings, as well as processed omics data (log₂-transformed). We have generated artificial IDs for each patient so that we can include all the requested data in an organized manner. A code template is also provided to replicate the main statistical results from this study. In addition, for readers interested in conducting analyses from the raw data, we have deposited the raw sequencing files and mass spectrometry data in GEO and Zenodo, as detailed in the ‘Data Availability’ section.
In Fig. 5B, the authors excluded the steatosis and fibrosis overlapped genes. Steatosis and fibrosis specific genes could simply reflect the outcomes rather than causes. In this case, the obtained results might not identify the gene signatures related to fibrosis initiation.
We appreciate this comment, but we do not fully understand the reviewer’s point since we did not exclude overlapped genes in our analysis, and it was unclear to us whether excluding overlapping genes has anything to do with causality of both processes.
In Figure 5B, we identified the gene signatures associated with steatosis and fibrosis after adjusting for potential confounders such as age, sex, BMI and diabetes status. Our results showed that these signatures were relatively independent, sharing a limited number of genes. We then examined genes uniquely associated with each process by additional adjustment (e.g., adjusting steatosis models for fibrosis grades). To us this was not an unreasonable approach, given that steatosis precedes fibrosis in most cases, especially in morbid obesity.
We nevertheless agree with the reviewer’s point that the gene expression changes we identified represent statistical associations without warranting causality. To specifically address fibrosis initiation mechanisms within the limitation of the current study design, we performed a separate comparative analysis between patients with fibrosis+steatosis versus those with steatosis alone (Table S11), which still identified GTPase regulation as a potential key mechanism in fibrosis initiation (Figure 6B).
In Fig. 6D, the authors used 3D liver spheroid to validate their findings. However, there is no images showing the 3D liver spheroid formation before and after PPARa/d agonist treatment. It is not clear whether the 3D liver spheroid was successfully established.
There is extensive literature (>40 papers) from the Lauschke lab on 3D liver spheroid culture, including but not limited to PMIDs 27143246, 28264975, 32775153, 37870288 and 39605182. Images of the spheroids can be seen in Figure 1c of Adv. Sci. 2024, 2407572 and elafibrinor treatment did not affect the morphology of the spheroids.
The authors suggested that targeting LX-2 cells with Rac1 and Cdc42 inhibitors could reduce collagen production. Did the authors observe these two genes upregulated in mRNA and protein expression levels in their cohort when compared MASLD patients with and without fibrosis? Did the authors observe that the expression levels of Rac1 and Cdc42 are correlated with fibrosis progression in MASLD patients?
Regarding comments 7 and 8, we targeted Rac1 and Cdc42 in the LX-2 cell experiment as they are common and major GTPases. Protein-level data are not available in our dataset, but we examined their transcript-level expression. RAC1 and CDC42 expression levels were positively associated with fibrosis progression, with coefficients of 0.362 (q = 0.027) and 0.342 (q = 0.031), respectively. These results are presented in Table S5, and the corresponding boxplots are shown here.
Figure R1. RAC1 and CDC42 expression levels in individuals with different fibrosis *levels. *
Other studies have revealed several metabolite changes related to MASLD progression (PMID: 35434590, PMID: 22364559). However, the authors did not discuss the discrepancies between their findings with the previous studies.
Thank you for the suggestion. We have incorporated a discussion of the two studies into the Discussion section, highlighting the consistencies and discrepancies between our plasma metabolomic results and previous findings. The main differences may stem from variations in MASLD spectrum and the degree of obesity in the cohorts.
Reviewer #1 (Significance (Required)):
Overall, the current study might provide some new resources regarding transcriptomic and metabolomic data derived from obese patients with and without MASLD. The MASLD research community will be interested in the resource data.
We thank this reviewer for the positive and constructive evaluation of our manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary:
In this paper, Kaldis and collaborators investigate the molecular heterogeneity of a 109 morbidly obese patient cohort, focusing on liver transcriptomics and metabolomics analysis from liver and serum. The main finding (i.e. upregulation of GTPase-coding genes) was validated in spheroids and a human HSC cell line. As these proteins are involved in critical cellular functions related to metabolism and cytoskeleton dynamics, these findings shed light on their involvement in human liver pathology which so far has been poorly (or even not) documented to date. This is an interesting addition to the current knowledge about chronic liver pathology. However the manuscript suffers from the lack of a clear-cut definition of patient subgroups and the seemingly indistinct use of generic (MASLD, NAS score) and more granular terms (MASH, fibrosis) across the various analysis they performed.
We thank this reviewer of highlighting the novelty of our manuscript. We agree that mixing generic and granular terms can be confusing and we tried to use of terms consistently throughout, which has been further improved in the revised version.
Figure 1 and Table 1 provide comprehensive information regarding histological phenotypes, NAS scores, and patient characteristics. From Figure 2 onward, we specifically focused on steatosis and fibrosis as distinct histological features, identifying molecular signatures associated with each process.
The term ‘MASH’ was used only when referring to the ex vivo 3D spheroids derived from histologically confirmed MASH patients for validation purposes. As our primary cohort represents early disease stages, we did not characterize molecular features of MASH in that data set.
In this cohort, the term 'NAS' was mentioned only in Section 1 to characterize the disease spectrum. Additionally, in Figures 3A and 6A, we illustrated the association between gene expression levels and NAS in two external cohorts. This was due to the absence of steatosis grades in the two datasets. NAS is an additive measure of multiple scores (steatosis, inflammation and ballooning), but does not account for fibrosis grades.
Our study focuses on the molecular features of steatosis grades and fibrosis grades as the main histological processes, with all terminology aligned with this stated objective. This allows us to map the transcriptome and metabolome to pathologist-defined steatosis/fibrosis severity (i.e., 0,1,2,3) and identify genes/metabolites that are correlated with increasing steatosis/fibrosis score.
Major comments:
The conclusions are generally consistent with findings from numerous previous studies, as many of the genes identified and their associations with disease states have been previously reported. However, I found it difficult to discern which specific disease stages the authors are referring to throughout the manuscript. Terms such as MASLD (Fig. 1F), steatosis (Fig. 4A), MASH, fibrosis (Fig. 6), and the composite NAS score (Fig. 1G) are used interchangeably, without clearly explaining whether or how the patient cohort was stratified to distinguish between isolated steatosis, MASH, and MASH with or without fibrosis. It is also unclear whether subgroups were propensity score-matched.
As explained in our previous point, we believe that we did not carelessly use the terms interchangeably, but rather used them as they were available or pertinent to the comparisons in discussion. We have provided a comprehensive cohort description in the first section (Table 1, including all histological features and NAS scores), then focused specifically on steatosis and fibrosis in subsequent analyses. We identified distinct molecular processes underlying these two histological features and validated key fibrosis-related pathways.
Regarding the comment of ‘propensity score-matched subgroups’, we would like to clarify that the only “sub”-group analysis performed in this paper is the transition from steatosis to steatosis with fibrosis. We have consistently used linear regression as the association analysis framework, without binarization of outcomes. We recall that this is a cross-sectional study with challenging recruitment situation from a bariatric surgery clinic that naturally represents the spectrum of MASLD in obesity. We acknowledge that the sampling can always be biased in such a study. However, given the invasiveness of liver resection, the study is also limited by the reality that not all patients would agree to the study, nor it is feasible to form a perfect subgroup meeting 1:1 ratio as in large-scale epidemiology studies based on plasma samples.
In a related point, the authors mention that 76% of patients are non-fibrotic, introducing a marked imbalance between fibrotic (n=26) and non-fibrotic (n=83) samples. Given this disparity and potential inter-individual variability, it would be helpful to include observed fold changes or effect sizes to give readers a sense of the magnitude of the biological dysregulations being reported.
As explained in our previous response, our study design examines associations between histological and molecular features rather than using a case-control approach. For effect size quantification, we report standardized linear regression coefficients, i.e. the change in gene expression Z-score per one-point increase in steatosis or fibrosis grade. We also provided fold changes in our comparative analysis of steatosis+fibrosis versus fibrosis-free steatosis. These effect sizes were fully documented in the Supplemental Tables.
Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
The authors seem pretty enthusiastic about elafibranor, despite a failed phase 3 clinical trial. I would qualify elafibranor as a useful tool in preclinical model. We agree with the reviewer and indeed used elafibranor as a research tool for PPARa/d modulation rather than a clinically promising prospect. Discussion regarding elafibranor has been updated.
The authors should make clearly the pronounced sex bias in their study, which includes mostly women (and btw refer to sex and not gender in the manuscript). Thank you for this important point. We added "Notably, the cohort was predominantly female (76.1%)" to the 'Overview of the study' section in the manuscript. We also replaced all 'gender' with 'sex' throughout the manuscript. In this cohorts, individuals with previous gender reassignment were excluded (see Materials and Methods).
The "MASH" status of the spheroid model is overstated. As described in the text it is much closer to a lipotoxicity model (and even glucotoxicity as Glc concentration is 2g/L). The 3D cultures were established from cells isolated from patients with histologically confirmed MASH. Besides steatosis, we observe increased secretion of pro-inflammatory cytokines, activation of hepatic stellate cells and increased deposition of collagen, thus phenocopying the critical disease hallmarks. Additionally, unbiased omics profiling (transcriptomics, proteomics and lipidomics) reveals significant increases in collagen biosynthesis, inflammatory signaling and cholesterol biosynthesis in MASH patient-derived cultures compared to controls. These differences largely overlapped with the results from analyses of six MASH case-control cohort studies. All of these results have been published previously (PMID 39605182).
This is confusing with panel D in which the authors establish a relationship between fibrotic patients (F2/F3 vs F0/S0, so I guess "no MASLD liver?) and this model. Is the relationship maintained for steatotic-only patients?
In Figure 6D, we compared GTPase-related gene expression between patients with fibrosis grade 2/3 (n = 26) and those without fibrosis and steatosis (n = 24). Principal component regression resulted in a positive correlation (β = 9.97) between log2 fold changes in 3D spheroids and human fibrosis samples, indicating consistent directional changes in both systems.
To answer the question from the reviewer, we compared the expression levels of GTPase-related genes in patients with steatosis but no fibrosis (n = 18) to those without fibrosis and steatosis (n = 24), we observed a negative correlation (β = -10.91). This indicates that GTPase-related gene changes in our 3D spheroids do not align with steatosis-related changes in humans.
Therefore, under the assumption that fibrosis follows steatosis in the majority of the cases of MASLD progression, the result indicates that the alterations in GTPase-related gene expression in the 3D spheroid model specifically is reflective of fibrosis rather than steatosis.
Figure R2. Comparison of expression level changes in GTPase-related genes between this human cohort and an independent 3D spheroid system: (A) positive correlation with fibrosis grade 2/3 patients versus controls (left), and (B) negative correlation with steatosis-only patients versus controls (right).
I am not convinced that HSC and LX2 cells express significant levels of PPARα. However, did the authors check for this parameter in their LX2 cell line and assessed whether PPARα/b activation by elafibranor (and/or pemafibrate as it is PPARα selective) alter GTPase expression? Whether negative or positive, this could give a clue about possible intercellular crosstalk in the spheroid model.
We thank this reviewer to point this out. In response, we analysed the mRNA expression of all PPARs in LX-2 cells with and without Elafibranor treatment, respectively (see Figure R3, same as Figure S8G in the Supplemental Material). We confirmed PPARs are expressed in LX-2 cells at the mRNA level (Figure R3A). Elafibranor does not affect their mRNA levels, which is consistent with previous reports that its primary mechanism is through binding and altering the activity of PPAR proteins, not gene expression (PMID 33326461 and PMID 37627519).
*Figure R3. Gene signatures in LX-2 cells with and without Elafibranor treatment (n = 3). *
In addition, we assessed mRNA levels of selected GTPase-related genes in LX-2 cells with and without Elafibranor treatment (Figure R3B). Although statistical power was limited, we observed a consistent trend toward reduced RHOU, DOCK2, and RAC1 expression with Elafibranor. this preliminary signal suggests that Elafibranor may counter the elevated GTPase levels seen in MASH patient spheroids, potentially via crosstalk among hepatic cell types, including HSCs.
To further investigate intercellular crosstalk in GTPase regulation among hepatic cell types, we evaluated signature GTPase-related genes in LX-2 cells, spheroid co-cultures (hepatocytes, HSCs, Kupffer cells), and hepatocyte monocultures. As shown in Figure R4 (same as Figure S10 in the supplemental material), TGFB1 served as a positive control, exhibiting the most pronounced induction upon TGF-β1 treatment in hepatocytes. Despite varied alterations across the selected GTPase-related genes, TGF-β1 treatment produced a trend toward increased VAV1 and DOCK2 expression in co-culture, hepatocytes, and LX-2 cells, and this was reversed by the TGF-β inhibitor in co-culture and hepatocytes. Other GTPase genes, including RAC1, RAB32, and RHOU, displayed cell type–specific responses to TGF-β1. These observations suggest that the regulation of GTPases is mediated by multiple hepatic cell types, supporting the importance of intercellular crosstalk.
Figure R4. Expression of GTPase-related genes in spheroid co-culture, hepatocyte monoculture, and LX-2 cells (n = 3). Controls for each gene and experiment were normalized to 1 to enable comparison across treatment groups.
The experiment mentioned above is cheap (cell culture, RT-QPCR) and can be performed within a couple of weeks.
Are the data and the methods presented in such a way that they can be reproduced? Yes
Are the experiments adequately replicated and statistical analysis adequate? There is no indication of group size, number of replicates for in vitro experiments
Thank you for this suggestion. We have added the sample sizes to all relevant sections: ‘n = 4’ in the figure legends for 3D spheroid experiments and ‘n = 8–10’ for the LX-2 experiments. This information has also been incorporated into the corresponding experimental descriptions in the Methods section.
**Referees cross-commenting**
I believe there is a general consensus on this potentially interesting contribution to the field, with three main points: (1) the need for a careful group-by-group comparison that accounts for potential confounders, (2) a more rigorous exploitation/characterization of the spheroid system, and (3) the need to benchmark the authors' findings against the available literature.
Thank you for summarizing the main points. Our responses are as follows:
Reviewer #2 (Significance (Required)):
The authors identified GTPases as players in the progression of MASLD. This is an interesting preliminary report warranting further molecular investigations (in which liver cell types, which GTPase pathway(s) are involved, which functions are controlled through this pathway...)
This paper will have an impact in the hepatology field
I have expertise in the analysis of "MASLD" human cohorts and in the molecular biology of chronic liver diseases.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary:
Metabolic dysfunction associated liver disease (MASLD) describes a spectrum of progressive liver pathologies linked to life style-associated metabolic alterations (such as increased body weight and elevated blood sugar levels), reaching from steatosis over steatohepatitis to fibrosis and finally end stage complications, such as liver failure and hepatocellular carcinoma. Treatment options for MASLD include diet adjustments, weight loss, and the receptor-β (THR-β) agonist resmetirom, but remain limited at this stage, motivating further studies to elucidate molecular disease mechanisms to identify novel therapeutic targets. In their present study, the authors aim to identify early molecular changes in MASLD linked to obesity. To this end, they study a cohort of 109 obese individuals with no or early-stage MASLD combining measurements from two anatomic sides: 1. bulk RNA-sequencing and metabolomics of liver biopsies, and 2. metabolomics from patient blood. Their major finding is that GTPase-related genes are transcriptionally altered in livers of individuals with steatosis with fibrosis compared to steatosis without fibrosis.
Major comments:
Rather than just stating corrections, subgroup analyses should be performed (accompanied with designated statistical power analyses) to infer the degree to which these conditions contribute to the observations. I.e., major findings stating MASLD-associated changes should hold true in the subgroup of MASLD patients without diabetes/of female sex and so forth (testing for each of the significant differences between groups).
Our original statistical analysis employed linear regression to examine associations between molecular variables (genes/metabolites) and histological progression (steatosis and fibrosis), with adjustment for potential confounders including diabetic status, age, sex, and BMI. We specifically focused on these two histological features to elucidate the disturbed molecular processes during their progression. Regression coefficients represent the expected change in abundance levels (in units of standard deviation of the corresponding molecule) per one-unit increase in histological grades.
To address the reviewer's question, we conducted additional subgroup analyses to determine whether our major findings remain consistent in individuals with and without diabetes. We assessed linear associations between gene signatures and histological features separately in non-diabetic (n = 71) and diabetic individuals (n = 23). Statistical power was estimated by comparing the variance explained by the full regression model (y ~ x + a + b + c) against the reduced model (y ~ a + b + c), converting the incremental R² for x into Cohen's f², and applying pwr.f2.test with the corresponding degrees of freedom and sample size at α = 0.05.
For both steatosis and fibrosis, the results in the non-diabetic subgroup (n = 71) showed high consistency with findings in our original analysis (n = 94, adjusted for diabetes), indicating that our originally reported gene signatures, after correction for diabetic status, remain valid in non-diabetic individuals.
In contrast, for diabetic individuals (n = 23), associations between genes and histological features did not closely replicate our original findings. Notably, we observed larger estimate effects for fibrosis-associated genes in diabetic individuals, suggesting a potential interaction between diabetes and fibrosis progression.
Figure R5. Subgroup analysis of the association between gene expressions and steatosis grades
Figure R6. Subgroup analysis of the association between gene expressions and fibrosis grades
On the comment "degree to which these conditions contribute to the observations," our original analysis adjusted for diabetes status to identify molecular signatures independently associated with fibrosis without the confounding of diabetes status. Consequently, the reported gene signatures in the original analysis more closely reflect patterns in the non-diabetes group, as demonstrated in our subgroup analysis plots. We also comment that, unfortunately, we did not adjust for the interaction of fibrosis and diabetes in the original analysis.
Furthermore, our additional analyses revealed a close relationship between diabetes and liver fibrosis. Consistent with Figure 1C, hepatic fibrosis is significantly correlated with insulin resistance parameters in clinical assays, including blood insulin levels and HOMA2-IR. To explore this association further, we compared gene expression profiles between diabetic MASLD patients (n = 21) and non-diabetic MASLD patients (n = 43). Although few genes reached significance after multiple testing correction, 166 genes showed differential expression (p 0.32) between these groups.
We identified 55 genes as potential "diabetic markers" that both showed differential expression between diabetic and non-diabetic MASLD patients and were significantly associated with steatosis or fibrosis progression. These genes are predominantly downregulated metabolic genes (e.g., BAAT, G6PC1, SULT2A1, MAT1A), suggesting that diabetes may exacerbate metabolic suppression as fibrosis advances. Given the high prevalence of diabetes in the MASLD population, our analysis supports the hypothesis that diabetes worsens MASLD outcomes, likely through impaired metabolic capability during fibrosis progression.
Regarding the comment on the "subgroup of female sex," our original analysis also adjusted for sex as a potential confounder. Since our cohort is predominantly female (>76%), the majority of our findings likely holds true in the female sub-population, similar to what we observed in our diabetes subgroup analysis.
External validation
Additionally, to back up the major GTPase signature findings, it would be desirable to analyze an external dataset of (pre)diabetes patients (other biased groups) for alternations in these genes. It would be important to know if this signature also shows in non-MASLD diabetic patients vs. healthy patients or is a feature specific to MASLD. Also, could the matched metabolic data be used to validate metabolite alterations that would be expected under GTPase-associated protein dysregulation?
We appreciate the comments regarding the validation GTPase as a unique MASLD signature by external datasets. As shown in our previous analysis, after adjusting for diabetes status, the gene signatures remained largely preserved in the non-diabetes subgroup. Before we respond further, we also preface that publicly available liver tissue data, with appropriate and full-scale clinical metadata and sufficient sample sizes, are extremely rare. To the best of our knowledge, the public data sets we brought into our paper were the most prominent data of reliable quality.
In the paper, we benchmarked our RNAseq dataset against two datasets: the VA cohort and EU cohort (Figure 1). Our cohort focused primarily on early MASLD patients with obesity, which aligns more closely with the disease spectrum represented in the VA cohort (Figure 1G). Notably, in the published paper for the VA cohort, Hoang et al. highlighted Rho GTPase signaling as one of the top pathways in the fibrosis PPI network (Figure 1B from publication PMID 31467298).
We interrogated GTPase-related genes in both the VA and EU cohorts. As shown in Figure R7 (below), GTPase-related genes demonstrated a strong association with fibrosis grades in the VA cohort, as expected. The EU cohort comprises more advanced MASLD cases with higher fibrosis grades, and our re-analysis in this cohort specifically focused on MASH patients (as designated by the authors). In those MASH patients, GTPase-related genes did not show significant positive associations with fibrosis progression. This finding is consistent with our hypothesis that GTPase regulation is triggered more prominent during the early progression of fibrosis rather than at later stages.
Unfortunately, diabetes status was not available in the GEO repository for the VA cohort. Available liver tissue sequencing datasets with balanced representation of diabetic and nondiabetic patients are rare, especially those derived from obese individuals and reflecting the early-to-middle stages of MASLD. In our own cohort, for instance, only two diabetic patients without MASLD were recruited (Table 1). While we cannot rule out a role for insulin resistance in GTPase regulation, we will plan future experiments using mouse models to examine GTPase-mediated fibrosis under diabetic and nondiabetic conditions.
Regarding the comment ‘validate metabolite alterations that would be expected under GTPase-associated protein dysregulation,’ we note that GTPases are primarily involved in cytoskeletal organization, vesicle trafficking, and other cellular processes, with few well-established links to specific metabolite signatures. Nevertheless, in our partial correlation network integrating hepatic genes and metabolites, we observed co-regulated metabolites associated with GTPase-related genes (Figure R8). These included palmitoleoyl ethanolamide (N-acylethanolamine, an anti-inflammatory metabolite and PPARα ligand), phenylacetic acid (a phenylalanine metabolite), biotin (a coenzyme), arginine, lysine, melatonin (a tryptophan metabolite), and several lipid species such as PC 32:0 and CAR 20:1. While causal relationships cannot be inferred from this dataset, our integrative network highlights potential connections related to the trafficking of these metabolites that warrant further investigation.
Figure R7. Associations between GTPase-related genes to fibrosis in this study and two external cohorts. Asterisks denote significant associations with q value Figure R8. Integrative subnetwork of GTPase-related genes. Blue squares represent GTPase-related genes, red circles indicate metabolites connected to these genes, and the purple diamond denotes fibrosis, which is connected to RHOU.*
3D liver spheroid MASH model, Fig. 6D/E
This 3D experiment is technically not an external validation of GTPase-related genes being involved in MASLD, since patient-derived cells may only retain changes that have happened in vivo. To demonstrate that the GTPase expression signature is specifically invoked by fibrosis the LX-2 set up is more convincing, however, the up-regulation of the GTPase-related genes upon fibrosis induction with TGF-beta, in concordance with the patient data, needs to be shown first (qPCR or RNA-seq).
We agree with the reviewer that experiments in LX-2 (HSC) cells are important and as we have described under ‘Reviewer #2’ we have done this (Figure R3 and Figure R4). Because HSCs only comprise a minor cell population of liver cells, the signals observed in patient bulk RNA data are likely driven primarily by hepatocytes. Nevertheless, we have highlighted the importance of hepatic cell crosstalk in Figure R4 and in our response to Reviewer #2. Additionally, in Supplementary Figure S9B, we identify the potential cell types of origin for the GTPase signals (predominantly hepatocytes and HSCs) using a single-cell dataset from an independent study (PMID: 37962490).
Additionally, the description of the 3D model is too uncritical. The maintenance of functional human PHHs in 3D has only become available this year (PMID: 40240606) marking a break-through in the field. Since the authors did not use this system, I would strongly assume their findings are largely attributable to the mesenchymal cells in the 3D culture, and these limitations need to be stated.
We humbly disagree with the reviewer on the 3D liver spheroids. The paper that the reviewer is referencing is related to the proliferation of hepatocytes in organoids, not – at least not directly – their functional maintenance. Here, we use a spheroid model of mature fully differentiated cells, which is conceptually different from the organoid approach. Maintenance of such functional human hepatocytes for multiple weeks in culture has been possible for close to a decade (PMID 27143246). Moreover, particularly for the modeling of chronic liver disease, such as MASH, it is important to use directly patient-derived cells as short induction cycles (typically 1-2 weeks) of disease phenotypes in organoid models do not faithfully reproduce the molecular signatures that stem from chronic exposures in vivo.
The 3D liver spheroid model we used here is derived from livers from patients with a histologically confirmed diagnosis of MASH. The isolated cells are fully mature and thus do not require in vitro differentiation. There are no MSCs in the 3D cultures; rather the spheroids contain hepatocytes, stellate cells, Kupffer cells as well as various other immune cell types present in the liver at the time of isolation (T cells, B cells, NK cells). Furthermore, the model is extensively characterized at the transcriptomic, proteomic and lipidomic level (PMID 39605182).
Novelty / references
Similar studies that also combined liver and blood lipidomics/metabolomics in obese individuals with and without MASLD (e.g. PMID 39731853, 39653777) should be cited. Additionally, it would benefit the quality of the discussion to state how findings in this study add new insights over previous studies, if their findings/insights differ, and if so, why.
Thank you for the suggestion. We added the two papers into the discussion section. Specifically, we discussed the consistent findings (such as AKR1B10 in PMID 37037945 and mitochondrial dysfunction in PMID 39731853) and discrepancies (such as limited plasma metabolomic changes and circulating sphingolipid alterations in multiple human and mouse models) in comparison with previously published omics studies in MASLD patients. Also, we thoroughly discussed our findings (e.g., lipid dysregulation, dysregulated tryptophan metabolism, GTPase regulation) and potential mechanisms with extensive literature supports from of human, animal, and cell studies.
Minor comments:
For Figure S7C, we presented the correlation matrix of more than 200 GTPase-related genes along with the TGF-β genes TGFB1 and TGFB3. This illustrates the overall co-expression patterns of GTPase-related genes rather than displaying individual gene labels, with arrows now included to highlight TGFB1 and TGFB3.
Reviewer #3 (Significance (Required)):
The authors provide an overall sound study on the hepatic transcriptomic and metabolomic signatures in an Australian cohort of 109 obese non-to-early stage MASLD patients. They perform thorough analyses of metabolome and transcriptome in liver biopsies and metabolome in blood, using standard technologies such as RNA sequencing and mass spectrometry. Their key finding is a GTPase-associated gene signature related to fibrosis onset. Limitations of the study include potential cohort confounders (raising the need for expanded control experiments), limited discussion of similar studies, and limits in cell-type resolution, the latter of which is related to the molecular read out, and has in parts been started to be addressed by in vitro experiments in an immortalized HSC lines. Taken together, given additional control analyses will be performed, the results could be of interest to an expert community in the field of molecular hepatology and, while still descriptive, hold the potential to prompt mechanistic follow-up studies.
We thank this reviewer for a balanced, positive, and constructive evaluation of our manuscript.
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Summary:
Metabolic dysfunction associated liver disease (MASLD) describes a spectrum of progressive liver pathologies linked to life style-associated metabolic alterations (such as increased body weight and elevated blood sugar levels), reaching from steatosis over steatohepatitis to fibrosis and finally end stage complications, such as liver failure and hepatocellular carcinoma. Treatment options for MASLD include diet adjustments, weight loss, and the receptor-β (THR-β) agonist resmetirom, but remain limited at this stage, motivating further studies to elucidate molecular disease mechanisms to identify novel therapeutic targets.
In their present study, the authors aim to identify early molecular changes in MASLD linked to obesity. To this end, they study a cohort of 109 obese individuals with no or early-stage MASLD combining measurements from two anatomic sides: 1. bulk RNA-sequencing and metabolomics of liver biopsies, and 2. metabolomics from patient blood. Their major finding is that GTPase-related genes are transcriptionally altered in livers of individuals with steatosis with fibrosis compared to steatosis without fibrosis.
Major comments:
Minor comments:
The authors provide an overall sound study on the hepatic transcriptomic and metabolomic signatures in an Australian cohort of 109 obese non-to-early stage MASLD patients. They perform thorough analyses of metabolome and transcriptome in liver biopsies and metabolome in blood, using standard technologies such as RNA-sequencing and mass spectrometry. Their key finding is a GTPase-associated gene signature related to fibrosis onset. Limitations of the study include potential cohort confounders (raising the need for expanded control experiments), limited discussion of similar studies, and limits in cell-type resolution, the latter of which is related to the molecular read out, and has in parts been started to be addressed by in vitro experiments in an immortalized HSC lines. Taken together, given additional control analyses will be performed, the results could be of interest to an expert community in the field of molecular hepatology and, while still descriptive, hold the potential to prompt mechanistic follow-up studies.
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Summary:
Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).
In this paper, Kaldis and collaborators investigate the molecular heterogeneity of a 109 morbidly obese patient cohort, focusing on liver transcriptomics and metabolomics analysis from liver and serum. The main finding (ie upregulation of GTPase-coding genes) was validated in spheroids and a human HSC cell line. As these proteins are involved in critical cellular functions related to metabolism and cytoskeleton dynamics, these findings shed light on their involvement in human liver pathology which so far has been poorly (or even not) documented to date. This is an interesting addition to the current knowledge about chronic liver pathology. However the manuscript suffers from the lack of a clear-cut definition of patient subgroups and the seemingly indistinct use of generic (MASLD, NAS score) and more granular terms (MASH, fibrosis) across the various analysis they performed.
Major comments:
The conclusions are generally consistent with findings from numerous previous studies, as many of the genes identified and their associations with disease states have been previously reported. However, I found it difficult to discern which specific disease stages the authors are referring to throughout the manuscript. Terms such as MASLD (Fig. 1F), steatosis (Fig. 4A), MASH, fibrosis (Fig. 6), and the composite NAS score (Fig. 1G) are used interchangeably, without clearly explaining whether or how the patient cohort was stratified to distinguish between isolated steatosis, MASH, and MASH with or without fibrosis. It is also unclear whether subgroups were propensity score-matched.
In a related point, the authors mention that 76% of patients are non-fibrotic, introducing a marked imbalance between fibrotic (n=26) and non-fibrotic (n=83) samples. Given this disparity and potential inter-individual variability, it would be helpful to include observed fold changes or effect sizes to give readers a sense of the magnitude of the biological dysregulations being reported. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? - The authors seem pretty enthusiastic about elafibranor, despite a failed phase 3 clinical trial. I would qualify elafibranor as a useful tool in preclinical model.<br /> - The authors should make clearly the pronounced sex bias in their study, which includes mostly women (and btw refer to sex and not gender in the manuscript).<br /> - The "MASH" status of the spheroid model is overstated. As described in the text it is much closer to a lipotoxicity model (and even glucotoxicity as Glc concentration is 2g/L). This is confusing with panel D in which the authors establish a relationship between fibrotic patients (F2/F3 vs F0/S0, so I guess "no MASLD liver?) and this model. Is the relationship maintained for steatotic-only patients?<br /> - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. I am not convinced that HSC and LX2 cells express significant levels of PPARα. However, did the authors check for this parameter in their LX2 cell line and assessed whether PPARα/b activation by elafibranor (and/or pemafibrate as it is PPARα selective) alter GTPase expression? Whether negative or positive, this could give a clue about possible intercellular crosstalk in the spheroid model.<br /> - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
The experiment mentioned above is cheap (cell culture, RT-QPCR) and can be performed within a couple of weeks. - Are the data and the methods presented in such a way that they can be reproduced?
Yes - Are the experiments adequately replicated and statistical analysis adequate?
There is no indication of group size, number of replicates for in vitro experiments
Referees cross-commenting
I believe there is a general consensus on this potentially interesting contribution to the field, with three main points: (1) the need for a careful group-by-group comparison that accounts for potential confounders, (2) a more rigorous exploitation/characterization of the spheroid system, and (3) the need to benchmark the authors' findings against the available literature.
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Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from simple steatosis, steatohepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma. In the current study, the authors aimed to determine the early molecular signatures differentiating patients with MASLD associated fibrosis from those patients with early MASLD but no symptoms. The authors recruited 109 obese individuals before bariatric surgery. They separated the cohorts as no MASLD (without histological abnormalities) and MASLD. The liver samples were then subjected to transcriptomic and metabolomic analysis. The serum samples were subjected to metabolomic analysis. The authors identified dysregulated lipid metabolism, including glyceride lipids, in the liver samples of MASLD patients compared to the no MASLD ones. Circulating metabolomic changes in lipid profiles slightly correlated with MASLD, possibly due to the no MASLD samples derived from obese patients. Several genes involved in lipid droplet formation were also found elevated in MASLD patients. Besides, elevated levels of amino acids, which are possibly related to collagen synthesis, were observed in MASLD patients. Several antioxidant metabolites were increased in MASLD patients. Furthermore, dysregulated genes involved in mitochondrial function and autophagy were identified in MASLD patients, likely linking oxidative stress to MASLD progression. The authors then determined the representative gene signatures in the development of fibrosis by comparing this cohort with the other two published cohorts. Top enriched pathways in fibrotic patients included GTPas signaling and innate immune responses, suggesting the involvement of GTPas in MASLD progression to fibrosis. The authors then challenged human patient derived 3D spheroid system with a dual PPARa/d agonist and found that this treatment restored the expression levels of GTPase-related genes in MASLD 3D spheroids. In conclusion, the authors suggested the involvement of upregulated GTPase-related genes during fibrosis initiation. Overall, the current study might provide some resources regarding transcriptomic and metabolomic data derived from obese patients with and without MASLD. However, several concerns should be carefully addressed.
Overall, the current study might provide some new resources regarding transcriptomic and metabolomic data derived from obese patients with and without MASLD. The MASLD research community will be interested in the resource data.
Reviewer #2 (Public review):
Summary:
In this manuscript, Zhu, Emanuelli, and colleagues describe a novel pharmacological activator of the Integrated Stress Response kinase GCN2. The work is conclusive and biochemically solid. This work significantly adds to the pharmacological arsenal targeting the ISR and, in particular, GCN2.
Strengths:
Strong biochemistry, novel molecular activator of GCN2 (GCN1 independent).
Weaknesses:
The rationale for the screen is not exploited in the results (e.g., pathogenic GCN2 mutants), and lots of cell-based read-outs are not endogenous.
Major points
(1) Regarding the justification of the work. Since the authors justify the screen for GCN2 activators with loss-of-function mutants associated with diseases, it would be of interest to evaluate whether the best compounds identified in the study are indeed able to prompt activation of those mutants (or at least of the most prevalent). This approach could actually go in parallel with the docking experiments carried out in the last figure of the manuscript, where mutants could be modelized as well.
(2) The compounds are only tested using « artificial » proximal signaling outputs. It would be interesting to evaluate whether the best identified compounds are capable of prompting endogenous eIF2alpha phosphorylation in cellular models.
(3) Other GCN2 activators (other than GCN2iB, e.g., HC-7366) were recently identified. In this context, it would be of interest to carry out a small benchmarking study to evaluate how the compounds identified in the current study perform against the previously identified molecules.
Reviewer #3 (Public review):
Summary:
In this manuscript, the authors describe the results of a high-throughput screen for small-molecule activators of GCN2. Ultimately, they find 3 promising compounds. One of these three, compound 20 (C20), is of the most interest both for its potency and specificity. The major new finding is that this molecule appears to activate GCN2 independent of GCN1, which suggests that it works by a potentially novel mechanism. Biochemical analysis suggests that each binds in the ATP-binding pocket of GCN2, and that at least in vitro, C20 is a potent agonist. Structural modeling provides insight into how the three compounds might dock in the pocket and generates testable hypotheses as to why C20 perhaps acts through a different mechanism than other molecules.
Strengths:
Of the 3 compounds identified by the authors, C20 is the most interesting, not just for its intriguing mechanistic distinction as being GCN1-independent (shown genetically in two distinct cell lines, CHO and 293T in Figure 4, and in contrast to other GCN2 activators) but also for its potency. In in-cellulo assays, compound 21 appears as more of an ISR enhancer than an activator per se, and although compound 18 and compound 21 lead to upregulation of the ISR targets (Figure 2), that degree of upregulation is probably not significantly different from that induced by those compounds in Gcn2-/- cells. For C20, the effect appears stronger (although it is unclear whether the authors performed statistical analysis comparing the two genotypes in Figure 2D). In Figure 3, only C20 activates the ISR robustly in both CHO and 293T. Ultimately, C20 might be a tool for providing mechanistic insight into the details of GCN2 activation and regulation, and could be exploited therapeutically.
Weaknesses:
There are some limitations to the existing work. As the authors acknowledge, they do not use any of the compounds in animals; their in vivo efficacy, toxicity, and pharmacokinetics are unknown. But even in the context of the in cellulo experiments, it is puzzling that none of the three compounds, including C20, has any effects in HeLa cells when Neratinib does. It's beyond the scope of this paper to address definitively why that is, but it would at least be reassuring to know that C20 activates the ISR in a wider range of cells, including ideally some primary, non-immortalized cells. In addition, the ISR is a complex, feedback-regulated response whose output varies depending on the time point examined. The in cellulo analysis in this paper is limited to reporter assays at 18 hours and qRT-PCR assays at 4 and 8 hours. A more extensive examination of the behavior of the relevant ISR mRNAs and proteins (eIF2, ATF4, CHOP, cell viability, etc.) for C20 across a more extensive time course would give the reader a clearer sense of how this molecule affects ISR output. I also find it a bit strange that the authors describe C20 as "demonstrat(ing) weak inhibition of ... PKR"-the measured IC50 is ~4 μM, which is right around its EC50 for GCN2 activation. This raises the confounding possibility that C20 would simultaneously activate GCN2 while inhibiting PKR. While perhaps inhibition of PKR is not relevant under the conditions when GCN2 would be activated either experimentally or therapeutically, examining in cells the effects of C20 on GCN2 and PKR across a dose range would shed light on whether this cross-reactivity is likely to be of concern.
Reviewer #3 (Public review):
Summary:
The manuscript by Raiola and colleagues entitled "Quantitative computerized analysis demonstrates strongly compartmentalized tissue deformation patterns underlying mammalian heart tube formation" takes a highly quantitative approach to interrogating the earliest stages of cardiogenesis (12 hours, from early cardiac crescent to early heart tube) in a new and innovative way. The paper presents a new computational framework to help identify both regional and temporal patterns of tissue deformation at cellular resolution. The method is applied to live embryo imaging data (newly generated and from the group's previous pioneering work). In the initial setup, the new model was applied directly to raw time-lapse data, and the results were compared to actual cell tracks identified manually, showing close correlations of the model with the manual tracking. Next, they integrated spatial and temporal information from different embryos to generate a new model for tissue movement, driven by parameters such as tissue growth and anisotropy. Key findings from their model suggest that there are distinct compartments of tissue deformation patterns as the bilateral cardiac crescent develops into the linear heart tube, and that the ventricular chamber forms by a defined expansion pattern, as a 'hemi-barrel shape', with the aterial and venous poles (IFT and OFT) acting as the harnessing belts constraining the expansion of the chamber further. Lastly, the model is tested for its ability to predict future residence of cardiac crescent cells in the heart tube, which it seems to be able to do successfully based on fate tracking validation experiments.
Strengths:
The manuscript provides an exceptionally careful analysis of a critical stage during heart development - that of the earliest stages of morphogenesis, when the heart forms its first tube and chamber structures. While numerous studies have interrogated this stage of heart development, few studies have performed time-lapse imaging, and, to my knowledge, no other report has performed such in in-depth quantitative analysis and modeling of this complex process. The computational model applied to normal heart development of the myocardium (labelled by Nkx2-5) has revealed multiple new and interesting concepts, such as the distinct compartments of tissue deformation patterns and the growth trajectories of the emerging ventricle. The fact that the model operates at cellular resolution and over a nearly continuous time period of approximately 12 hours allows for unprecedented depth of the analysis in a largely unbiased manner. Going forward, one can imagine such models revealing additional information on these processes, performing analyses of subpopulations that form the heart, and maybe most importantly, applying the model to various perturbation models (genetic or otherwise). The manuscript is very well written, and the data display is accessible and transparent.
Weaknesses:
No major weaknesses are noted with the study. It would have been very exciting to see the model applied to any kind of perturbation, for example, a left-right defect model, or a model with compromised cardiac progenitor populations. However, the amount of live imaging required for such analyses renders this out of scope for the current study.
Reviewer #1 (Public review):
Summary:
The authors demonstrate the stereoselective role of D-serine in 1C metabolism, showing that D-serine competes with L-serine and inhibits mitochondrial L-serine transport. They observe expression of 1C metabolites in their metabolomics approach in primary cortical neurons treated with L-serine, D-serine, and a mixture of both. Their conclusions are based on the reduction in levels of glycine, polyamines, and their intermediates and formate. Single-cell RNA sequencing of N2a cells showed that cells treated with D-serine enhanced expression of genes associated with mitochondrial functions, such as respiratory chain complex assembly, and mitochondrial functions, with downregulation of genes related to amino acid transport, cellular growth, and neuron projection extension. Their work demonstrates that D-serine inhibits tumor cell proliferation and induces apoptosis in neural progenitor cells, highlighting the importance of D-serine in neurodevelopment.
Strengths:
D-amino acids are a marvel of nature. It is fascinating that nature decided to make two versions of the same molecule, in this case, an amino acid. While the L-stereoisomer plays well-known roles in biology, the D-stereoisomer seems to function in obscurity. Research into these novel signaling molecules is gathering momentum, with newer stereoisomers being discovered. D-serine has been the most well-studied among the different stereoisomers, and we still continue to learn about this novel neurotransmitter. The roles of these molecules in the context of metabolism is not well studied. The authors aim to elucidate the metabolic role of D-serine in the context of neuronal maturation with implications for 1C metabolism and in cell proliferation. The metabolic role of these molecules is just beginning to be uncovered, especially in the context of mammalian biology. This is the strength of the manuscript. The authors have done important work in prior publications elucidating the role of D-amino acids. The advancement of the field of D-amino acids in mammalian biology is significant, as not much is known. The presentation of RNA seq data is a valuable resource to the community, however, with caveats as mentioned below.
Weaknesses:
The following are some of the issues that come out in a critical reading of the manuscript. Addressing these would only strengthen and clarify the work.
(1) Kinetic assessment of D-serine versus L-serine: While the authors mention that D-serine is not a good substrate for SHMT2 compared to L-serine, the kinetic data are presented for only D-serine. In a substrate comparison with an enzyme, data must be presented for L-serine as well to make the conclusion about substrate specificity and affinity. Since the authors talk about one versus another substrate, there needs to be a kinetic comparison of both with Km (affinity). (Ref Figure 2 panel).
(2) Molecular Dynamics simulations, while a good first step in modeling interactions at the active site, rely on force fields. These force fields are approximations and do not represent all interactions occurring in the natural world. Setting up the initial conditions in the simulations can impact the final results in non-equilibrium scenarios. The basic question here is this: Is the simulated trajectory long enough so that the system reaches thermodynamic equilibrium and the measured properties converge? Prior studies have shown mixed results with the conclusion that properties of biological systems tend to converge in multi-second trajectories (not nanosecond scales as reported by the authors) and transition rates to low probability conformations require more time. (Ref Figure 2C).
(3) The authors use N2a cell line to demonstrate D-serine burden on primary cortical neurons. N2a is an immortalized cell line, and its properties are very different from primary neurons. The authors need to mention a rationale for the use of an immortalized cell line versus primary neurons. The transcriptomic profile of an immortalized cell line is different compared to a primary cell. Hence, the response to D-serine may vary between the two different cell types.
(4) In Figure 4D, the authors mention that D-serine activates the cleavage of caspase 3. Figure 4D shows only cleaved caspase 3 as a single band. They need to show the full blot that contains the cleaved fragments along with the major caspase 3 band.
(5) In Figure panel 4, the authors use neural progenitor cells (NPCs). They need to demonstrate that the population they are working with is NPCs and not primary neurons. There must be a figure panel staining for NPC markers like SOX2 and PAX6. Also, Figure S5 needs to be properly labeled. It is confusing from the legend what panels B-E refer to? Also, scale bars are not indicated.
(6) In Supplementary Figure panel 7F, the authors mention phosphatidyl L-serine and phosphatidyl D-serine. A chromatogram of the two species would clarify their presence as they used 2D-HPLC. On an MS platform, these 2 species are not distinguishable. Including a chromatogram of the 2 species would be helpful to the readers.
(7) The authors mention about enantiomeric shift of serine metabolism during neural development, which appears to be a discussion of prior published data from Hubbard et al, 2013, Burk et al, 2020, and Bella et a,l 2021 in Supplementary Figure panels 8 A-E. This should not be presented as a figure panel, as it gives the false impression that the authors have performed the experiment, which is clearly not the case. However, its discussion can well serve as part of the manuscript in the discussion section.
(8) The entire presentation of the section on enantiomeric shift of serine metabolism during neural development (lines 274-312) is a discussion and should be part of the discussion section and not in the results section. This is misleading.
(9) The discussion section is not well written. There is no mention of recent work related to D-serine that has a direct bearing on its metabolic properties. In the discussion section, paragraph 1, the authors mention that their work demonstrates the selective synthesis of D-serine in mature neurons as opposed to neural progenitor cells. This concept has been referred to in prior publications:
(a) Spatiotemporal relationships among D-serine, serine racemase, and D-amino acid oxidase during mouse postnatal development. PMID:14531937.
(b) D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain. PMID:34556581.
(10) In the abstract, in lines 101 and 102, the authors mention "how D-serine contributes to cellular metabolism beyond neurotransmission remains largely unknown". In 2023, a paper in Stem Cell Reports by Roychaudhuri et al (PMID:37352848) showed that D and L-serine availability impacts lipid metabolism in the subventricular zone in mice, affecting proliferative properties of stem-cell derived neurons using a comprehensive lipidomics approach. There is no mention of this work even in the discussion section, as it bears directly on L and D-serine availability in neurons, which the authors are investigating. In the discussion section in lines 410-411, the authors mention the role of D-serine in neurogenesis, but surprisingly don't refer to the above reference. The role of D-serine in neurogenesis has been demonstrated in the Sultan et al (lines 855-857) and Roychaudhuri et al references.
(11) Both D-serine and the structurally similar stereoisomer D-cysteine (sulfur versus oxygen atom) have a bearing on 1C metabolism and the folate cycle. With reference to the folate cycle, Roychaudhuri et al in 2024 (PMID:39368613) have shown in rescue experiments in mice that supplementing a higher methionine diet provides folate cycle precursors to rescue the high insulin phenotype in SR-deficient mice. Since 1C metabolism is being discussed in this manuscript, the authors seem to overlook prior work in the field and not include it in their discussion, even when it is the same enzyme (SR) that synthesizes both serine and cysteine. Since the field of D-amino acid research is in its infancy, the authors must make it a point to include prior work related to D-serine at least, and not claim that it is not known. The known D-stereoisomers are not many, hence any progress in the area must include at least a discussion of the other structurally related stereoisomers.
(12) Racemases (serine and aspartate) in general are promiscuous enzymes and known to synthesize other stereoisomers in addition to D-serine, D-cysteine, and D-aspartate. A few controls, like D-aspartate, D-cysteine, or even D-alanine must be included in their study to demonstrate the specific actions of D-serine, especially in the N2a cell treatment experiments. Cysteine and Serine are almost identical in structure (sulfur versus oxygen atom), and both are synthesized by serine racemase (published). Cysteine has also been very recently shown to inhibit tumor growth and neural progenitor cell proliferation. (PMIDs: 40797101 and 34556581). How the authors' work relates to the existing findings must be discussed, and this would put things in perspective for the reader.
Reviewer #2 (Public review):
Summary:
This study by Suzuki et al. reports an interesting stereo-selective role of D-serine in regulating one-carbon metabolism during neurodevelopment to adapt the functional transition, probably through the competition with mitochondrial transport of L-serine. The authors provide a multi-layered set of evidence, including metabolomics, enzyme assays, mitochondrial transport competition, and functional assays in immature/neural progenitor cells, to build up a conceptual integration of D-serine as both a neurotransmitter and a metabolic regulator in the central neural system, which raises a broad potential interest to the neuroscience and metabolism communities.
Strengths:
This work provides a conceptual advance that D-serine not only serves as a traditional neurotransmitter in the central neural system but also critically contributes to metabolic regulation of neural cells. The authors performed solid metabolomic assays to validate the suppressive effect of D-serine on the one-carbon metabolic pathway, providing some evidence that D-serine competitively inhibits mitochondrial serine transport, but not directly impairs SHMT2 enzymatic activity. All these data indicate a critical role of D-serine synthesis during neural maturation and suggest a potential translational strategy for targeting serine metabolism in neural tumors.
Weaknesses:
(1) The detailed mechanism by which D-serine competes with L-serine for its mitochondrial transport is not investigated. For example, although the authors made some discussion, they did not provide direct genetic or biochemical evidence linking these effects to the specific transporters, such as SFXN1.
(2) Unlike tumor cells, where SHMT2 usually plays a predominant role in catalyzing serine/THF-derived one-carbon metabolism, normal cells may employ both SHMT1 and SHMT2 to do the work. Even under certain conditions that SHMT2-mediated one-carbon metabolism is suppressed, the activity of SHMT1 could be elevated for compensation. Thus, it is important to investigate whether D-serine affects SHMT1 activity or changes the balance between SHMT1- and SHMT2-mediated one-carbon metabolism. To this aim, the authors are strongly encouraged to perform a metabolic flux assay (MFA) by using 13C-labeled L-serine in the model cells in the presence and absence of D-serine.
(3) A defect in serine-derived one-carbon metabolism may cause multiple cellular stress responses. It is valuable to detect whether cellular NADPH/NADH, GSH, or ROS is altered before and after D-serine treatment.
(4) The physiological relevance between D-serine and neural cell maturation/death should be further tested and discussed, since the dosage of D-serine used in the in vitro assay is much higher than that in physiological conditions.
Reviewer #3 (Public review):
Summary:
This manuscript presents a comprehensive and well-executed investigation into the metabolic role of D-serine in the central nervous system. The authors provide solid evidence that D-serine competitively inhibits mitochondrial L-serine transport, thereby impairing one-carbon metabolism. This stereoselective mechanism reduces glycine and formate production, suppresses cellular proliferation, and induces apoptosis in immature neural cells and glioblastoma stem cells. Developmental analyses further reveal a physiological enantiomeric shift in serine metabolism during neurogenesis, aligning with the transition from proliferation to maturation. Overall, the study bridges developmental neurobiology, cancer metabolism, and amino acid transport, uncovering a previously unrecognized metabolic function of D-serine beyond its role in neurotransmission.
Strengths:
(1) The discovery that D-serine inhibits one-carbon metabolism by competing for mitochondrial L-serine transport-rather than through enzymatic inhibition or receptor-mediated signaling-represents a significant and previously underappreciated mechanism. This finding has broad implications for understanding metabolic regulation during neurodevelopment and offers potential relevance for targeting metabolic vulnerabilities in cancer.
(2) The authors integrate metabolomics, mitochondrial transport assays, molecular dynamics simulations, genetic and pharmacologic perturbations, transcriptomics, and both in vitro and ex vivo models. The breadth of experimental approaches, combined with the coherence of the findings across systems, provides strong support for the central conclusions and enhances the overall impact of the study.
(3) The temporal shift in D-/L-serine levels during neurodevelopment is elegantly linked to the transition from proliferative to mature neuronal states. The selective vulnerability of neural progenitors and tumor cells-contrasted with the resistance of mature neurons-highlights a biologically meaningful and potentially targetable metabolic distinction.
Weaknesses:
(1) While the authors attribute D-serine's metabolic effects to competition with mitochondrial L-serine transport, the specific identity of the transporter(s) mediating this process remains undefined. This represents a meaningful mechanistic gap, as the central conclusion depends on D-serine limiting mitochondrial L-serine availability to inhibit one-carbon metabolism.
(2) The effective concentrations of D-serine used in vitro (IC₅₀ ≈ 1-2 mM) exceed typical brain levels (~0.3 mM). While the authors acknowledge this, a more focused discussion on whether higher local D-serine concentrations could arise in specific microenvironments - such as synaptic compartments, tumor niches, or pathological states-would help contextualize the in vitro findings and strengthen their physiological relevance. For example, disruptions in D-serine clearance or altered expression of serine racemase and transporters in disease contexts could lead to localized accumulation. Moreover, differences between extracellular and intracellular D-serine pools - and the mechanisms governing their regulation - may further influence its metabolic impact in vivo.
(3) While the manuscript focuses on neural stem/progenitor cells and neural tumors, it remains unclear whether the anti-proliferative effects of D-serine are specific to neural lineages or extend to other highly proliferative non-neural cell types. A brief discussion addressing this point would help clarify the scope of D-serine's metabolic impact and whether its mechanism of action reflects a unique vulnerability in neural cells or a more general feature of proliferative metabolism. This distinction is particularly relevant for assessing the broader therapeutic potential of targeting mitochondrial L-serine transport.
Reviewer #2 (Public review):
Summary:
The authors aim to address whether nuclear pore complex components localize and function at PD in plant cells to mediate cell-to-cell communication.
Strengths:
(1) Novelty and Significance:<br /> The core hypothesis, drawing parallels between PD and NPC transport, is highly original and addresses a critical gap in understanding plant intercellular communication. The idea that phase-separated domains formed by FG-NUPs could act as diffusion barriers at PD offers a plausible and sophisticated explanation for their complex transport properties, including size exclusion and facilitated translocation. This could fundamentally change how we view PD function.
(2) Comprehensive Evidence:<br /> The study employs a rigorous and diverse set of experimental approaches, including a comprehensive bioinformatic analysis of both moss and Arabidopsis NUPs in available PD proteomic datasets, extensive imaging analysis of Nup localization in vivo, and functional transport assays using a loss-of-function nup mutant (cpr5). The transport assay is particularly important to provide functional evidence linking CPR5 to PD-mediated transport. The finding that callose levels were not significantly different in cpr5 mutants under these conditions is helpful and supports a distinct, callose-independent mechanism of transport regulation.
(3) Objectivity:<br /> The authors are forthright in discussing the limitations and potential artifacts of their own data, clearly distinguishing between observations and definitive conclusions.
Weaknesses:
While the claims are generally justified as hypotheses or consistent observations, the authors themselves extensively detail the caveats, which are worth reiterating for clarity:
(1) Potential Overexpression Artifacts in Localization:<br /> Although efforts were made to control expression levels, the authors acknowledge that transient overexpression could still lead to NUP accumulation at PD, either as a physiologically relevant accumulation under excess conditions or due to mis-targeting, or even as storage depots. The resolution of confocal microscopy also does not allow for a definitive conclusion on the nature of the location.
(2) Proteomics Purity:<br /> The authors note that the presence of NUPs in PD fractions/proteomics cannot definitively rule out contamination, as PD cannot currently be purified to absolute homogeneity and is often contaminated with other organelles, including the nucleus.
(3) CPR5 Mutant Interpretation:<br /> While cpr5 mutants exhibited reduced macromolecular transport, the authors state that they cannot exclude that the reduced transport is due to secondary effects in the cpr5 mutants, which show rather severe phenotypic defects. This is an important distinction, as CPR5 has known roles in defense responses and hormone signaling that could indirectly influence PD integrity, independent of callose deposition. The lack of effect on small molecule transport is a good control, but the broader pleiotropic effects of cpr5 mutants remain a consideration.
(4) Conceptual Distinction between NPC and PD:<br /> The authors correctly point out that while similarities exist, the physical assembly of NUPs at PD must differ from that at the NPC due to the presence of the desmotubule and smaller cytoplasmic sleeve width at PD. Moreover, nucleocytoplasmic transport depends on karyopherin proteins that interact with the NPC central channel to complete the transport. Yet the role of karyopherins in this case is not clear. Therefore, the proposed "PD pore complex" may bear some NPC features, but not be identical.
Reviewer #3 (Public review):
Summary:
This manuscript presents a step towards testing the hypothesis that plasmodesmata have homology to nuclear pores. The similarities between the two structures have long been noted as both structures allow the transport of proteins and nucleic acids, and both structures are composed of curved membranes. The manuscript has identified nuclear pore proteins (NUPs) in plasmodesmal protein fractions and uses live imaging in a non-endogenous system and functional assays of a mutant to propose that this might be a bona fide association.
The conclusions the authors seek to draw are that: NUPs are present in plasmodesmal protein fractions; NUPs localise at plasmodesmata; NUPs might form a pore-gating complex at plasmodesmata, regulating non-specific (2xGFP) and specific (SHR) transport through plasmodesmata
The authors then use these conclusions to propose the possibility that phase separation mediates transport through plasmodesmata. If there is phase separation at plasmodesmata or a nuclear pore-like complex, it would revolutionise the community. However, this data is insufficient to act as a cornerstone for such a discovery.
Strengths:
The strength of the manuscript lies in the boldness and novelty of the idea.
Weaknesses:
The weaknesses lie in the lack of informative controls. The authors' own assessments of their data suggest they agree with this - in their abstract alone, they point out that the transport defects they observe might be off-target effects, and suggest there is a requirement in the future to determine whether the NUPs are bona fide PD components.
Across the proteomic and live imaging experiments, the conclusions could be stronger if they compared the NUP localisation and accumulation with ER proteins - the question of whether NUPs behave like other ER proteins is not addressed. As NUPs reside in the nuclear envelope, continuous with the ER, and the ER traverses plasmodesmata, a comparison between the NUPs and ER proteins would be extremely informative.
Regarding the proteomic identification of NUPs in plasmodesmal fractions, the authors place significant weight on their own metric for PD enrichment, the PD score. As I understand it, this a metric derived from addition of two factors: a two component enrichment score that is the difference between intensity of peptides of a given protein in the PD fraction and cell wall fraction, added to the difference between intensity of peptides of a given protein in the PD fraction and total cell fraction, and a feature score that is a factor that describes representation of protein domains contained in said given protein in the plasmodesmal fraction relative to the representation of that domain in proteins in the whole proteome. The features chosen for analysis are not indicated, and the feature factor, as I understand it, is a score common to all proteins with a given feature. While each of the factors carries a measure of meaning and information, I do not understand how adding them is mathematically or biologically meaningful.
Reviewer #3 (Public review):
Summary:
Mutations that result in consistent RAS activation constitute a major driver of cancer. Therefore, RAS is a favorable target for cancer therapy. However, since normal RAS activity is essential for the function of normal cells, a mechanism that differentiates aberrant RAS activity from normal one is required to avoid severe adverse effects. To this end, the authors designed and optimized a synthetic gene circuit that is induced by active RAS-GTP. The circuit components, such as RAS-GTP sensors, dimerization domains, and linkers. To enhance the circuit selectivity and dynamic range, the authors designed a synthetic promoter comprised of MAPK-responsive elements to regulate the expression of the RAS sensors, thus generating a feed-forward loop regulating the circuit components. Circuit outputs with respect to circuit design modification were characterized in standard model cell lines using basal RAS activity, active RAS mutants, and RAS inactivation.
This approach is interesting. The design is novel and could be implemented for other RAS-mediated applications. The data support the claims, and while this circuit may require further optimization for clinical application, it is an interesting proof of concept for targeting of aberrant RAS activity. I therefore recommend accepting this paper.
Strengths:
Novel circuit design, through optimization and characterization of the circuit components, solid data.
Weaknesses:
This manuscript could significantly benefit from testing the circuit performance in more realistic cell lines, such as patient-derived cells driven by RAS mutations, as well as in corresponding non-cancer cell lines with normal RAS activity. Furthermore, testing with therapeutic output proteins in vitro, and especially in vivo, would significantly strengthen the findings and claims.
Summary:
Given the revision made, I would recommend a minor revision that discusses the specificity limitations of this experimental setup.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
The manuscript by Senn and colleagues presents a comprehensive study on the developing synthetic gene circuits targeting mutant RAS-expressing cells. This study aims to exploit these RAS-targeting circuits as cancer cell classifiers, enabling the selective expression of an output protein in correlation with RAS activity. The system is based on the bacterial two-component system NarX/NarL. A RAS-binding domain, the RBDCRD domain of the RAS effector protein CRAF, is fused to the histidine kinase domain, which carries an inactivating amino acid exchange either in its ATP-binding site (N509A) or in its phosphorylation site (H399Q). Dimerization or nanocluster formation of RAS-GTP reconstitutes an active histidine kinase sensor dimer that phosphorylates the response regulator NarL. The phosphorylated DNA-binding protein NarL, fused to the transcription activator domain VP48, binds its responsive element and induces the expression of the output protein. In comparison to mutated RAS, the effect of the RAS activator SOS-1 and the RAS inhibitor NF1 on the sensing ability as well as the tunability of the RAS sensor were examined. A RAS targeting circuit with an AND gate was designed by expressing the RAS sensor proteins under the control of defined MAPK response elements, resulting in a large increase in the dynamic range between mutant and wild-type RAS. Finally, the RAS targeting circuits were evaluated in detail in a set of twelve cancer cell lines expressing endogenous levels of mutant or wild-type RAS or oncogenes affecting RAS signaling upstream or downstream.
Strengths:
This proof-of-concept study convincingly demonstrates the potential of synthetic gene circuits to target oncogenic RAS in tumor cell lines and to function, at least in part, as an RAS mutant cell classifier.
Weaknesses:
The use of an appropriate "therapeutic gene" might revert the oncogenic properties of RAS mutant cell lines. However, a therapeutic strategy based on this four-plasmid-based system might be difficult to implement in RAS-driven solid cancers.
Thank you for the insightful comments. We agree that the delivery of a four-plasmid system represents a major challenge for translating RAS-targeting circuits into therapeutic applications. Reducing the number of plasmids –ideally consolidating all components onto a single vector– will be critical for clinical implementation.
Viral delivery is generally the most efficient strategy for DNA-based therapies, but viral vectors have limited packaging capacities, which differ by virus type[1]. The RAS_sensor_F.L.T. circuit under the EF1α promoter requires ~7.7 kb for the sensing components alone, excluding the output gene. This exceeds the packaging limit of adeno-associated virus (AAV) and is at the upper boundary for lentiviral vectors but could potentially be accommodated by larger vectors such as γ-retroviruses, poxviruses, or herpesviruses¹. Co-transduction with dual AAVs [2] or ongoing engineering to expand packaging capacity [3] may also offer future solutions. An additional route to reduce construct size could be alternative splicing, especially given redundancy between the two NarX fusion proteins[4].
An advantage of our current architecture is that synthetic response elements replace constitutive promoters, reducing construct size. For example, the MAPK-driven PY2_NarX&NarL circuits range between 4.9 and 5.2 kb depending on the transactivation domain, bringing them within AAV packaging limits for the sensor module[5], though co-delivery of the output gene would still be necessary. For lentiviruses, this is within the packaging capacity of 8 kb<sup>1</sup> and would allow for inclusion of ~3 kb output genes.
Still, assembling multiple modules onto a single vector introduces new challenges, including possible crosstalk or interference between neighboring promoters [6]. For example, placing the output gene too close to MAPK response elements may trigger unwanted MAPKdependent expression, potentially bypassing the intended AND-gate logic. Moreover, expressing three genes under separate response elements may shift expression ratios and reduce circuit functionality. Nonetheless, the absence of constitutive promoters and the RAS-dependence of MAPK response elements could provide partial robustness, since even unintended activation would still reflect RAS signaling to some extent. Further, our data (Fig. 1d) show that some deviation in component levels can be tolerated, provided all parts are sufficiently expressed. Nonetheless, assembling the circuit on a single vector will require careful design and rigorous validation to ensure optimal performance.
While addressing this is beyond the scope of the current study, we agree that future efforts should focus on vector consolidation and delivery strategies. We now include a paragraph discussing these challenges in the revised manuscript.
Reviewer #2 (Public review):
The manuscript describes an interesting approach towards designing genetic circuits to sense different RAS mutants in the context of cancer therapeutics. The authors created sensors for mutant RAS and incorporated feed-forward control that leverages endogenous RAS/MAPK signaling pathways in order to dramatically increase the circuits' dynamic range. The modularity of the system is explored through the individual screening of several RAS binding domains, transmembrane domains, and MAPK response elements, and the author further extensively screened different combinations of circuit components. This is an impressive synthetic biology demonstration that took it all the way to cancer cell lines. However, given the sole demonstrated output in the form of fluorescent proteins, the authors' claims related to therapeutic implications require additional empirical evidence or, otherwise, expository revision.
Thank you very much for the thoughtful evaluation, precise critique, and constructive suggestions.
As correctly noted, our study initially focused on developing and optimizing input sensors and processing units for synthetic gene circuits targeting mutated RAS. To address the concern regarding therapeutic relevance, we have now incorporated functional validation using a clinically relevant output protein: herpes simplex virus thymidine kinase (HSV-TK), which converts ganciclovir into a cytotoxic compound. We replaced the mCerulean reporter with HSV-TK and tested the resulting RAS-targeting circuits in both RAS-mutant and wild-type cancer cell lines. The results, now presented in a new chapter (Figure 8 and Supplementary Fig. 14), demonstrate robust killing of RAS-mutant cells and support the potential therapeutic utility of these circuits.
Major comments:
"These therapies are limited to cancers with KRASG12C mutations" is technically accurate. However, in this fast-moving field, there are examples such as MRTX1133 which holds the promise to target the very G12D mutation that is the focus of this paper. There are broader efforts too. It would help the readers better appreciate the background if the authors could update the intro to reflect the most recent landscape of RAS-targeting drugs.
Thank you for this helpful suggestion. We have updated the introduction to reflect the rapidly evolving landscape of RAS-targeting therapies, including the development of inhibitors for nonG12C mutations such as KRASG12D (e.g., MRTX1133). Given the pace and breadth of these advances, we also refer readers to a recent comprehensive review that provides an in-depth overview of current RAS-targeting strategies.
Only KRASG12D was used as a model in the design and optimization work of the genetic circuits. Other mutations should be quite experimentally feasible and comparisons of the circuits' performances across different KRAS mutations would allow for stronger claims on the circuits' generalizability. Particularly, the cancer cell line used for circuit validation harbored a KRASG13D mutation. While the data presented do indeed support the circuit's "generalizability," the model systems would not have been consistent in the current set of data presented.
To further support the generalizability of our RAS sensor, we titrated plasmid doses for a panel of oncogenic RAS variants, including multiple KRAS mutants as well as HRAS<sup>G12D</sup and NRAS<sup>G12D</sup. Across all tested variants, we observed concentration-dependent activation of the RAS sensor. At 1.67 ng/well, the sensor output for all oncogenic RAS variants was at least as high as that for KRAS<sup>G12D</sup>, suggesting that the behavior observed in our initial design and optimization is representative of a broader set of RAS mutations.
We also noted that high overexpression of wildtype HRAS and NRAS can lead to substantial activation of the sensor, exceeding that observed with wildtype KRAS. This underscores the importance of considering all RAS isoforms when assessing circuit specificity and avoiding potential off-target activation in healthy cells.
In Figure 2a, the text claims that "inactivation of endogenous RAS with NF1 resulted in a lower YFP/RBDCRD-NarX expression," but Figure 2a does not show a statistically significant reduction in expression of SYFP (measured by "membrane-to-total signal ratio [RU]).
Thank you for pointing this out. We repeated the experiment to reassess the effect of NF1 on RBDCRD-NarX-SYFP2 expression and were able to confirm statistical significance. Accordingly, we have replaced Figure 2a with updated data. To facilitate better visual comparison across conditions, we also standardized the y-axis range across all relevant flow cytometry plots.
The therapeutic index of the authors' systems would be better characterized by a functional payload, other than florescent proteins, that for example induce cell death, immune responses, etc.
Thank you for this insightful comment. We agree that fluorescent reporters are limited to approximating expression levels, and that a functional output protein is more appropriate for assessing therapeutic potential. To address this, we replaced mCerulean with the therapeutic suicide-gene, HSV-TK, and tested the circuits in RAS-mutant and wild-type cancer cell lines. These experiments demonstrate that our circuits can express functional proteins and induce cell death in two RAS-mutant cell lines while showing low toxicity in a RAS wild type cell line (new chapter including Fig. 8 and Supplementary Fig.14).
Comparing confluence of cells transfected with the RAS-targeting circuits to cells transfected with non-toxic GFP-output negative control or the constitutively expressed EF1αHSV-TK positive control allowed us to estimate the killing-strength of the circuits in each cell line. In RAS-mutant HCT-116 the confluence curves were similar to the positive control, indicating effective killing (Fig. 8b). At lower DNA dose in HCT-116, or in SW620 with lower transfection efficiency, the killing of transfected RAS-driven cancer cells was less pronounced, falling approximately midway between the controls (Fig. 8g&j). In the RAS wild type cell line, Igrov-1, the RAS circuits showed continued growth similar to the non-toxic negative control (Fig. 8d), suggesting low toxicity.
While this may indicate low circuit activation in Igrov-1, an alternative explanation for the low toxicity could also be insufficient transfection efficiency. Testing in SW620 –which had similar transfection efficiency as Igrov-1 (Supplementary Fig. 14a)– showed that this moderate transfection efficiency was sufficient for RAS-circuit-dependent killing (Fig. 8d & 8g), supporting the notion of low activation in Igrov-1 and selective cytotoxicity in RAS-driven cancer cells.
Nonetheless, it is important to note that comparisons between the cell lines need to be interpreted cautiously because of inter-cell line differences in transfection, growth, and HSV-TK/ganciclovir (GCV)-sensitivity (Supplementary Fig. 14) and further validation will be essential.
A conclusive assessment will require more efficient delivery strategies, such as viral vectors (as discussed above). Efficient delivery would allow to investigate selectivity in a more realistic setting with patient-derived RAS-mutant cancer and healthy cells as well as testing in an vivo model. While beyond the scope of the current study, we view it as a critical direction for future work and have therefore added a paragraph about this to our discussion.
Regarding data presented in "Mechanism of action" (Figure 2), the observations are interesting and consistent across different fluorescent reporters. However, with regard to interpretations of the underlying molecular mechanisms, it is not clear whether the different output levels in 2b, 2c, and 2d are due to the pathway as described by the authors or simply from varied expression levels of RBDCRD-NarX itself (2a) that is nonlinearly amplified by the rest of the circuit. From a practical standpoint, this caveat is not critical with respect to the signal-to-noise ratios in later parts of the paper. From a mechanistic interpretation standpoint, claims made forth in this section are not clearly substantiated. Some additional controls would be nice. For example, if the authors express NarXs that constitutively dimerize on the membrane, what would the RasG12Dresponsiveness look like? Does RasG12D alter the input-output curve of NarL-RE? How would Figure 4f compare to a NaxR constitutively dimerized control that only relies on transcriptional amplification of the Ras-dependent promoters?
This is a great point. We agree that the observed differences in output levels (Fig. 2) could arise from non-linear amplification due to increased expression of RBDCRD-NarX, rather than RAS binding or dimerization alone. To further investigate this possibility, we performed titrations of KRAS<sup>G12D</sup> in combination with the functional RAS sensor and a series of constitutively active and inactive control constructs (Supplementary Fig. 4).
Inactive controls lacking NarX dimerization showed only a modest increase in output expression, similar to direct mCerulean expression under the EF1α promoter. Transfection of the output plasmid alone, with NarL, or with NarL and non-RAS-binding RBD<sup>R89L</sup> CRD<sup>C168S</sup> -NarX, resulted in minimal RAS-dependent increases (Supplementary Fig. 4a). Importantly, after normalization using the EF1α-driven mCherry transfection control, these effects were fully or even slightly over-compensated (Supplementary Fig. 4b), showing that we don’t include the effect of EF1α-dependent increased leakiness in the data presented throughout the manuscript, but also that –due to the normalization– we potentially underestimate the dynamic range of the RAS-targeting circuits.
In contrast, constitutively dimerizing NarX controls (both membrane-bound and cytosolic dimerized via the FKBP–FRB system) exhibited a more pronounced RAS-dependent increase in output –even after normalization– confirming the presence of non-linear amplification (up to 3–4fold). However, this effect was still lower than that achieved with the functional RAS-binding sensor (8-fold at 1.67 ng/well KRAS<sup>G12D</sup>; 14-fold at 5–15 ng/well), indicating that the increase in expression of the sensor parts is not the full explanation of the effect we see. Instead, RAS binding and dimerization further amplify the response and are necessary for full activation (Supplementary Fig. 4b).
We also addressed the reviewer’s suggestion by testing the MAPK response elements used in Fig. 4f with constitutively dimerizing NarX. These controls generally showed lower fold changes between KRAS<sup>G12D</sup>; and KRAS<sup>WT</sup> than the corresponding RAS-binding circuits (Supplementary Fig. 7), with one exception: the combination of SRE_NarX and PY2_NarL-VP48.
Together, these data show that non-linear amplification via increased expression and dimerization contributes to output activation. However, RAS binding and induced dimerization of the NarX sensor are required for full functionality and enhanced signal strength. This underscores that integrating the MAPK response elements with the binding-based RAS sensor into RAS-targeting circuits generally improves the distinction between cells with KRAS<sup>G12D</sup>; and KRAS<sup>WT</sup> and that it was the combination that allowed to reach maximal fold changes.
It's also possible that these Ras could affect protein production at the post-transcriptional or even post-translational levels, which were not adequately considered.
Thank you for this comment. We now mention in the manuscript the potential mechanisms by which (over-)activated RAS or MAPK signaling can increase protein synthesis. We cite relevant reports of the mechanisms we found, including upregulation of translational initiation and machinery[10] and ribosomal biogenesis[11].
The text claims that "in contrast to what we saw in HEK293 overexpressing RAS (Figure 5d), the "AND-gate" RAS-targeting circuits do not generate higher output than the EF1a-driven, bindingtriggered RAS sensor in HCT-116. Instead, the improved dynamic range results from decreased leakiness in HCT- 116k.o." Comparing the experiment from Figure 5d, which looks at activation in KRASG12D and KRASWT, to the experiments in Figure 6b-d, which looks at activation in HCT-116WT and HCT-116KO is misleading. In Fig 5d., cells are transfected with KRASG12D and KRASWT to emulate high levels of mutant RAS and high levels of wild-type RAS. In Figures 6b-d, HCT-116WT has endogenous levels of mutant RAS, while the KCT-116KO is a knock-out cell line, and does not have mutant or WT RAS. Therefore, the improved dynamic range or "decreased leakiness in HCT-116KO" in comparison to Figure 5d. is more comparable to the NF1 condition from Figure 2, which deactivates endogenous RAS. While this may not be feasible, the most accurate comparison would have been an HCT-116KO line with KRASWT stably integrated.
Thank you for this input. We understand that comparing the results from HEK293 cells transfected with KRAS<sup>G12D</sup>; or KRAS<sup>WT</sup> (Fig. 5d) to those from HCT-116<sup>WT</sup> and HCT-116<sup>k.o</sup>. cells (Fig. 6b–d) may be misleading if interpreted as a direct comparison of RAS signaling levels. Our intent was not to compare HEK293 with KRAS<sup>WT</sup> directly to HCT-116<sup>k.o</sup>.., but rather to contrast the behavior of the EF1α-driven RAS sensor and the MAPK-responsive RAS-targeting circuits within each cell line context.
Specifically, we observed that in HEK293 cells expressing KRAS<sup>G12D</sup>, the MAPK-based RAS-targeting circuits produced higher output than the EF1α-expressed RAS sensor. In contrast, in HCT-116<sup>WT</sup> cells, the EF1α-expressed RAS sensor resulted in higher output levels than the RAS-targeting circuits. Despite this, the MAPK-driven circuits showed an improved dynamic range compared to the EF1α-expressed RAS sensor in HCT-116, due to the reduced background expression in the HCT-116<sup>k.o</sup>.. cells. We have revised the manuscript text to clarify this distinction.
We agree that an HCT-116<sup>k.o</sup> cell line with stable integration of KRAS<sup>WT</sup> would provide a more direct comparison. Nonetheless, HCT-116<sup>k.o</sup>.. cells still express endogenous NRAS and HRAS, both of which are capable of activating the RAS sensor (as shown in Fig. 1g). Therefore, we believe that HCT-116<sup>k.o</sup>. cells are more comparable to HEK293 with KRAS<sup>WT</sup> than to the NF1 condition in Fig. 2, in which all endogenous RAS isoforms are inactivated.
We couldn't locate the citation or discussion of Figure 4d in the text. Conversely, based on the text description, Figure 6g would contain exciting results. But we couldn't find Figure 6g anywhere ... unless it was a typo and the authors meant Figure 6f, in which case the cool results in Figure S8 could use more elaboration in the main text.
Thank you for this helpful observation. The figure references were indeed incorrect due to a typo. The results discussed in the text refer to Figure 6f (not 6g), which is now Figure 7a in the revised version. To further highlight these findings, we have added a new Figure 7b that better illustrates how different MAPK response elements enabled us to identify, for each RAS-mutant cell line, a RAS-targeting circuit that showed stronger activation than in all RAS wild-type lines. We have also expanded the corresponding section in the main text to elaborate on these results and their significance.
Reviewer #3 (Public review):
Summary:
Mutations that result in consistent RAS activation constitute a major driver of cancer. Therefore, RAS is a favorable target for cancer therapy. However, since normal RAS activity is essential for the function of normal cells, a mechanism that differentiates aberrant RAS activity from normal one is required to avoid severe adverse effects. To this end, the authors designed and optimized a synthetic gene circuit that is induced by active RAS-GTP. The circuit components, such as RAS-GTP sensors, dimerization domains, and linkers. To enhance the circuit selectivity and dynamic range, the authors designed a synthetic promoter comprised of MAPK-responsive elements to regulate the expression of the RAS sensors, thus generating a feed-forward loop regulating the circuit components. Circuit outputs with respect to circuit design modification were characterized in standard model cell lines using basal RAS activity, active RAS mutants, and RAS inactivation.
This approach is interesting. The design is novel and could be implemented for other RASmediated applications. The data support the claims, and while this circuit may require further optimization for clinical application, it is an interesting proof of concept for targeting aberrant RAS activity.
Strengths:
Novel circuit design, through optimization and characterization of the circuit components, solid data.
Weaknesses:
This manuscript could significantly benefit from testing the circuit performance in more realistic cell lines, such as patient-derived cells driven by RAS mutations, as well as in corresponding non-cancer cell lines with normal RAS activity. Furthermore, testing with therapeutic output proteins in vitro, and especially in vivo, would significantly strengthen the findings and claims.
Thank you very much for the thoughtful and supportive comments. We fully agree with the reviewer’s suggestions for improving the translational potential of the RAS-targeting circuits.
As a first step toward therapeutic relevance, we replaced the fluorescent reporter with HSV-TK, a clinically validated suicide gene, and demonstrated killing in RAS-mutant cancer cell lines. This is described above and in the new section of the manuscript (Figure 8).
We also agree that testing in patient-derived cancer cells and especially healthy cells with wild-type RAS activity will be essential. However, testing in primary or patient-derived cells presents delivery challenges: transient transfection of our current four-plasmid system is unlikely to achieve sufficient expression. As discussed in our response to Reviewer #1, development of a more efficient delivery strategy –such as viral vector-based delivery– is a necessary next step.
Once a delivery system is established, identifying relevant off-target tissues throughout the body with high physiological RAS signaling will be key to assessing selectivity. While comparative data on RAS activation across healthy tissues are scarce[12,13], recent atlases of transcription factor activity[14,15] provide insights to identify off-target cells with high activation of RAS-dependent transcription factors and may even approximate RAS activity across healthy tissue. Alternatively, our single-input sensors for RAS and MAPK pathway activity could be used in vivo to identify off-target cells based on endogenous activity.
Once relevant target and off-target cells have been identified, patient-derived cancer and healthy cells can help select and adapt cancer-specific RAS-targeting circuits and nominate therapeutic candidates for further safety and efficacy assessment[6,8].
Reviewer #1 (Recommendations for the authors):
For the most part, the data in this study are very convincing and very well presented. The cartoons make it easier to understand the complex experimental setups.
(1) Did the authors use wild-type Sos-1 or a constitutively active membrane-bound catalytic domain in their studies? How is SOS-1 activated when in case Sos-1 wild-type was used?
Thank you for this feedback. We used the constitutively active catalytic domain of Sos-1 (AA5641049; PDB ID 2II0).
(2) Figure 1f: In case of KRAS-G12D, it looks like the output expression does not really correlate with the RAS-GTP level. Can the authors give an explanation?
Thank you for this interesting question. We believe the observed discrepancy arises primarily from differences in the sensitivity and readout dynamics of the two assays. The RAS-GTP pulldown ELISA appears insufficiently sensitive to detect small changes in RAS-GTP levels at lower KRAS<sup>G12D</sup> plasmid doses (0.19, 0.56, or 1.67 ng). Only at 5 ng and 15 ng do we observe clear increases in RAS-GTP signal (25% and 700%, respectively). In contrast, the RAS sensor shows strong activation already in the 0.56–5 ng range but begins to saturate at higher doses (see Figure 1f and Figure 1e).
Beyond the differing technical sensitivities of the ELISA (plate reader) and flow cytometry, an important conceptual distinction may further explain this behavior: the RAS sensor likely integrates RAS signaling over time. Once NarX binds RAS-GTP and dimerizes, it activates NarL, triggering mCerulean expression. If the rate of mCerulean production exceeds its degradation, signal accumulates throughout the assay duration. Thus, the flow cytometry readout reflects time-integrated signaling, allowing small differences in RAS-GTP to be amplified into measurable differences in output—especially at low input levels. This may explain why flow cytometry detects circuit activation earlier and more steeply than the pulldown assay, which provides a snapshot of RAS-GTP abundance at a single time point and saturates less readily at high input levels.
Together, these factors likely explain the observed differences in signal dynamics: the RAS sensor exhibits steep activation followed by saturation at high plasmid doses (flow cytometry), while the ELISA shows limited sensitivity at low doses but a broader linear range at higher doses.
(3) Figure 2b: It appears that even in the case of KRAS-G12D and Sos-1, only a few cells are positive. Does this result depend on low cell density, low transfection efficiency, or a wide range of the expression level? As a control, nuclear staining could be shown.
Thank you for this question. In the experiment shown in Figure 2b, our goal was to assess the membrane localization of the RBD^CRD-NarX-SYFP2 construct, which serves as a proxy for RAS-bound sensor. To enable accurate computational segmentation and separation of membrane signal from adjacent cells, we intentionally reseeded cells at low density in glassbottom plates for confocal imaging.
The observed variability in signal likely reflects a combination of transient transfection and heterogeneous expression levels. While the overall transfection efficiency was approximately 70%, expression varied between individual cells. To account for this, we analyzed the membrane-to-total signal ratio per cell, which internally normalizes the membrane signal to the total cellular expression of SYFP2 and controls for differences in transfection efficiency.
In response to the reviewer’s suggestion, we have updated the figure to include nuclear staining to aid interpretation. We would like to emphasize, however, that the images are intended to illustrate subcellular localization per cell, not expression frequency or intensity across the population.
Minor points
(1) Figure 1b: "The third plasmid expresses NarL, .." should be changed to "The third plasmid expresses NarL-VP48, .."
Done
(2) Figure 1c, right part: The orange arrow should be labeled NarX-H399Q (not N509A).
Done
(3) Supplementary Table 6 and 7: [cells/wells] - should probably be [cells 10*3/well].
Thank you for these points, we updated the manuscript accordingly
Reviewer #2 (Recommendations for the authors):
Minor comments:
(1) N509A seems mislabeled in Figure 1b.
(2) It would help the readers if the authors could elaborate a bit on what is known about the RBD and CRD mutations used here.
Thank you for the input, we added a paragraph in the paper to expand on the effect of these commonly used mutations.
(3) The KRASWT&Sos1 condition is not explained within the text for Figure 1f, which is the first figure with the KRASWT&Sos1 condition, but rather later on for Figure 2a. Adding a description of this condition to the discussion of Figure 1f would add clarity to this figure.
Thank you, we corrected this.
(4) Citing AlphaFold2 structural predictions as having "revealed that longer linkers between the sensor's RBDCRD and NarX-derived domains could bring the NarX domains into closer proximity" is probably an overstatement. AlphaFold2 generally has low confidence in the placement of long flexible linkers, and the longer linkers in the illustration could facilitate NarX and NarL being even farther apart than they are in the original design.
Thank you for this input. We agree that AlphaFold2 predictions generally have low confidence in the placement of long, flexible linkers, and we did not intend to imply that the structural models were predictive of actual linker conformations. Rather, the models were used heuristically to generate the hypothesis that longer linkers might facilitate better positioning of the NarX domains for dimerization.
As described in the Methods, we manually rotated the flexible linker regions to explore plausible conformations. These exploratory models showed that with a short (1x GGGGS) linker, it was more challenging to bring the NarX domains into close proximity, whereas longer linkers allowed greater positional flexibility. This modeling exercise provided a structural rationale for experimentally testing longer linkers. We have revised the manuscript text to clarify that the structural predictions were used to motivate linker design –not to validate or predict structural outcomes.
(5) Figure 3b shows that the fold change (KRASG12D/KRASWT) is higher at shorter linker lengths and lower at longer linker lengths, and that the output expression of mCerulean is lower at shorter linker lengths and higher at longer linker lengths. Having a bar plot with the output expression mCerulean levels comparing KRASG12D and KRASWT next to each other would be a significantly more informative representation of this data. In particular, the readers might be interested in understanding the effect of linker length on off-target activation from the sensor, which is not clear from this figure.
Thank you for the suggestion. We adapted Figure 3b to better present this.
(6) While it is implied that the sentence "Among the tested binding domains, the Ras association domain (RA) of the natural RAS effector Rassf5, the RAS association domain 2 (RA2) of the phospholipase C epsilon (PLCe)33, and the synthetic RAS binder K5534 showed a slightly higher or similar dynamic range." is comparing these RAS binding domains to RBDCRD, for clarity it should be noted what the point of reference is for this "slightly higher or similar dynamic range."
(7) Claims are made throughout the text that require supporting data, and thus require a reference to a figure, but there are a few instances where the reference is several sentences after the discussion of data and findings begins. For example, the discussion of Figure 3c begins with the claim "Among the tested binding domains, the Ras association domain (RA) of the natural RAS effector Rassf5, the RAS association domain 2 (RA2) of the phospholipase C epsilon (PLCe)33, and the synthetic RAS binder K5534 showed a slightly higher or similar dynamic range," but there is no reference to the data or figure being discussed until the end of the discussion of Figure 3c. This formatting is also present in Figure 3d and Figure 6f.
Thank you for mentioning these imprecisions and inconsistencies, we addressed them in the manuscript.
(8) In Figures 5d and 5e, the formatting of underscores and dashes is occasionally inconsistent within the text. (ex. "PY2_NarX_FLT or PY2_NarL-FLT" on page 13.).
Thank you for this precise observation. The formatting differences were intentional and reflect distinct design principles. Specifically:
An underscore (e.g., PY2_NarX_FLT) denotes that two separate proteins are expressed –here, PY2-driven RBDCRD-NarX and EF1α-driven NarL-F.L.T.
A dash (e.g., PY2_NarL-F.L.T.) indicates a fusion protein –i.e., PY2-driven NarL-F.L.T. combined with EF1α-driven RBDCRD-NarX.
This notation is used to distinguish expression sources and fusion constructs while avoiding redundancy with the base circuit (EF1α_NarX + EF1α_NarL-VP48). We hope the included schematic diagrams in each relevant figure helps the reader interpret these combinations.
(9) The text claims that "loss-of-function mutations in RBDCRD decreased activation. However, the dynamic range was only 3-fold" and attributes this claim to Figure 6a. For a claim about specific fold-change activation, one would expect a corresponding figure with quantitative measurements of this fluorescence to be referenced.
Thank you for this remark. We made a supplementary figure (Supplementary Fig. 11) to show the quantitative measurement of the 3-fold dynamic range between HCT-116<sup>WT</sup> and HCT-116<sup>k.o</sup>. when using the EF1a-expressed RAS sensor with NarL-VP48.
(10) The claim of this Figure 2d is that the effect of RAS-GTP levels on mCerulean output is amplified in comparison to Figures 2a, 2b, and 3c, representing expression, RAS binding, and dimerization respectively. While visually this might be true from the figure, the readers might be confused by the lack of significance between the control and the NF1 condition, alongside the variation between the triplicates. Could this experiment be repeated to gain clearer data and to support their claim more effectively?
Thank you for this important observation. To address the concern regarding variability and statistical significance in Figure 2d, we repeated the experiment using 24-well plates to increase the number of cells analyzed per condition. This improved the consistency of the data and allowed us to reduce variability across replicates. As a result, we now observe a statistically significant difference between the control and the NF1 condition. The updated results are shown in the revised Figure 2.
(11) The readers might be less familiar with the concept of "composability" than "modularity" and it would be good to explain it if the authors did intend to use the former.
Thank you for this comment. We changed it to modularity to avoid confusion.
References
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Reviewer #3 (Public review):
Summary:
This manuscript reports the discovery of new compounds that selectively inhibit SMARCA4/SMARCA2 ATPase activity and have pronounced effects on uveal melanoma cell proliferation. They induce apoptosis and suppress tumor growth, with no toxicity in vivo. The report provides biological significance by demonstrating that the drugs alter chromatin accessibility at lineage specific gene enhancer regions and decrease expression of lineage specific genes, including SOX10 and SOX10 target genes.
Strengths:
The study provides compelling evidence for the therapeutic use of these compounds and does a thorough job at elucidating the mechanisms by which the drugs work. The study will likely have a high impact on the chromatin remodeling and cancer fields. The datasets will be highly useful to these communities.
Weaknesses:
The authors have addressed all my concerns.
Author response:
The following is the authors’ response to the current reviews.
Reviewer 1:
While BAP1 mutant UM cell lines were included for some of the experiments, it seems the in-vivo data mentioned in the response to the reviewers comment is missing? The authors stated that "MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor." But the CDX model data shown in Figure 4 is from 92.1 cells. If this data is available, then the manuscript would benefit from its addition.
We thank the reviewer for bringing this to our attention. As the reviewer mentioned, we show 92-1 CDX model in our manuscript. Additionally, strong tumor growth inhibition in MP-46 CDX model treated with our BAF ATPase inhibitor can be found in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/).
Reviewer 3:<br /> Supplementary Figure 2C<br /> Is the T910M mutation in the parental MP41 cells heterozygous? If so, the authors should indicate this in the figure legend. If this is a homozygous mutation, the authors should explain how the inhibitors suppress SMARCA4 activity in cells that have a LOF mutation.
We thank the reviewer for bringing this to our attention. We updated the figure legend accordingly to reflect the genotype of the mutations highlighted in the table.
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Summary:
The presented study by Centore and colleagues investigates the inhibition of BAF chromatin remodeling complexes. The study is well-written, and includes comprehensive datasets, including compound screens, gene expression analysis, epigenetics, as well as animal studies. This is an important piece of work for the uveal melanoma research field, and sheds light on a new inhibitor class, as well as a mechanism that might be exploited to target this deadly cancer for which no good treatment options exist.
Strengths:
This is a comprehensive and well-written study.
Weaknesses:
There are minimal weaknesses.
We thank the reviewer for the positive comments.
Reviewer #2 (Public Review):
Summary:
The authors generate an optimized small molecule inhibitor of SMARCA2/4 and test it in a panel of cell lines. All uveal melanoma (UM) cell lines in the panel are growth-inhibited by the inhibitor making the focus of the paper. This inhibition is correlated with the loss of promoter occupancy of key melanocyte transcription factors e.g. SOX10. SOX10 overexpression and a point mutation in SMARCA4 can rescue growth inhibition exerted by the SMARCA2/4 inhibitor. Treatment of a UM xenograft model results in growth inhibition and regression which correlates with reduced expression of SOX10 but not discernible toxicity in the mice. Collectively the data suggest a novel treatment of uveal melanoma.
Strengths:
There are many strengths of the study including the strong challenge of the on-target effect, the assays used, and the mechanistic data. The results are compelling as are the effects of the inhibitor. The in vivo data is dose-dependent and doses are low enough to be meaningful and associated with evidence of target engagement.
Weaknesses:
The authors introduce the field stating that SMARCA4 inhibitors are more effective in SMARCA2 deficient cancers and the converse. Since the desirable outcome of cancer therapy would be synthetic lethality it is not clear why a dual inhibitor is desirable. Wouldn't this be associated with more side effects? It is not known how the inhibitor developed here impacts normal cells, in particular T cells which are essential for any durable response to cancer therapies in patients. Another weakness is that the UM cell lines used do not molecularly resemble metastatic UM. These UM most frequently have mutations in the BAP1 tumor suppressor gene. It is not clear if the described SMARCA2/4 inhibitor is efficacious in BAP1 mutant UM cell lines in vitro or BAP1 mutant patient-derived xenografts in vivo.
We thank the reviewer for their insightful and constructive comments. As we demonstrate in Fig. 1d, uveal melanoma cells are selectively and deeply sensitive to BAF ATPase inhibition, and provides a therapeutic window. This is confirmed in Fig. 4a-c, as we demonstrated robust tumor growth inhibition, achieved at a dose well-tolerated in xenograft study. FHD-286, a dual BRM/BRG1 inhibitor similar to FHT-1015 with optimized physical properties, has been evaluated in a Phase I trial in patients with metastatic uveal melanoma (NCT04879017) and manuscript describing results of this clinical trial is currently in preparation.
As the reviewer mentioned, BAP1 loss is a signature of metastatic uveal melanoma. MP38 is a BAP1 mutant uveal melanoma cell line, and we demonstrated growth inhibition and robust caspase 3/7 activity in response to FHT-1015 (Supplementary Fig. 3a and 3f). MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor.
Reviewer #3 (Public Review):
Summary:
This manuscript reports the discovery of new compounds that selectively inhibit SMARCA4/SMARCA2 ATPase activity that work through a different mode as previously developed SMARCA4/SMARCA2 inhibitors. They also demonstrate the anti-tumor effects of the compounds on uveal melanoma cell proliferation and tumor growth. The findings indicate that the drugs exert their effects by altering chromatin accessibility at binding sites for lineage-specific transcription factors within gene enhancer regions. In uveal melanoma, altered expression of the transcription factor, SOX10, and SOX10 target gene underlies the anti-proliferative effects of the compounds. This study is significant because the discovery of new SMARCA4/SMARCA2 inhibitory compounds that can abrogate uveal melanoma tumorigenicity has therapeutic value. In addition, the findings provide evidence for the therapeutic use of these compounds in other transcription factor-dependent cancers.
Strengths:
The strengths of this manuscript include biochemical evidence that the new compounds are selective for SMARCA4/SMARCA2 over other ATPases and that the mode of action is distinct from a previously developed compound, BRM014, which binds the RecA lobe of SMARCA2. There is also strong evidence that FHT1015 suppresses uveal melanoma proliferation by inducing apoptosis. The in vivo suppression of tumor growth without toxicity validates the potential therapeutic utility of one of the new drugs. The conclusion that FHT1015 primarily inhibits SMARCA4 activity and thereby suppresses chromatin accessibility at lineage-specific enhancers is substantiated by ATAC-seq and ChIP-seq studies.
Weaknesses:
The weaknesses include a lack of more precise information on which SMARCA4/SMARCA2 residues the drugs bind. Although the I1173M/I1143M mutations are evidence that the critical residues for binding reside outside the RecA lobe, this site is conserved in CHD4, which is not affected by the compounds. Hence, this site may be necessary but not sufficient for drug binding or specifying selectivity. A more precise evaluation of the region specifying the effect of the new compounds would strengthen the evidence that they work through a novel mode and that they are selective. Another concern is that the mechanisms by which FHT1015 promotes apoptosis rather than simply cell cycle arrest are not clear. Does SOX10 or another lineage-specific transcription factor underlie the apoptotic effects of the compounds?
We thank the reviewer for the valuable comments.
We believe that our dual ATPase inhibitor is selective and additional insights into binding specificity and selectivity for earlier stage compounds of this series were recently published in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/).
The reviewer also poses a great question regarding the mechanism of apoptosis. The mechanism of apoptosis is extremely complex, but we observed a decrease in pro-survival BCL-2 protein expression in response to FHT-1015, in the experiment corresponding to Supplementary Fig. 5e. In the experiment described in Fig. 3k, we also monitored caspase 3/7 activity over time, and SOX10 overexpression rescued 92-1 cells from FHT-1015 induced apoptosis. This suggests the role of SOX10 as an important mediator of response to BAF ATPase inhibition, including apoptosis induced by FHT-1015.
Additional Reviews:
The referees would like to draw the authors' attention to the following issues that would best benefit from additional revision.
The clinical relevance of the study would be strengthened by the use of uveal melanoma cell lines with BAP1 mutations that better represent metastatic uveal melanoma. The use of patient-derived xenografts would also be pertinent and would be a useful addition. Similarly, attention to the effects of the inhibitor on non-cancerous proliferative cells such as blood/T/immune cells would also strengthen the manuscript. As the study reports the administration of one of the inhibitors in mice for the xenograft experiments, it would be important to assess any potential effects on blood cell counts and better discuss the eventual toxicity or lack of toxicity and how it was assessed.
The authors should better explain how SOX10 over expression can rescue viability in the presence of the inhibitor. Similarly given the critical roles of BRG1, SOX10, and MITF in cutaneous melanoma some specific discussion on the sensitivity of cutaneous melanoma cells to the inhibitor should be considered, and potential differences with uveal melanoma highlighted.
Aside from these issues, the authors are urged to consider the other points mentioned below.
Reviewer #1 (Recommendations For The Authors):
Figure 1d, as well as the text in the manuscript referring to this figure, would benefit from indicating specific cell lines used for UM. The same for the sentence in line 153.
We thank the reviewer for bringing this to our attention. We have added the cell line names and updated the manuscript accordingly.
For any of the studies conducted, is there any link with the genetics of UM? E.g. BAP1 wildtype/BAP1 mutant?
As addressed above in the public review section, MP38 is a BAP1 mutant uveal melanoma cell line, and we demonstrated growth inhibition and robust caspase 3/7 activity in response to FHT-1015 (Supplementary Fig. 3a and 3f). MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor.
Row 191 - How were peaks classified as enhancer-occupied?
We used annotatePeaks function of HOMER package to annotate genomic locations, as well as H3K27ac ChIP-seq to annotate peaks as enhancer-occupied. We thank the reviewer to pointing it out and have updated the manuscript accordingly to include this information.
Row 259, the two cell lines should be named, also in Figure 3i.
We have added the cell line names and updated the manuscript accordingly.
Reviewer #2 (Recommendations For The Authors):
As a proof of concept, this study is truly excellent and the authors should be commended. However, it is desirable that new knowledge in cancer is translated to the clinic. To this end there are a few things needed to strengthen the study.
I am rephrasing my statements from the public review to say that I would recommend testing the inhibitor in T cells (side effects) and BAP1 mutant cell lines (for clinical relevance).
As addressed in the public review section, MP38 is a BAP1 mutant uveal melanoma cell line, and we demonstrated growth inhibition and robust caspase 3/7 activity in response to FHT-1015 (Supplementary Fig. 3a and 3f). MP46 (Supplementary Fig. 3a) is BAP1-null uveal melanoma cell line with no detectable protein expression (Amirouchene-Angelozzi et al., Mol Oncol 2014), and we have observed strong tumor growth inhibition in this CDX model with our BAF ATPase inhibitor.
Regarding concerns for any potential side effect on T cells, we observed an increase in both CD4 and CD8 T-cell populations in the peripheral blood and the spleen, when naïve, non-tumor bearing CD-1 mice were dosed with SMARCA2/4 dual ATPase inhibitor FHD-286 once daily for 14 days. FHD-286 is a compound similar to FHT-1015 described in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/). In addition, FHD-286 has been tested in tumor bearing syngeneic models. When B16F10 tumor bearing C57BL/6 were dosed with FHD-286 for 10 days, we observed an increase in CD69+ activated CD8 T-cell infiltration in the tumor microenvironment (doi:10.1136/jitc-2022-SITC2022.0888).
Reviewer #3 (Recommendations For The Authors):
(1) Determine drug binding by crystal structure or generate additional SMARCA4 or SMARCA2 mutations in the region near I1173/I1143 that are not conserved in CHD4 and test them in an ATPase assay for effects on drug inhibition. For example, Q1166 in SMARCA4 and Q1136 in SMARCA4 could be changed to Alanine as in CHD4. Would this abrogate drug inhibition?
We believe that our dual ATPase inhibitor is selective and additional insights into binding specificity and selectivity for earlier stage compounds of this series were recently published in Vaswani et al., 2025 (PMID:39801091, https://pubmed.ncbi.nlm.nih.gov/39801091/).
(2) The finding that SOX10 can rescue the antiproliferative effects of FHT1015 suggests that SMARCA4 is primarily needed for SOX10 expression. However, the co-occupancy of SMARCA4 and SOX10 at enhancers suggests that they cooperate to promote chromatin accessibility. It is unclear how over-expression of SOX10 can promote chromatin accessibility in drug-inhibited cells since SOX10 does not have chromatin remodeling activity. ATAC-seq in cells over-expressing SOX10 and treated with the drug could identify SOX10-dependent targets that do not require SMARCA4 activity and clarify the mechanism. It would also be informative to determine if SOX10 over-expression abrogates the effects of FHT1015 on both cell cycle and apoptosis, helping to resolve whether it is a partial or complete rescue of proliferation.
We agree that running ATAC-seq in cells overexpressing SOX10 would clarify this mechanism. However, shifts in corporate strategy deprioritized any further experiments for this project. One potential mechanism that SOX10 overexpression can partially rescue BAF inhibition phenotype is through overexpressed SOX10 localizing to open chromatin regions (mostly promoters) across the genome. We know from our ATAC-seq data (Fig. 2) that BAF inhibition leads to loss of chromatin accessibility at SOX10 enhancer sites, while promoter regions are only partially affected. Therefore, we think that overexpression of SOX10 would allow upregulation of its target genes via binding to the promoter regions. In this model, the enhancer-driven SOX10 target genes are likely to remain silenced.
(3) Although the in vivo studies indicate that the drugs are well-tolerated, additional in vitro studies to determine the effects of the drug on the proliferation/survival of non-cancerous cells would further validate their therapeutic utility.
Author Response: The reviewer raises a critical question. FHD-286, a dual BRM/BRG1 inhibitor similar to FHT-1015 with optimized physical properties, has been evaluated in a Phase I trial in patients with metastatic uveal melanoma (NCT04879017), and it was well tolerated at continuous daily dose of up to 7.5 mg QD and at intermittent dose of up to 17.5 mg QD. Manuscript describing results of this clinical trial is currently in preparation.
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Reviewer #1 (Public review):
The manuscript "Heterozygote advantage cannot explain MHC diversity, but MHC diversity can explain heterozygote advantage" explores two topics. First, it is claimed that the recently published conclusion by Mattias Siljestam and Claus Rueffler (in the following referred to as [SR] for brevity) that heterozygote advantage explains MHC diversity does not withstand even a very slight change in ecological parameters. Second, a modified model that allows an expansion of the MHC gene family shows that homozygotes outperform heterozygotes. This is an important topic and could be of potential interest to readers if the conclusions are valid and non-trivial.
Let me first comment on the second part of the manuscript that describes the fitness advantage of the 'gene family expansion'. I think this, by itself, is a totally predictable result. It appears obvious that with no or a little fitness penalty, it becomes beneficial to have MHC-coding genes specific to each pathogen. A more thorough study that takes into account a realistic (most probably non-linear in gene number) fitness penalty, various numbers of pathogens that could grossly exceed the self-consistent fitness limit on the number of MHC genes, etc, could be more informative. Yet, as I understood the narrative of the manuscript, the expansion of the gene family serves as a mere counter-example to the disputed finding of [SR], rather than a systematic study of the eco-evolutionary consequences of this process.
Now to the first part of the manuscript, which claims that the point made in [RS] is not robust and breaks down under a small change in the parameters. An addition or removal of one of the pathogens is reported to affect "the maximum condition", a key ecological characteristic of the model, by an enormous factor 10^43, naturally breaking down all the estimates and conclusions made in [RS]. This observation is not substantiated by any formulas, recipes for how to compute this number numerically, or other details, and is presented just as a self-standing number in the text. The only piece of information given in the manuscript is that, unlike in [SR], the adjustable parameter c_{max} is kept constant when the number of pathogens is changed.
In my opinion, the information provided in the manuscript does not allow one to conclude anything about the relevance and the validity of its main claim. At the same time, the simulations done in [SR] are described with a fair amount of detail. Which allows me to assume that the conclusions made in [SR] are fairly robust and, in particular, have been demonstrated not to be too sensitive to changes in the main "suspect', c_{max}. Let me briefly justify my point.
First, it follows from Eqs (4,5) in the main text and (A12-A13) in the Appendix that c_{max} and K do not independently affect the dynamics of the model, but it's rather their ratio K/c_max that matters. It can be seen by dividing the numerator and denominator of (5) by c_max. Figure 3 shows the persistent branching for 4 values of K that cover 4 decades. As it appears from the schemes in the top row of Figure 3, those simulations are done for the same positions and widths/virulences of pathogens. So the position of x* should be the same in all 4 cases, presumably being at the center of pathogens, (x*,x*) = (0,0). According to the definition of x* given in the Appendix after Eqs (A12-A13), this means that c_max remains the same in all 4 cases. So one can interpret the 4 scenarios shown in Figure 3 as corresponding not to various K, but to various c_max that varied inversely to K. That is, the results would have been identical to those shown in Figure 3 if K were kept constant and c_max were multiplied by 0.1, 1, 10, and 100, or scaled as 1/K. This begs the conclusion that the branching remains robust to changes in c_max that span 4 decades as well.
Naturally, most, if not all, the dynamics will break down if one of the ecological characteristics changes by a factor of 10^43, as it is reported in the submitted manuscript. As I wrote above, there is no explanation behind this number, so I can only guess that such a number is created by the removal or addition of a pathogen that is very far away from the other pathogens. Very far in this context means being separated in the x-space by a much greater distance than 1/\nu, the width of the pathogens' gaussians. Once again, I am not totally sure if this was the case, but if it were, some basic notions of how models are set up were broken. It appears very strange that nothing is said in the manuscript about the spatial distribution of the pathogens, which is crucial to their effects on the condition c. In [SP], it is clearly shown where the pathogens are.
Another argument that makes me suspicious in the utility of the conclusions made in the manuscript and plays for the validity of [SP] is the adaptive dynamics derivation of the branching conditions. It is confirmed by numerics with sufficient accuracy, and as it stands in its simple form of the inequality between two widths, the branching condition appears to be pretty robust with respect to reasonable changes in parameters.
Overall, I strongly suspect that an unfortunately poor setup of the model reported in the manuscript has led to the conclusions that dispute the much better-substantiated claims made in [SD].
Reviewer #2 (Public review):
Summary:
This study addresses the population genetic underpinnings of the extraordinary diversity of genes in the MHC, which is widespread among jawed vertebrates. This topic has been widely discussed and studied, and several hypotheses have been suggested to explain this diversity. One of them is based on the idea that heterozygote genotypes have an advantage over homozygotes. While this hypothesis lost early on support, a reason study claimed that there is good support for this idea. The current study highlights an important aspect that allows us to see results presented in the earlier published paper in a different light, changing strongly the conclusions of the earlier study, i.e., there is no support for a heterozygote advantage. This is a very important contribution to the field. Furthermore, this new study presents an alternative hypothesis to explain the maintenance of MHC diversity, which is based on the idea that gene duplications can create diversity without heterozygosity being important. This is an interesting idea, but not entirely new.
Strengths:
(1) A careful re-evaluation of a published model, questioning a major assumption made by a previous study.
(2) A convincing reanalysis of a model that, in the light of the re-analysis-loses all support.
(3) A convincing suggestion for an alternative hypothesis.
Weaknesses:
(1) The statement that the model outcome of Siljestam and Rueffler is very sensitive to parameter values is, in this form, not correct. The sensitivity is only visible once a strong assumption by Siljestam and Rueffler is removed. This assumption is questionable, and it is well explained in the manuscript by J. Cherry why it should not be used. This may be seen as a subtle difference, but I think it is important to pin done the exact nature of the problem (see, for example, the abstract, where this is presented in a misleading way).
(2) The title of the study is very catchy, but it needs to be explained better in the text.
Reviewer #3 (Public review):
This manuscript describes a careful and thorough evaluation of an evolutionary simulation model published previously. The model and this report address the question, whether heterozygote advantage (HA) by itself as a selection mechanism can explain a substantial level of allelic diversity as it is often seen in MHC immune genes. Despite decades of research on the topic of pathogen-mediated selection for MHC diversity, it remains an open question by which specific selection mechanisms this exceptional allelic diversity is maintained.
The previously published paper posits, in contrast to various previous studies, that HA is, in fact, able to maintain a level of allelic diversity as seen in many populations, just by itself, given certain conditions. The current manuscript now challenges this conclusion by highlighting that the previous model results only hold under very narrow parameter ranges.
Besides criticizing some of the conceptual points of the previous paper, the author carefully rebuilt the previously published model and replicated their results, before then evaluating the robustness of the model results to reasonable variation in different parameters. From this evaluation, it becomes clear that the previously reported results hinge strongly on a certain scaling or weighing factor that is adjusted for every parameter setting and essentially counteracts the changes induced by changing the parameters. The critical impact of this one parameter is not clearly stated in the previous paper, but raises serious doubts about the generalizability of the model to explain MHC allelic variation across diverse vertebrate species.
Given the fact that the MHC genes are among the most widely studied genes in vertebrates, and that understanding their evolution will shed light on their association with various complex diseases, the insights from this report and the general discussion of how MHC diversity evolved are of interest to at least some of the community. The manuscript is very well written and makes it easy to follow the theoretical and methodological details of the model and the arguments. I have only a few minor comments that I am detailing below. Furthermore, I would be very interested to read a response by the previous authors, especially on the relevance of this scaling/weighing factor that they introduced into their model, as it is possible that I might have missed something about its meaning.
eLife Assessment
This valuable study reveals that the GSK-3 inhibitor AZD2858 inhibits the formation of TOPBP1 condensates and hence DNA damage responses in colorectal cancer cells. The evidence supporting the claims of the authors is convincing, although uncovering how this drug blocks bio-condensate formation would have strengthened the study. The work will be of interest to cancer researchers searching for synergistic drug combination strategies.
[Editors' note: this paper was reviewed by Review Commons.]
Reviewer #3 (Public review):
Summary:
The authors have extended their previous research to develop TOPBP1 as a potential drug target for colorectal cancer by inhibiting its condensation. Utilizing an optogenetic approach, they identified the small molecule AZD2858, which inhibits TOPBP1 condensation and works synergistically with first-line chemotherapy to suppress colorectal cancer cell growth. The authors investigated the mechanism and discovered that disrupting TOPBP1 assembly inhibits the ATR/Chk1 signaling pathway, leading to increased DNA damage and apoptosis, even in drug-resistant colorectal cancer cell lines.
Comments on latest version:
This reviewer does not have further comments to the paper.
Author response:
The following is the authors’ response to the original reviews
Reviewer #1:
Comments on revised version:
I have reviewed the revised manuscript and read the rebuttal. The authors have carefully addressed my concerns. There is however one point that needs further work:
This follows up on my major point #1 in my initial review. I had I asked the authors to demonstrate that FOLFIRI + AZD are less toxic to untransformed colorectal cells than colorectal cancer cell lines. It is good to see that the authors took my advice and show effects of the drug treatments on the untransformed colorectal cell line CCD841. It seems to be less sensitive to AZD and FOLFIRI in the figure in the rebuttal. What surprises me is that I cannot find these new figures anywhere in the revised manuscript. Also, the data seem preliminary, because I do not see any standard errors in the graphs, and I cannot find a description of the time of drug incubation. I ask the authors to make sure that the CCD841 data are reproducible, and make sure they incorporate the data in the revised manuscript.
We thank the reviewer for this insightful comment. In the initial revised version of the manuscript, we did not include results from the untransformed colorectal cell line CCD841, as those experiments had only been performed once and were considered preliminary. However, we fully agree with the reviewer on the importance of including these data.
To address this, we have repeated the experiments in CCD841 cells to ensure reproducibility. We now report the results from three independent experiments testing the combination of AZD2858 and FOLFIRI on healthy epithelial colon cells. These results are shown in Supplementary Figure S7, where blue matrices represent cell viability and black matrices reflect the level of synergy between AZD2858 and FOLFIRI.
Our results confirm that, individually, each drug has little to no effect on healthy cells, and no consistent synergistic interaction was observed, except in Experiment 1, which could not be reproduced. Importantly, the drug concentrations used were identical to those applied in the cancer cell experiments, allowing for direct comparison between normal and malignant cell responses.
Reviewer #2:
Comments on latest version:
Morano et al. have revised their manuscript in response to the points raised by reviewer #3 as follows.
(1) Fig. 2E: Correcting the previously erroneous labelling of this Fig. makes it match the textual description.
(2) Figs 3A and B: The revised textual description of the flow cytometry BrdU incorporation is now precise.
(3) Fig. 3E: Removing the suspect WB images is a pragmatic decision that does not significantly affect the overall conclusions of the paper.
(4) Fig. 3D: Despite its puzzling appearance this data is now described accurately in the text as "DSBs remained elevated after the combined treatment" rather than "increased after the combined treatment. A more convincing increase in the presumed damaged DNA band is evident in Fig. 4D when AZD2858 is combined with a much lower concentration of SN38 (1.5nM) which could mean that the concentration used in Fig. 3D (300nM) induced maximal damage that could not be further enhanced.
We thank the reviewer for their thoughtful comments and constructive feedback, which have helped us improve the clarity and rigor of the manuscript.
Reviewer #3:
Comments on latest version:
The authors have addressed most of the concerns that I raised in the first round of revision and I have no further questions. I appreciate the authors's efforts in carrying out an preliminary in vivo work, although as the authors pointed out the compound seems to be not effective in vivo. Future work is desired to address this to clarify the significance of the work.
We thank the reviewer for acknowledging our efforts in addressing the previous concerns. We also appreciate the recognition of our preliminary in vivo work. While these results suggest limited in vivo efficacy of the compound at this stage, we agree that additional studies will be necessary to fully evaluate its therapeutic relevance. We consider this an important next step and are committed to pursuing it in future work.
Reviewer #1 (Public review):
Summary:
The authors use Dyngo-4a, a known Dynami inhibitor to test its influence on caveolar assembly and surface mobility. They investigate whether it incorporates into membranes with Quartz-Crystal Microbalance, they investigate how it is organized in membranes using simulations. Finally, they use lipid-packing sensitive dyes to investigate lipid packing in the presence of Dyngo-4a, membrane stiffness using AFM and membrane undulation using fluorescence microscopy. They also use a measure they call "caveola duration time" to claim that something happens to caveolae after Dyngo-4a addition and using this parameter, they do indeed see an increase in it in response to Dyngo-4a, which is reduced back to the baseline after addition of cholesterol.
Overall, the authors claim: 1) Dyngo-4a inserts into the membrane and this 2) results in "a dramatic dynamin-independent inhibition of caveola scission". 3) Dyngo-4a was inserted and positioned at the level of cholesterol in the bilayer and 4) Dyngo-4a-treatment resulted in decreased lipid packing in the outer leaflet of the plasma membrane 5) but Dyngo-4a did not affect caveola morphology, caveolae-associated proteins, or the overall membrane stiffness 6) acute addition of cholesterol counteracts the block in caveola scission caused by Dyngo-4a.
Overall, in this reviewers opinion, claims 1, 3, 4, 5 are well-supported by the presented data from electron and live cell microscopy, QCM-D and AFM.
However, there is no convincing assay for caveolar endocytosis presented besides the "caveola duration" which although unclearly described seems to be the time it takes in imaging until a caveolae is not picked up by the tracking software anymore in TIRF microscopy.
Since the main claim of the paper is a mechanism of caveolar endocytosis being blocked by Dyngo-4a, a true caveolar internalization assay is required to make this claim. This means either the intracellular detection of not surface connected caveolar cargo or the quantification of caveolar movement from TIRF into epifluorescence detection in the fluorescence microscope. Otherwise, the authors could remove the claim and just claim that caveolar mobility is influenced.
Significance:
A number of small molecule inhibitors for the GTPase dynamics exist, that are commonly used tools in the investigation of endocytosis. This goes as far that the use of some of these inhibitors alone is considered in some publications as sufficient to declare a process to be dynamin-dependent. However, this is not correct, as there are considerable off-target effects, including the inhibition of caveolar internalization by a dynamin-independent mechanism. This is important, as for example the influence of dynamin small molecule inhibitors on chemotherapy resistance is currently investigated (see for example Tremblay et al., Nature Communications, 2020).
The investigation of the true effect of small molecules discovered as and used as specific inhibitors and their offside effects is extremely important and this reviewer applauds the effort. It is important that inhibitors are not used alone, but other means of targeting a mechanism are exploited as well in functional studies. The audience here thus is besides membrane biophysicists interested in the immediate effect of the small molecule Dyngo-4a also cell biologists and everyone using dynamic inhibitors to investigate cellular function.
Comments on revised version:
Please include the promised data on caveolar internalization and remove the above mentioned claim on membrane undulations from the text.
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors probe the mechanisms by which Dyngo-4a, a dynamin inhibitor used to block endocytosis, disrupts caveolae dynamics. They provide compelling evidence that Dyngo-4a inhibits caveolae dynamics and endocytosis (as well as several other aspects of plasma membrane dynamics) by a dynamin-independent mechanism. They also provide strong computational and experimental data showing that Dyngo-4a inserts into membranes and decreases lipid packing in the outer leaflet of the plasma membrane. Finally, they demonstrate that the addition of excess cholesterol to cells reverses the effects of Dyngo-4a on caveolae dynamics, presumably by reversing lipid packing defects. Based on these findings they conclude that lipid packing regulates caveolae dynamics and endocytosis in a cholesterol-dependent manner.
This work should be of value to cell biologists interested in plasma membrane remodeling and membrane trafficking, biophysicists that study small molecule/membrane interactions and membrane remodeling processes, and chemists interested in designing drugs to target membrane trafficking machinery and pathways.
Strengths:
This work addresses the important topic of how a widely used endocytic inhibitor actually works. In the process of addressing this question, the authors uncover unexpected connections between how lipids are packed in cell membranes and membrane dynamics. The methods are appropriate and many of the claims made in this work are well supported by data.
Weaknesses:
I appreciate that the manuscript has already gone through one round of revisions and that many of the concerns from the previous reviewers appear to have been addressed. However, as an interested reader, I would like to offer several additional comments for the authors to consider.
(1) It is not clear based on the data presented whether the effects of Dyngo-4a on lipid packing give rise to defects in caveolae dynamics or if these effects are merely correlated. To show this more definitively, one might expect additional experimental approaches to be used to perturb lipid packing. I appreciate this is probably beyond the scope of the current study. However, it seems important for the manuscript to be clear about how far this interpretation can be pushed in the absence of additional independent lines of evidence.
(2) On a related note, it is not obvious how changes in lipid packing in the outer leaflet could impact caveolae dynamics. It would be helpful to include a cartoon illustrating how this might work.
(3) The authors note that Dyngo-4a inhibits several dynamic processes including generalized plasma membrane mobility (Fig 4A&B), transferrin uptake (Fig S4C), and fusion of fusogenic liposomes (Fig S4G). This clearly indicates there is a major disruption of the plasma membrane going on here that is not limited to caveolae. They go on to show that the addition of cholesterol reverses the effects of Dyngo-4a on caveolae dynamics. However, they do not discuss whether adding back cholesterol has similar effects on plasma membrane mobility and transferrin uptake. This information could help to further pinpoint whether the mechanisms of action are shared, and if the role of cholesterol is more general in controlling these events or is instead specific to caveolae.
(4) In Fig 4C, the morphology of the neck region of the Dyngo04a treated caveolae structure appears to be "pinched" compared to the control. I appreciate that more EM studies are underway. It would be useful to specifically compare the morphology of the caveolae as part of those studies.
(5) In Line 91, a statement is made that 8S complex formation requires cholesterol. This is debatable, as they appear to form in E. coli in the absence of cholesterol (reference 14).
Author response:
General Statements
In this paper we demonstrate that the lipid packing of the plasma membrane has a huge impact on the stability of caveolae. By using interdisciplinary techniques, we show that the widely used dynamin inhibitor Dyngo-4a adsorbs and inserts to lipid bilayers leading to a decreased lipid packing and hence reduced caveolae dynamics and internalization even in cells lacking dynamin. We have added experiments that validates that Dyngo-4a treatment does not result in fragmentation or disassembly of the caveolae. A FRAP assay of cytosolic caveolae has been employed to address questions concerning scission. Moreover, as suggested by the reviewers, we have also included new simulation data that show and expand on the fact that Dyngo-4a positions in the lipid leaflet similar to cholesterol and preferentially associates with cholesterol clusters, affecting the spatial distribution of cholesterol in the membrane. We believe that these added data have greatly improved the paper and strengthened our conclusions that the lipid packing is a critical determinant in the balance between internalization and stable plasma membrane association of membrane vesicles.
As requested, we have expanded the introduction to provide more detailed information about previous findings in the field. Changes and addition to the text has been highlighted in red for easier tracking.
Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity):
The authors use Dyngo-4a, a known Dynami inhibitor to test its influence on caveolar assembly and surface mobility. They investigate, whether it incorporates into membranes with Quartz-Crystal Microbalance, they investigate how it is organized in membranes using simulations. Finally, they use lipid-packing sensitive dyes to investigate lipid packing in the presence of Dyngo-4a, membrane stiffness using AFM and membrane undulation using fluorescence microscopy. They also use a measure they call "caveola duration time" to claim that something happens to caveolae after Dyngo-4a addition and using this parameter, they do indeed see an increase in it in response to Dyngo-4a, which is reduced back to the baseline after addition of cholesterol.
Overall, the authors claim: 1) Dyngo-4a inserts into the membrane and this 2) results in "a dramatic dynamin-independent inhibition of caveola scission". 3) Dyngo-4a was inserted and positioned at the level of cholesterol in the bilayer and 4) Dyngo-4a-treatment resulted in decreased lipid packing in the outer leaflet of the plasma membrane 5) but Dyngo-4a did not affect caveola morphology, caveolae-associated proteins, or the overall membrane stiffness 6) acute addition of cholesterol counteracts the block in caveola scission caused by Dyngo-4a.
Overall, in this reviewers opinion, claims 1, 3, 4, 5 are well-supported by the presented data from electron and live cell microscopy, QCM-D and AFM.
However, there is no convincing assay for caveolar endocytosis presented besides the "caveola duration" which although unclearly described seems to be the time it takes in imaging until a caveolae is not picked up by the tracking software anymore in TIRF microscopy.
Since the main claim of the paper is a mechanism of caveolar endocytosis being blocked by Dyngo-4a, a true caveolar internalization assays is required to make this claim. This means either the intracellular detection of not surface connected caveolar cargo or the quantification of caveolar movement from TIRF into epifluorescence detection in the fluorescence microscope. Otherwise, the authors could remove the claim and just claim that caveolar mobility is influenced.
We thank the reviewer for the nice constructive comments, and we very much appreciate the positive critique. We have now included a FRAP experiment of endocytic Cav1-GFP supporting the effect on internalization. In addition, we are currently preforming CTxB HRP experiments to quantify the number of caveolae at PM using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long.
Reviewer #1 (Significance):
A number of small molecule inhibitors for the GTPase dynamics exist, that are commonly used tools in the investigation of endocytosis. This goes as far that the use of some of these inhibitors alone is considered in some publications as sufficient to declare a process to be dynamin-dependent. However, this is not correct, as there are considerable off-target effects, including the inhibition of caveolar internalization by a dynamin-independent mechanism. This is important, as for example the influence of dynamin small molecule inhibitors on chemotherapy resistance is currently investigated (see for example Tremblay et al., Nature Communications, 2020).
The investigation of the true effect of small molecules discovered as and used as specific inhibitors and their offside effects is extremely important and this reviewer applauds the effort. It is important that inhibitors are not used alone, but other means of targeting a mechanism are exploited as well in functional studies. The audience here thus is besides membrane biophysicists interested in the immediate effect of the small molecule Dyngo-4a also cell biologists and everyone using dynamic inhibitors to investigate cellular function.
Reviewer #2 (Evidence, reproducibility and clarity):
This manuscript uses the small molecule dynamin inhibitors dynasore and dyngo to show that in dynamin triple knockout cells that these inhibitors impact lipid packing and organization in the plasma membrane. Data showing that dyngo affects caveolin dynamics using tirf microscopy is also shown and is interpreted to reflect inhibition of caveolae scission from the membrane.
This data showing that dyngo and dynasore target membrane order is quite compelling and argues that the effects of these inhibitors is not dynamin specific and that inhibition of endocytosis by these small molecule inhibitors is dynamin-independent. The in vitro and in vivo data they provide is convincing.
Similarly, the data showing that dynasore and dyngo affect caveolin dynamics and clathrin endocytosis (transferrin) is quite convincing and argues that altered lipid packing is impacting membrane dynamics at the plasma membrane.
What is less convincing is the conclusion that dyngo is preventing caveolae scission from the membrane. Study of caveolae endocytosis is based on a TIRF assay that has inherent limitations:
- Caveolae are defined as bright cav1-positive spots in diffraction limited TIRF and their disappearance presumed to be endocytic events. Cav1 spots are presumed to be caveolae but the authors do not consider that they may be flat non-caveolar oligomers. The diffraction limited TIRF approach interprets the large structures as caveolae but evidence to that effect is lacking.
This is a valid comment and to address this we have now included data showing colocalization of cavin1 and EHD2 to the Cav1-GFP spots. We can however not determine if they are flat or invaginated. We do have extensive experience imaging caveolae using TIRF microscopy and carefully chose cells that display low expression of fluorescently labelled caveolin to avoid non-caveolar structures.
- The analysis (and the diagram presented in figure 4) considers that caveolae can either diffuse laterally in the membrane or internalize and does not consider that caveolae can flatten and possibly fragment in the membrane. Is it not possible that loss of Cav1 spots is a fragmentation event and not necessarily a scission event?
This is a good question, yet, fragmentation and disassembly would result in shorter track durations and this is not what is observed in data. We have now also included data showing that cavin1 is persistently associated with the Cav1 spots identified as caveolae during Dyngo-4a treatment indicating that these are caveolae. Furthermore, IF stainings showing colocalization of Cav1GFP with cavin1 or EHD2 after Dyngo-4a treatment have also been added. We have now also expanded on the different interpretations of the data in the results section.
- The analysis is based on overexpression of Cav1-GFP that may alter the stoichiometry between Cav1 and cavin1 such that while caveolae may be expressed, larger non-caveolar structures may accumulate.
Yes, this is correct, we have specifically imaged cell expressing low levels of Cav1-GFP to avoid accumulated non-caveolar structures that can be spotted in cells with high expression.
- Cav1 has been shown to be internalized via the CLIC pathway (Chaudary et al, 2014) and if dyngo is impacting clathrin then maybe it is also impacting CLIC endocytosis and thereby Cav1 endocytosis via this pathway?
Dyngo-4a has been shown to not affect CLIC endocytosis (McCluskey et al., 2013) and in our data we do not see internalization following Dyngo-4a treatment.
- The longer Cav1 TIRF track time and shorter displacement with dyngo is consistent with inhibition of caveolae scission. However, as the authors discuss, could not reduced membrane undulations due to dyngo's impact on membrane order be responsible for the longer tracks? Alternatively, perhaps the altered lipid packing is corralling Cav1 movement and reducing non-caveolar Cav1 endocytosis, resulting in shorter tracks of longer duration? The proposed interaction of dyngo with cholesterol could prevent scission but also stabilize large (flat?) Cav1 oligomers in the membrane, perhaps reducing Cav1 oligomer fragmentation.
We completely agree that membrane undulations contribute to instability of the TIRF-field and therefore disruption of cav1-GFP tracks as we discuss in the results section and have been described in previous work (Larsson et al., 2023). Yet, we have also shown that internalization of caveolae results in shorter tracks (Hubert et al., 2020; Larsson et al., 2023; Mohan et al., 2015). Furthermore, the tracked Cav1-GFP spots are persistently positive for cavin1 both with and without Dyngo-4a treatment showing that the majority do not disassemble become internalized by other pathways. Additionally, the added IF stainings after 30 min Dyngo-4a treatment also show that the Cav1-GFP spots remain positive for cavin1 and EHD2 just as ctrl-treated cells.
My point here is not to discredit the data but only to suggest that the TIRF approach used is an indirect measure of caveolae scission from the membrane that requires substantiation using other approaches.
We appreciate these comments and have tried to address these by adding new data and discussions on the interpretation of the tracking data in the results section.
Dyngo is certainly generally affecting lipid packing via cholesterol and thereby affecting Cav1 dynamics in the plasma membrane. The claim of caveolae scission should be qualified and alternative possibilities considered and discussed. If the authors persist in arguing that dyngo is affecting caveolae scission then the effect should be substantiated by accumulation of caveolae by quantitative EM and high spatial and temporal resolution imaging of Cav1 and cavin1 to define the endocytic events. As the latter represents a new, and potentially very challenging, line of experimentation, I would suggest that it is beyond the scope of the current study. As indicated above the additional experiments are not necessary and qualification of the claims would be sufficient.
We have now included a FRAP experiment of endocytic Cav1-GFP supporting the effect on internalization. We are also currently preforming CTxB HRP experiments to quantify the number of caveolae at the PM using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long.
Other points
Figure 1C - Cav1 positive spots cannot be interpreted to be caveolae from diffraction limited confocal images. Same comment applies to Fig 4G - caveola? duration.
We completely agree with this and that the claims should be qualified. We have added IF stainings showing that the Cav1-GFP structures are also positive for cavin1. We have now clarified that we cannot distinguish between flat or different curved states of caveolae using this methodology. We have also changed the labelling of Fig. 4G.
Figure 4C - it is not clear why this EM data is not quantified - for both the number of caveolae and clathrin coated pits - as this would help clarify the interpretation of the effect reported.
We are currently preforming CTxB HRP experiments to quantify the number of caveolae using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long.
Figure 4D - the AFM experiments should perhaps be repeated as the non-significant effect of dyngo on the Young's modulus may be a result of insufficient n values.
We would like to clarify that to ensure the robustness of our AFM measurements, we performed the experiments with sufficient biological and technical replicates. Specifically, each data point shown in Figure 4D represents a Young’s modulus value averaged from approximately sixty force-distance curves per cell. For each condition, we collected force-distance maps on eight to nine individual cells, obtained from two separate petri dishes per day. We repeated this process on two independent days. In total, we analysed thirty-one cells for the DMSO control and thirty-three cells for the Dyngo-4a treatment. We performed the “student’s t-test with Welch’s correction” to access the statistical significance between the two conditions, as described in the main text. We believe that the sample size and statistical approach are sufficient to support the conclusions presented. Furthermore, we also analysed cell stiffness by calculating the slope of the linear portion of the force-distance curves. This analysis also did not reveal any statistically significant differences between the conditions (data not shown), further supporting our conclusion that Dyngo-4a treatment does not significantly alter the Young’s modulus under our experimental setup (or conditions).
Reviewer #2 (Significance):
This data showing that dyngo and dynasore target membrane order is quite compelling and argues that the effects of these inhibitors is not dynamin specific and that inhibition of endocytosis by these small molecule inhibitors is dynamin-independent. The in vitro and in vivo data they provide is convincing.
Similarly, the data showing that dynasore and dyngo affect caveolin dynamics and clathrin endocytosis (transferrin) is quite convincing and argues that altered lipid packing is impacting membrane dynamics at the plasma membrane.
What is less convincing is the conclusion is that dyngo is preventing caveolae scission from the membrane.
Reviewer #3 (Evidence, reproducibility and clarity):
Larsson et al present experimental and computational data on the role of Dyngo4a (a compound that was developed to inhibit dynamin) on the dynamics of caveolae. The manuscript mostly documents effects of Dyngo on caveolae, with one experiment to suggest a mechanism for this result. This one rather unconvincing result forms the focus of the manuscript contributing to a disconnect between the data and the presentation. Additionally, there are concerns with data interpretation. The writing could also benefit from revision to address grammar mistakes, strengthen referencing, and increase precision. Overall, the manuscript requires substantial revisions before being considered for publication. The central claim, in particular, needs stronger evidence to support the proposed mechanism.
We thank the reviewer for the thorough review and for experimental suggestions that we believe has strengthened our data further.
Significant issues (in approximate order of importance):
(1) The data supporting the central mechanistic explanation appears limited. There is no evidence that Dyngo remains in one leaflet
The simulations show that the energy barrier for moving in between bilayers is very high. Furthermore, simulations of C-Laurdan has shown that it does not readily flip in between membrane leaflets (Barucha-Kraszewska et al., 2013) supporting that it reports on the outer lipid leaflet when added to cells. We have however now changed this and state that Dyngo-4a decreased the lipid order in the plasma membrane.
- the GP of the PM is very low compared to previous measurements,
The absolute GP-values will vary between setups depending on what filters are used so they are not comparable between laboratories. What is of importance is that we found a significant change in the relative GP-values in cells treated with Dyngo-4a and control cells. It is this change that we report. We have not performed any GP-measurements on this cell type earlier so it is unclear what previous measurements reviewer #3 are referring to.
- effects on other membranes are not explored,
The order of the intracellular membranes is as expected lower than that of the plasma membrane. Differentiating different intracellular membranes of interest like endocytotic vesicles from other intracellular membranes would be very difficult but, more importantly, our study is focused on what is happening in the plasma membrane where caveolae reside and would be of minor interest for plasma membrane dynamics.
- dynamin-directed effects of Dyngo are not considered,
In the discussion section we discuss the difficulties with disentangling dynamin-direct and indirect effects.
(2) The QCM-D measurements and claims require explanation as several aspects remains unclear. In Fig S2, the 'softness' (what does this mean?) changes by 4-fold with DMSO alone (what does this mean?), then fractionally more with Dyngo. Then fractionally more again when Dyngo is removed (why?). Then it remains somewhat higher when both Dyngo and DMSO are removed, which is somehow interpreted as Dyngo remaining in the bilayer, but not DMSO.
We understand the confusion of the reviewer and hope our explanations provide clarity. QCM-D measurements are based on an oscillating quartz crystal sensor. Specifically, alterations in oscillation frequency (ΔF) and the rate of energy dissipation from the sensor surface (ΔD) are what is measured. Allowing the measurement of: 1) materials adsorbing to the sensor surface, 2) changes in the viscoelastic properties of a solution in contact with the sensor surface, 3) changes in the material adsorbed to the sensor surface upone exposure to different solutions. The ratio of ΔD/-ΔF reports the mechanical softness or rigidity of an adsorbed material, in this case the SLB.
A “buffer shift” is the term used when there is not an adsorption to the sensor surface, but rather an effect from altering the solution above the sensor surface. One reason is because different solutions can have different densities (e.g., a DMSO-buffer mixture vs buffer alone), which impacts the oscillations of the sensor. It was observed that the DMSO-buffer mixture alone gave a large buffer shift in comparison to the adsorption of the Dyngo-4a into the SLB, thereby muddling the data interpretation. Thus, in Fig. S2 the system was first equilibrated with the DMSO-buffer mixture prior to addition of the Dyngo-4a solution to allow for clearer visualization of the two events. In QCMD to assess if something has made a permeant change to the system you change back to the solutions used before the addition, thus first we washed with a DMSO-Buffer mixture followed by buffer alone. Control experiments were carried out in which no Dyngo-4a was added (also shown in Fig. S2). The control shows the same “buffer shift” from the DMSO-buffer mixture occurs in both systems and that upon returning to a buffer only condition there is no permanent change to the system caused from exposure to the DMSO. In contrast, once the system that received Dyngo-4a is changes back to a buffer only system we see that mass has been added to the system (ΔF) with little change to the dissipation (ΔD), thereby resulting in a lower ratio of ΔD/-ΔF, which is to say that the SLB after the adsorption of Dyngo-4a was more rigid that the SLB without Dyngo-4a.
These interpretations are difficult to grasp, as the authors seem to be implying simple amphiphilic partitioning into the membrane, which should all be removable by efficient washing.
Amphiphilic partitioning is not fully reversible by “efficient washing” it depends on partitioning coefficients.
I do not doubt that this compound interacts with membranes, but the quantifications appear ambiguous. A bilayer with 16 mol% (or worse, 30% if all in one leaflet) Dyngo is very unlikely (to remain a bilayer). Even if such a bilayer was conceivable, the authors are claiming an ADDITION of Dyngo that would INCREASE the area of one leaflet by 30%, which needs explanation as it appears unlikely.
We understand that in our attempt provide numbers in the results section for the amount of binding observed in QCM-D, this can easily be interpreted as this is what is observed to insert into the PM. However, as discussed in the discussion, we also see aggregations of Dyngo-4a that associate with the membrane in the simulations which likely could contribute to the binding observed in QCM-D prior to washing. The precise amount of membrane inserted Dyngo-4a is difficult to measure as we discuss in the text. In order to make this clearer, we have now moved all these details to the discussion section where we elaborate on this. Furthermore, since Dyngo-4a, like cholesterol, is intercalating in between the head groups of the lipids the area would not increase in direct proportion to the mol%.
Also, there are no replicates shown, so unclear how reproducible these effects are?
For clarity, only single experiments are shown. However, multiple experiments were performed and the range in measured values for 3 technical repeats can be observed in the standard deviations found in the main text (e.g., 6 ± 2 mol%).
(3) The simulations are insufficiently described and difficult to interpret. How big are these systems? Why do the figures show the aqueous system with lateral boundaries?
There are no explicit boundaries used in the simulations, periodic boundary conditions are applied in all three dimensions. The lateral boundaries observed in the figures correspond to the simulation box edges and are a visual artifact of 2D projections with QuickSurf representation. No artificial wall or constraints were introduced laterally. Additional technical details, including the system size and periodic boundary conditions have now been added to the methods section.
It seems quite important that multiple Dyngo molecules aggregate rather than partition into membranes - is this likely to occur in experiment?
Yes, this is important and with the additional simulation experiments suggested by Reviewer #3 it has been clarified that they contribute a great deal to the change in lipid packing of lipid bilayers containing cholesterol. However, it is hard to test aggregation is the cellular system, but we believe that this happens and contribute to the effect on membranes. We have now emphasized the effect of the aggregates in the text.
PMF simulations are strongly suggesting that Dyngo does not spontaneously cross membranes, which is inconsistent with its drug-like amphiphilicity (cLogP~2.5 is optimally suited for membrane permeation) and known effects on intracellular proteins. This suggests an artefact in these PMFs.
As stated in the submitted version of the manuscript, logP was used to validate the topology and the observed value was in a very good agreement with cLogP. Moreover, this validation complemented the standard procedure of CHARMM-GUI ligand modelling, that provided a reasonable penalty score (around 20) for the Dyngo-4a topology. POPC and cholesterol molecules are standard in the force field and validated by numerous studies. The parameters used for the membrane simulations and AWH in particular are very common for this type of studies. Thus, we do not see what may cause any artifacts in the free energy profile construction. In fact, amphiphilicity of the molecule may be one of the key reasons that Dyngo-4a molecule remains at the aqueous interface of the membrane and does not cross the membrane spontaneously. Also, we believe that the energy barrier of 40-60 kJ/mol is not prohibitively high and Dyngo-4a molecules may still overcome the barrier eventually, though we expect majority to reside in the upper leaflet.
The authors should experimentally measure the permeation of Dyngo through bilayers (or lack thereof), to more robustly support their finding that Dyngo does not cross membranes spontaneously.
We thank the reviewer for the suggestion, however this if very technically challenging and would require establishment of precise systems which is beyond the scope of this manuscript.
(4) Why not measure effect of Dyngo on lipid packing directly and more broadly in model membranes?
With the added modelling experiments supporting the previous simulations and the calculated GP values from the C-Laurdan experiments on cellular plasma membrane, we do not find it necessary to include more model membranes experiments than the already existing ones on lipid monolayers and supported lipid bilayers.
(5) Statistics should not be done on individual cells (n>26), but rather on independent experiment (N=3?)
We have performed the statistics on live cell particle tracking according to previous literature on similar systems (Boucrot et al., 2011; Larsson et al., 2023; Shvets et al., 2015; Stoeber et al., 2012).
(6) Fig 1G is important but rather unclear. Firstly, these kymographs are an odd way to show that the caveolae are not moving. More importantly, caveolae in normal cells have been shown to be quite stable and immobile (eg doi: 10.1074/jbc.M117.791400), yet here they are claimed to be very mobile.
Although this might be an odd and unconventional way to depict dynamic processes, we believe that this is a very illustrative way to show track stability over time in bulk rather than just a kymograph over a few structures in a cell. Furthermore, we are not claiming that caveolae are very mobile but rather the opposite very stable in agreement with previous work (Boucrot et al., 2011; Larsson et al., 2023; Mohan et al., 2015). We have now edited the text to make this even clearer.
Also, if Dyngo prevents caveolae scission, there should be more of them at the membrane - why no quantification like Fig 1C to show accumulation of caveolae upon Dyngo treatment? Or directly counting caveolae via EM, as in Fig 4C?
We are currently preforming CTxB HRP experiments using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long. However, Dynasore has previously been shown, by EM, to increase the number of caveolae at the PM (Moren et al., 2012; Sinha et al., 2011).
(7) The writing can be made more precise and referencing could be strengthened.
The introduction was written in a short format, and we have now extended this and made it more precise.
Some examples:
(a) 'scissoned' is not a word in English,
Thanks, we have now changed this.
(b) what is meant by "Cav1 assembly is driven by high chol content"? There are many types of caveolin assemblies.
We agree that this can be made more precise and have now clarified this in the introduction.
(c) "This generates a unique membrane domain with distinct lipid packing and a very high curvature." Unclear what 'this' refers to and there is no reference here, so what is the evidence for either of these claims? Caveolin-8S oligomers are not curved. Perhaps 'this' is caveolae, but they are relatively large and also not very highly curved and I am unaware of measurements of lipid packing therein.
Caveolae are around 50 nm which in biology is a very high curvature of a membrane. It has been extensively proven that caveolae have a distinct lipid composition highly enriched in cholesterol and sphingolipids, which thereby also will generate a unique lipid packing as compared to the surrounding membrane. Yet, the reviewer is correct that lipid packing has not been measured in a caveola for obvious technical challenges. Thus, we have now changed the text to “special lipid composition”.
The sentence following that one again makes a specific, but unreferenced, claim.
(d) intro claims that lipid packing is critical for fission, but it is unclear quite what is meant by this claim. The references do not help, as they are often about the basic biophysics of lipids, rather than how packing affects fission.
We have now edited the text.
(e) intro strongly implies that caveolae remain membrane attached because of stalled scission. How strong is the evidence for this? The fact that EHD2 is at the neck is not definitive,
We used the term stalled scission to describe that all omega shaped membrane invaginations do not scission in the same automatic way as clathrin coated vesicles. We have now changed this in the text. Caveolae are shown to be released (undergo scission) and be detected as internal caveolae if the protein EHD2 is removed. Hence this must be interpreted as if EHD2 stalls scission. The evidence includes data compiled over the last 12 years from others and us which include for example: 1) Caveolae with EHD2 have a longer duration time (Larsson et al., 2023; Mohan et al., 2015; Moren et al., 2012; Stoeber et al., 2012), Knock down of EHD2 results in more internalized caveolae as measured by CTxB HRP using EM (Moren et al., 2012) and shorter duration time at the PM (Hubert et al., 2020; Larsson et al., 2023; Mohan et al., 2015; Stoeber et al., 2012). 2) EHD2 overexpression results in less internalized caveolae as measured by CTxB HRP using EM (Stoeber et al., 2012). Furthermore, 3) overexpression or acute addition of purified EHD2 via microinjection counteracts lipid induced scission of caveolae and hence result in caveolae stabilization at the PM (Hubert et al., 2020). It is very hard to see that the release and internalization of caveolae could result from anything else than that these have undergone scission. EHD2 has been found around the rim of caveolae (Matthaeus et al., 2022) and overexpression of EHD2 oligomerizing mutants have been shown to expand the caveola neck (Hoernke et al., 2017; Larsson et al., 2023).
(f) unclear what is meant by 'lipid packing frustration' and how Dyngo supposedly induces it.
Lipid packing frustration refers to what is usually referred to as lipid packing defect, but since lipid membranes are describe as a fluid system it should not have defects whereby, we believe that lipid packing frustration is more accurate. However, we have now changed the text and use “decreased lipid packing” or “decreased lipid order” more thoroughly to describe the effect on the plasma membrane.
(8) IF of Cav1 is insufficient to claim puncta as caveolae. Co-stained puncta of caveolin with cavin are much stronger evidence. Same issue for Cav1-GFP puncta.
We agree and have now provided IF showing cavin1 and EHD2 colocalization to Cav1GFP in non and Dyngo-4a-treated cells.
(9) Fig 3E claims that "preferred position of Dyngo-4a was closer to the head groups" but the minimum looks to be in similar place as Fig 3B without cholesterol. Response:
We appreciate the reviewer’s observation. The PMF minima in the POPC and POPC:Chol membranes are indeed close in absolute position (~1.1–1.2 nm from the bilayer center). However, as clarified in the revised text, the presence of cholesterol leads to a slight shift of Dyngo-4a closer to the headgroup region and broadens the positional distribution. This is also evident from the added density profiles (Fig. S3A) and is now described more precisely in the manuscript.
Critically, these results do not support the notion that Dyngo affects lipid packing sufficiently, which is not measured in the simulations (though could be).
We thank the reviewer for the excellent suggestion. In response, we have now included a detailed analysis of Dyngo-4a’s effect on lipid packing in the simulations. As described in the revised manuscript, we measured deuterium order parameters, area per lipid (APL), and lipid–Dyngo–cholesterol spatial distributions (Figs. 3-H, S3C-E). The results demonstrate that Dyngo-4a decreases lipid order in POPC:Chol membranes. Both single molecules and clusters reduce the order parameter by up to 0.04 units, particularly in the upper leaflet, where Dyngo-4a reside.The reduction is most pronounced in the midchain region of the sn1 tail and around the double bond of the sn2 tail. These effects were accompanied by increased APL in POPC:Chol membranes and by colocalization of Dyngo-4a near cholesterol-rich regions. Together, these data confirm that Dyngo-4a perturbs membrane organization and lipid packing in a composition-dependent manner. We believe these additions directly address the concern and demonstrate that the simulations indeed support the conclusion that Dyngo-4a modulates lipid packing.
Finally, the simulation data do not show "that Dyngo-4a is competing with cholesterol"; it is unclear what 'competition' means in this context, but regardless, the data only shows that Dyngo sits at a similar location as cholesterol.
We agree with the reviewer that “competition” was an imprecise term. We have rephrased the relevant sections to clarify that Dyngo-4a and cholesterol localize to overlapping regions and exhibit spatial coordination. As now stated in the manuscript, cholesterol appears to partially displace Dyngo-4a from its preferred depth seen in pure POPC, broadens its membrane distribution, and alters lipid packing. According to the order parameters there is an interplay between chol and Dyngo-4a and the heatmaps show that the distribution of chol in the membrane gets less uniform in the presence of Dyngo-4a. These interactions suggest that Dyngo-4a perturbs cholesterol-rich domains.
As new analysis routines were added to the study, we have now also added the details on those to the Methods section of the text.
(10) AFM measures the stiffness of the cell (as correctly explained in Results section) not "overall stiffness of the PM" as stated in the Discussion.
We thank the reviewer for pointing this out, we have now altered this in the discussion section.
(11) Fig2A: what was the starting lipid surface pressure? How does Dyngo insertion depend on initial lipid packing?
The starting pressure lipid pressure was 20 mN m<sup>-1</sup which we now have incorporated in the figure legend. We performed several such experiments with a starting pressure ranging from 20-23 mN m<sup>-1</sup> showing consistent results which we described in the materials and methods section. Given that we also performed QCMD analysis and simulations on bilayers showing that Dyngo-4a adsorbed and inserted respectively, we have not performed a titration of starting pressures resulting in a MIP of Dygo-4a.
(12) Fig 4B is a strange approach to measure membrane motion. Why not RMSD or some other displacement based method? As its shown, it implies that the area of the cell changes.
The method that we used to quantify the area of the cell which is attached (or close to) the glass and thereby is visible in TIRF microscopy. This is area indeed changes over time which has been frequently observed and used to describe and quantify the mobility, lamellipodia and filopodia formation among other things. We agree that RMSD can also be used to analyze the data before and after treatments and we have now included RMSD analysis in the manuscript.
Reviewer #3 (Significance):
The title, abstract, and introduction of the manuscript are largely framed around lipid packing, but most of the data investigate other unexpected effects of treating cells with Dyngo4a. The only measurement for lipid packing (or any other membrane properties) is Fig 4E-F. Therefore, this paper is effectively an investigation of an artefact of a common reagent, which itself could be a valuable contribution. However, the mechanism to explain its effect requires stronger evidence, and its broad biological significance needs further exploration.
Overall, the impact of documenting the effects of Dyngo4a on membranes appears modest but may be valuable to the membrane trafficking community.
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Hoernke, M., J. Mohan, E. Larsson, J. Blomberg, D. Kahra, S. Westenhoff, C. Schwieger, and R. Lundmark. 2017. EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation. Proc Natl Acad Sci U S A. 114:E4360-E4369.
Hubert, M., E. Larsson, N.V.G. Vegesna, M. Ahnlund, A.I. Johansson, L.W. Moodie, and R. Lundmark. 2020. Lipid accumulation controls the balance between surface connection and scission of caveolae. Elife. 9.
Larsson, E., B. Moren, K.A. McMahon, R.G. Parton, and R. Lundmark. 2023. Dynamin2 functions as an accessory protein to reduce the rate of caveola internalization. J Cell Biol. 222.
Matthaeus, C., K.A. Sochacki, A.M. Dickey, D. Puchkov, V. Haucke, M. Lehmann, and J.W. Taraska. 2022. The molecular organization of differentially curved caveolae indicates bendable structural units at the plasma membrane. Nat Commun. 13:7234.
McCluskey, A., J.A. Daniel, G. Hadzic, N. Chau, E.L. Clayton, A. Mariana, A. Whiting, N.N. Gorgani, J. Lloyd, A. Quan, L. Moshkanbaryans, S. Krishnan, S. Perera, M. Chircop, L. von Kleist, A.B. McGeachie, M.T. Howes, R.G. Parton, M. Campbell, J.A. Sakoff, X. Wang, J.Y. Sun, M.J. Robertson, F.M. Deane, T.H. Nguyen, F.A. Meunier, M.A. Cousin, and P.J. Robinson. 2013. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic. 14:1272-1289.
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Reviewer #1 (Public review):
Summary:
This study uses a novel DNA origami nanospring to measure the stall force and other mechanical parameters of the kinesin-3 family member, KIF1A, using light microscopy. The key is to use SNAP tags to tether a defined nanospring between a motor-dead mutant of KIF5B and the KIF1A to be integrated. The mutant KIF5B binds tightly to a subunit of the microtubule without stepping, thus creating resistance to the processive advancement of the active KIF1A. The nanospring is conjugated with 124 Cy3 dyes, which allows it to be imaged by fluorescence microscopy. Acoustic force spectroscopy was used to measure the relationship between the extension of the NS and force as a calibration. Two different fitting methods are described to measure the length of the extension of the NS from its initial diffraction-limited spot. By measuring the extension of the NS during an experiment, the authors can determine the stall force. The attachment duration of the active motor is measured from the suppression of lateral movement that occurs when the KIF1A is attached and moving. There are numerous advantages of this technology for the study of single molecules of kinesin over previous studies using optical tweezers. First, it can be done using simple fluorescence microscopy and does not require the level of sophistication and expense needed to construct an optical tweezer apparatus. Second, the force that is experienced by the moving KIF1A is parallel to the plane of the microtubule. This regime can be achieved using a dual beam optical tweezer set-up, but in the more commonly used single-beam set-up, much of the force experienced by the kinesin is perpendicular to the microtubule. Recent studies have shown markedly different mechanical behaviors of kinesin when interrogated by the two different optical tweezer configurations. The data in the current manuscript are consistent with those obtained using the dual-beam optical tweezer set-up. In addition, the authors study the mechanical behavior of several mutants of KIF1A that are associated with KIF1A-associated neurological disorder (KAND).
Strengths:
The technique should be cheaper and less technically challenging than optical tweezer microscopy to measure the mechanical parameters of molecular motors. The method is described in sufficient detail to allow its use in other labs. It should have a higher throughput than other methods.
Weaknesses:
The experimenter does not get a "real-time" view of the data as it is collected, which you get from the screen of an optical tweezer set-up. Rather, you have to put the data through the fitting routines to determine the length of the nanospring in order to generate the graphs of extension (force) vs time. No attempts were made to analyze the periods where the motor is actually moving to determine step-size or force-velocity relationships.
Reviewer #2 (Public review):
Summary:
This work is important because it complements other single-molecule mechanics approaches, in particular optical trapping, which inevitably exerts off-axis loads. The nanospring method has its own weaknesses (individual steps cannot be seen), but it brings new clarity to our picture of KIF1A and will influence future thinking on the kinesins-3 and on kinesins in general.
Strengths:
By tethering single copies of the kinesin-3 dimer under test via a DNA nanospring to a strong binding mutant dimer of kinesin-1, the forces developed and experienced by the motor are constrained into a single axis, parallel to the microtubule axis. The method is imaging-based, which should improve accessibility. In principle, at least, several single-motor molecules can be simultaneously tested. The arrangement ensures that only single molecules can contribute. Controls establish that the DNA nanospring is not itself interacting appreciably with the microtubule. Forces are convincingly calibrated, and reading the length of the nanospring by fitting to the oblate fluorescent spot is carefully validated. The excursions of the wild-type KIF1A leucine zipper-stabilised dimer are compared with those of neuropathic KIF1A mutants. These mutants can walk to a stall plateau, but the force is much reduced. The forces from mutant/WT heterodimers are also reduced.
Weaknesses:
The tethered nanospring method has some weaknesses; it only allows the stall force to be measured in the case that a stall plateau is achieved, and the thermal noise means that individual steps are not apparent. The nanospring does not behave like a Hookean spring - instead linearly increasing force is reported by exponentially smaller extensions of the nanospring under tension. The estimated stall force for Kif1A (3.8 pN) is in line with measurements made using 3-bead optical trapping, but those earlier measurements were not of a stall plateau, but rather of limiting termination (detachment) force, without a stall plateau. More confidence in the 3.7 pN stall plateau determined in the current work could be obtained by demonstrating that a stall at a higher force is obtained using the nanospring method on kinesin-1, which stalls at >7 pN in single bead optical trapping.
Résumé vidéo [00:47:45][^1^][1] - [01:05:30][^2^][2]:
La troisième partie de la vidéo traite de la question de faire vivre les valeurs de la République en établissement scolaire, avec les témoignages de trois intervenants : une professeure d'histoire-géographie, un principal de collège et une chercheuse en sciences de l'éducation. Ils partagent leurs expériences, leurs pratiques et leurs réflexions sur les enjeux et les moyens de transmettre et d'incarner ces valeurs auprès des élèves, des personnels, des parents et des partenaires extérieurs.
Points forts : + [00:47:45][^3^][3] L'école comme parenthèse pour les élèves * Leur faire sentir qu'ils peuvent vivre les valeurs républicaines à l'école * Leur faire découvrir la mixité sociale, l'égalité, la culture * Leur proposer des sorties pédagogiques, des projets, des actions + [00:49:01][^4^][4] L'interconnaissance avec les acteurs extérieurs à l'école * Développer des partenariats avec le monde associatif, la politique de la ville, etc. * Coproduire des projets avec ces acteurs pour prendre en charge le jeune dans sa globalité * Former les enseignants et les chefs d'établissement avec ces acteurs pour avoir un référentiel commun + [00:53:05][^5^][5] La question du public allophone * Lever la barrière de la langue pour faire vivre les valeurs de citoyens français * Profiter de la grande diversité et de la fascination pour la France des élèves allophones * Faire partie de collectifs pour penser et débattre ensemble + [00:58:51][^6^][6] La synthèse graphique de la table ronde * Représenter le parcours professionnel des intervenants comme un parcours d'équilibriste * Identifier les valeurs qui les animent, les publics qu'ils touchent, les actions qu'ils mènent * Souligner l'importance du collectif, de la politique, de la mixité sociale harmonieuse
Résumé vidéo [00:23:45][^1^][1] - [00:47:41][^2^][2]:
La deuxième partie de la vidéo parle de comment faire vivre les valeurs de la République en établissement scolaire, à travers des expériences, des dispositifs et des instances. Les intervenants partagent leurs pratiques, leurs réflexions et leurs témoignages sur les enjeux de l'éducation à la citoyenneté, à la santé, à l'égalité, à la laïcité et à la fraternité.
Points forts: + [00:24:14][^3^][3] L'articulation entre l'individuel et le collectif * Comment chacun se positionne sur ses valeurs * Comment on travaille avec l'ensemble des acteurs * Comment on crée du lien et du sens entre les actions + [00:24:26][^4^][4] Le comité d'éducation à la santé, à la citoyenneté et à l'environnement (CESCE) * Une instance de réflexion, d'observation, de veille et de proposition * Une instance qui associe les parents, les partenaires, les élus, les référents et les professeurs * Une instance qui définit la politique de prévention et d'éducation dans les domaines de la citoyenneté, de la santé, de la lutte contre les violences, de l'égalité fille-garçon, etc. + [00:31:00][^5^][5] L'expérience du collège Berlios à Paris * Un établissement qui accueille une grande diversité d'élèves * Un établissement qui a mis en place une mixité harmonieuse et une bienveillance éducative * Un établissement qui a amélioré son climat scolaire, sa réussite et son ouverture + [00:38:16][^6^][6] L'éducation universelle en classe * Une approche pédagogique qui vise à lever les obstacles à l'apprentissage * Une approche qui s'interroge sur les besoins éducatifs particuliers des élèves * Une approche qui favorise l'équité et la réussite de tous les élèves
Résumé vidéo [00:00:01][^1^][1] - [00:23:42][^2^][2]:
Cette vidéo est la première partie d'une table ronde sur le thème "Faire vivre les valeurs en établissement". Trois intervenants partagent leurs expériences et leurs réflexions sur les questions d'éducabilité, de réussite, d'égalité et de citoyenneté à l'école. Ils abordent également les enjeux de l'éducation inclusive, de la mixité sociale, du climat scolaire et du bien-être des élèves et des personnels.
Points forts: + [00:00:01][^3^][3] Présentation de la table ronde et des intervenants * Sandrine Benevkir, conseillère technique établissement vie scolaire au cabinet de la rectrice de l'académie de Lille * Faride Boualifa, proviseur au lycée professionnel Le Chatelier à Marseille * Anne-Lore Perrin, conseillère pédagogique de circonscription et docteure en psychologie sociale + [00:07:52][^4^][4] Intervention de Sandrine Benevkir * Elle évoque son parcours personnel et professionnel marqué par les questions d'éducabilité, de réussite et d'égalité * Elle présente les différents dossiers qu'elle porte au niveau académique, comme la vie lycéenne, l'égalité fille-garçon, la prévention de la radicalisation ou les valeurs de la République * Elle insiste sur l'importance de l'appartenance à des collectifs pour transmettre et promouvoir ces valeurs + [00:11:47][^5^][5] Intervention de Faride Boualifa * Il raconte son parcours de CPE puis de chef d'établissement dans des zones difficiles, animé par la volonté de lutter contre les déterminismes sociaux * Il relate son expérience de la mixité sociale à travers une expérimentation de montée alternée entre deux collèges aux profils sociologiques opposés * Il souligne le rôle du climat scolaire, du bâti, des projets, des sorties et du management dans la réussite et l'épanouissement des élèves + [00:18:18][^6^][6] Intervention d'Anne-Lore Perrin * Elle expose ses recherches en psychologie sociale sur les attitudes des enseignants à l'égard de l'éducation inclusive * Elle montre que les valeurs personnelles et organisationnelles des enseignants influencent leur perception de l'éducation inclusive * Elle révèle que le terme de réussite peut avoir des effets contradictoires sur les attitudes des enseignants, selon qu'il est défini et compris
il s'agit de l'atelier 2 autour du bien-être à l'école de l'atelier 3 du coup je vous le fais de têtes alors 00:57:45 que j'avais des des notes en tout cas c'est l'atelier 3 et l'atelier 4 les ateliers donc 2 3 et 4 sont annulés en revanche il reste des places dans les autres ateliers du coup vous pouvez vous y rendre de façon spontanée et vous 00:57:58 ajouter sur les listes d'émargements donc il reste le 1 euh valeur de la République avec du coup l'équipe de collègues Carole Janine et j'ai oublié le nom de la troisème personne le 00:58:11 l'atelier 5 l'Escape game autour de l'inclusion le 6 euh le 6 les réseaux sociaux merci beaucoup autour des réseaux sociaux avec le Clémi 00:58:22 euh l'atelier 7 euh qui est autour de l'interculturalité justement du plurilinguisme et l'atelier 8 avec la question de l'expérience du débat en classe
Résumé de la vidéo [00:00:00][^1^][1] - [00:19:25][^2^][2]:
La vidéo présente une avocate spécialisée dans la défense des enfants atteints de troubles neurodéveloppementaux.
Elle explique son approche flexible en droit pour s'adapter aux besoins spécifiques de ses clients, souvent confrontés à des diagnostics erronés et à des prises en charge inadaptées.
Elle souligne l'importance de lutter contre la stigmatisation et de promouvoir des soins adaptés, en mettant l'accent sur la formation des professionnels et l'écoute des parents.
Points forts: + [00:00:11][^3^][3] Défense des enfants atypiques * Troubles TDAH, TSA * Syndrome d'alcoolisation fœtale + [00:01:00][^4^][4] Flexibilité en droit * Droit diversifié * Centré sur le neurodéveloppement + [00:02:01][^5^][5] Lutte contre les diagnostics erronés * Responsabilité médicale * Prises en charge inadaptées + [00:03:02][^6^][6] Droits des handicapés * Allocations via MDPH * Prise en charge adaptée + [00:04:01][^7^][7] Importance de l'accompagnement humain * AESH individuels * Besoin d'attention soutenue + [00:05:03][^8^][8] Contentieux en droit administratif * Éducation nationale * Conseils de discipline + [00:07:00][^9^][9] Responsabilité pénale des jeunes * Discernement * Alternatives à la prison + [00:09:01][^10^][10] Protection de l'enfance * Formations obsolètes * Importance du diagnostic + [00:11:01][^11^][11] Rôle des parents * Observations parentales * Collaboration avec professionnels + [00:12:01][^12^][12] Risques de placement abusif * Informations préoccupantes * Mesures administratives + [00:15:46][^13^][13] Assistance éducative * Évaluations sociales et psychologiques * Importance des soins adaptés + [00:17:00][^14^][14] Départ des CMP * Risque d'IP pour défaut de soins * Importance de la prise en charge en libéral + [00:19:00][^15^][15] Écoute des parents * Non-toxicité * Besoin de répit, pas de retrait Video summary [00:20:00][^1^][1] - [00:39:54][^2^][2]:
La vidéo présente une avocate spécialisée dans la défense des enfants avec des troubles neurodéveloppementaux.
Elle explique son approche flexible en droit pour s'adapter aux besoins spécifiques de ses clients et aborde les défis liés à la responsabilité médicale, les droits des handicapés, et la protection de l'enfance.
Highlights: + [00:20:00][^3^][3] Défense des enfants * Troubles neurodéveloppementaux * Flexibilité en droit + [00:21:01][^4^][4] Responsabilité médicale * Diagnostics inadaptés * Culpabilisation des parents + [00:22:26][^5^][5] Droits des handicapés * Allocations via MDPH * Prise en charge adaptée + [00:24:02][^6^][6] Protection de l'enfance * Évaluation des besoins * Formations pour professionnels + [00:27:44][^7^][7] Enjeux du placement * Impact sur les familles * Importance du diagnostic + [00:30:03][^8^][8] Rôle du juge des enfants * Lien entre services et familles * Compréhension des troubles
Document d'Information : Synthèse de la 18ème Journée du Refus de l'Échec Scolaire
Résumé
La 18ème Journée du Refus de l'Échec Scolaire (JRES), organisée par l'AFEV, a porté sur le thème "Jeunesses populaires rurales et urbaines : même combat face aux inégalités éducatives ?".
L'événement a mis en lumière les convergences et les divergences entre ces deux jeunesses, souvent opposées dans le discours public via la formule "jeunesse des tours, jeunesse des bourgs".
Les débats ont révélé que, bien que confrontées à des défis spécifiques liés à leur territoire (mobilité pour les ruraux, discriminations pour les urbains), ces jeunesses partagent des problématiques communes profondément ancrées dans les inégalités sociales.
Une enquête exclusive de Trajectoires Réflex, menée auprès de 1500 lycéens, a démontré la prépondérance du milieu social sur le lieu de vie dans la détermination des parcours scolaires et des aspirations.
Les jeunes des classes populaires, qu'ils soient ruraux ou urbains, s'orientent davantage vers des filières courtes, tandis que les jeunes de classes supérieures visent des études longues. L'étude souligne également une anxiété partagée face à l'orientation, notamment à cause de Parcoursup.
Les intervenants, incluant le sociologue Benoît Coquard, des élus locaux, des acteurs associatifs et des chercheurs, ont unanimement appelé à dépasser les oppositions stériles.
Ils ont souligné l'importance de reconnaître les réalités diverses des territoires, les dynamiques d'autocensure, la complexité de la mobilité (partir, rester, revenir) et l'impact de l'isolement.
Un consensus s'est dégagé sur l'urgence de refonder des politiques publiques décloisonnées, de repenser le modèle de la méritocratie et de valoriser toutes les formes de réussite et d'engagement pour reconstruire un "destin commun" et garantir une véritable égalité des chances pour tous les jeunes.
1. Thématique Centrale : Dépasser l'Opposition "Tours vs. Bourgs"
La 18ème JRES a été initiée pour analyser l'articulation entre les jeunesses populaires rurales et urbaines, un parallèle popularisé par la formule "jeunesse des tours, jeunesse des bourgs".
Les intervenants ont largement critiqué l'instrumentalisation politique et médiatique de cette opposition.
• Origine de la réflexion (Eunice Mangado-Lunetta, AFEV) : L'AFEV, historiquement ancrée dans les Quartiers Prioritaires de la politique de la Ville (QPV), a constaté que "la France qui va mal, on la retrouve partout".
L'association a remis en question le discours opposant un "bloc métropolitain" riche à un "bloc périphérique" en difficulté, discours souvent utilisé pour critiquer l'investissement public dans les QPV au détriment d'une "France qui souffre silencieusement dans les campagnes".
• Perspective sociologique (Benoît Coquard) : Le sociologue, parrain de l'événement, a souligné que cette formule est un slogan efficace mais "en partie trompeur".
Il a expliqué que l'enjeu politique actuel est de se réclamer de la ruralité, devenue un "faire-valoir" et un "écran de fumée" masquant les questions sociales.
Il insiste sur la nécessité de parler des "ruralités" au pluriel, car il existe des écarts de richesse et des dynamiques démographiques très variés entre les campagnes, tout comme dans les villes.
• Objectif de la journée : L'objectif n'était pas d'opposer ces jeunesses ni de mettre un signe égal entre elles, mais de "chercher du commun" tout en reconnaissant ce qui diffère, afin d'éviter les "oppositions stériles". L'enquête présentée visait spécifiquement à superposer le filtre territorial et le filtre social pour une analyse plus juste.
2. Enquête Trajectoires Réflex : Données Clés sur les Lycéens
Une enquête a été réalisée de mai à juillet 2025 auprès de plus de 1500 lycéens de filières générales, technologiques et professionnelles.
L'échantillon comprenait 41% de jeunes de communes rurales et 26% de jeunes de petites villes. L'analyse croise le lieu de vie avec le milieu social (défini par le diplôme et la CSP des parents).
2.1 Orientation et Projections d'Études Le milieu social apparaît comme le facteur prépondérant dans les choix d'orientation, plus que le lieu de vie.
• Rapport au lycée :
◦ 94% des jeunes disent avoir choisi leur filière.
◦ 70% ressentent de la pression liée aux cours et aux résultats.
◦ Plus d'un tiers déclare avoir des difficultés d'apprentissage, une part plus importante chez les jeunes de classes populaires.
• Orientation post-bac : ◦ 61% se sentent bien informés, mais 58% sont inquiets.
◦ Le stress est principalement causé par les démarches Parcoursup et le flou du projet d'orientation.
• Aspirations :
◦ 80% souhaitent poursuivre des études supérieures.
◦ L'influence du milieu social est nette : les jeunes urbains de classe supérieure privilégient les études longues (Master, Grande École), tandis que les jeunes ruraux de classe populaire ciblent davantage les filières courtes pour une insertion plus rapide sur le marché du travail.
Type d'études Jeunes de classe populaire RURALE Jeunes de classe populaire URBAINE Filière courte (BTS/BUT) 32% 16% Niveau Master 38% 55%
• Sens donné aux études : Pour les jeunes de classe supérieure, la "réussite personnelle" prime. Pour ceux des milieux populaires, c'est avant tout le fait de "gagner de l'argent".
2.2 Mobilité, Attachement et Avenir
• Attachement au territoire : 82% des jeunes partagent un fort attachement à leur lieu de vie. Cet attachement est plus marqué chez les jeunes ruraux (85%) et encore plus chez les jeunes ruraux de classe populaire (90%).
• Projet de départ : ◦ 69% se préparent à partir de chez leurs parents pour leurs études.
◦ Ce sont les jeunes de milieux favorisés et les jeunes ruraux qui sont les plus nombreux à se préparer à partir.
◦ Cependant, le départ est plus souvent subi par les ruraux : 17% des jeunes ruraux parlent d'une "contrainte à quitter le domicile familial", contre seulement 9% des jeunes citadins.
• Bien-être et difficultés :
◦ 84% se sentent bien au quotidien, mais 64% témoignent de leur anxiété.
◦ 1 jeune sur 5 se sent isolé. Ce sentiment est plus prononcé chez les jeunes de classe populaire rurale (21%) que chez les urbains (15%).
◦ Les difficultés financières sont plus fréquentes chez les jeunes de milieu populaire (40% vs 14% chez les enfants de Bac+5).
• Projection sociale :
◦ Seuls 36% des citadins et 25% des ruraux pensent avoir une meilleure vie que leurs parents. 45% ne savent pas.
◦ Les jeunes de milieu populaire semblent plus optimistes, espérant une ascension sociale. Cependant, un clivage territorial existe : 42% des citadins de milieu populaire ont une image positive de leur avenir, contre 31% des ruraux de milieu populaire.
2.3 Mobilité Quotidienne et Déplacements Les difficultés de déplacement sont significativement plus marquées pour les jeunes ruraux.
• Seule la moitié (50%) des jeunes de classe populaire rurale déclare qu'il est facile pour eux de se déplacer, contre 90% des jeunes de classe populaire urbaine.
• Dépendance à la voiture : 53% des jeunes ruraux utilisent la voiture au quotidien (contre 12% des citadins). Le permis est jugé indispensable pour 80% des ruraux (contre 55% des citadins).
2.4 Rapport aux Institutions et à l'Engagement
• Discriminations :
◦ 36% des jeunes déclarent en avoir été victimes, principalement à l'école (94% des cas).
◦ Le sentiment est plus marqué chez les jeunes urbains (43% vs 31% des ruraux).
◦ Les motifs varient : pour les citadins de classe populaire, les discriminations sont liées à l'origine, l'apparence et les convictions. Pour les ruraux de classe populaire, elles sont liées à l'apparence, la classe sociale et l'état de santé.
• Confiance dans les institutions : La confiance est plus forte chez les jeunes de classe supérieure et les jeunes ruraux.
◦ Police : 83% des jeunes ruraux de milieu modeste font confiance à la police, contre 58% des jeunes urbains de milieu modeste.
◦ Une méfiance généralisée est partagée envers les médias (42% de confiance), les réseaux sociaux (21%) et surtout la classe politique (16%).
• Rapport au vote et à l'engagement : ◦ Le rapport au vote est socialement inégal : 60% des jeunes de milieu favorisé pensent voter, contre 46% des enfants de non-diplômés.
◦ 44% des jeunes ne savent pas s'ils comptent s'engager plus tard. Ce doute est plus fort chez les jeunes ruraux.
3. Parcours de Vie et Analyses Croisées
3.1 Le Témoignage de Benoît Coquard : Du Rural à la Sociologie
Le sociologue Benoît Coquard a partagé son parcours personnel, emblématique des barrières et des aléas qui façonnent les destins.
• Origines : Vient d'un village de Haute-Marne, un département avec très peu de cadres. Ses parents, "petits fonctionnaires", ont quitté l'école à 14-15 ans. Il n'avait "pas de modèle estudiantin".
• Bifurcation : Doit sa mobilité sociale à "l'intervention un peu divine, un peu aléatoire" de sa sœur qui l'a poussé vers les études supérieures.
Il souligne que sans l'inexistence de Parcoursup à l'époque, il n'aurait pas pu s'inscrire à l'université avec son "mauvais dossier scolaire".
• Rapport à l'école : Mauvais élève, il a raté son bac. L'école était pour lui "la seule scène sociale sur laquelle [il était] parfois dévalorisé". Il décrit la construction d'une "culture anti-école" où l'on développe des modèles de reconnaissance alternatifs (sport, sociabilité locale).
• Analyse sociologique : Son travail de recherche, notamment Ceux qui restent, l'a amené à étudier son propre milieu.
Il met en évidence que les jeunes ruraux croient davantage en leurs "ressources d'autochtonie" (réseau local, piston) pour s'en sortir, ce qui peut les détourner de l'école.
À l'inverse, les jeunes des quartiers, souvent issus de l'immigration, surinvestissent l'école car leurs parents n'ont pas ce capital d'autochtonie.
3.2 Le Témoignage d'Yvon Atonga : Destins Divergents en Banlieue
Yvon Atonga, co-auteur de Petit frère, a raconté son histoire et celle de son frère Wilfried, qui a grandi dans le même environnement à Villiers-le-Bel mais a connu un destin tragique.
• Points de bifurcation : Il identifie plusieurs moments clés qui ont séparé leurs trajectoires :
1. L'intervention de sa mère : Un jour, alors que ses amis venaient le chercher, sa mère leur a dit qu'il n'était pas là car il faisait ses devoirs. Ses amis sont partis commettre un braquage et ont été condamnés à de lourdes peines.
2. Un voyage au Congo : À 15 ans, un voyage dans son pays d'origine a été un "électrochoc", lui faisant prendre conscience des opportunités qu'il avait en France.
• La loyauté au quartier : Il décrit une "loyauté indiscutable" au groupe d'amis et au quartier, qui représente une "deuxième famille". Partir est souvent perçu comme une "trahison".
Il insiste sur le fait qu'il revient aujourd'hui au quartier via son association pour "ne pas trahir" et "transmettre aux petits frères et aux petites sœurs".
3.3 Regards des Acteurs de Terrain
• Élus locaux (Fabrice Bossui, Driss Étaoui) : Ils dénoncent le manque de concertation sur des sujets cruciaux comme la carte scolaire, qui impose des temps de transport démesurés aux jeunes ruraux.
Ils soulignent l'évitement scolaire massif vers le privé qui accentue la ségrégation et la concentration de la pauvreté dans certains établissements publics.
• ANCT (Corine de la Maîtrise) : Rappelle que si le milieu social reste un "déterminant majeur", le territoire module les trajectoires.
L'objectif est de "lutter contre les inégalités de destin territorial".
Elle souligne que 30% des QPV se situent aujourd'hui dans des petites ou moyennes villes, brouillant la frontière traditionnelle entre politique de la ville et ruralité.
Elle alerte sur le fait qu'il faut "12 générations pour sortir de la pauvreté quand on vient d'un QPV", contre 9 en moyenne en France.
• Acteurs associatifs (Salomé Berlou, Ashraf Manar) : Ils appellent à dépasser une vision "victimaire" des jeunes des territoires populaires.
Ces jeunes sont des acteurs engagés dans leurs localités, même si cet engagement n'est pas toujours formalisé ou reconnu.
Leurs associations (Rura, Destins Liés) visent à outiller ces jeunes pour qu'ils aient le "choix" de leur parcours, en luttant contre l'autocensure et en créant des ponts entre les territoires.
4. Conclusions et Perspectives • Le primat du social : La journée a réaffirmé que la question sociale est le fil rouge qui relie les difficultés des différentes jeunesses populaires, bien au-delà des spécificités territoriales.
• La complexité du "partir/rester" : Le départ n'est pas toujours un choix émancipateur mais souvent une contrainte, notamment pour les ruraux.
Rester n'est pas forcément un échec mais peut correspondre à un attachement profond, tout en étant parfois synonyme d'assignation à résidence.
• L'isolement et la santé mentale : La solitude, que ce soit dans un village éloigné ou dans une cité universitaire après le départ, est une problématique centrale et croissante qui impacte lourdement les parcours.
• La crise de la méritocratie : Les intervenants ont critiqué le modèle méritocratique qui invisibilise les déterminismes sociaux et territoriaux, et qui génère de la frustration.
La promesse "travaille bien à l'école et tu réussiras" n'est plus tenue.
• L'urgence de refonder du commun : La conclusion, portée par Benoît Coquard et Christophe Paris (AFEV), est un appel à dépasser l'individualisme et la concurrence entre territoires.
Il est crucial de reconstruire des "consciences collectives" et un "sentiment du nous" pour s'attaquer collectivement aux inégalités systémiques.
Cela passe par des politiques publiques qui valorisent toutes les formes de contribution au bien commun et qui garantissent à chaque jeune les moyens d'une "vraie autodétermination de son parcours".
Annotations on “An Antidote to Injustice” by Jennifer M. Morton
[Annotation 1] Line: “This is your gruelling daily routine. Now, ask yourself: what could philosophy do for you?” → Morton paints a vivid picture of hardship to ground philosophy in lived experience. The rhetorical question links abstract theory with real-life struggles, making philosophy feel practical and relevant.
[Annotation 2] Line: “They are strivers, seeking an education…while holding onto jobs and taking care of families.” → The word strivers highlights perseverance and determination. Morton emphasizes the resilience of her students, portraying their pursuit of education as a meaningful effort despite heavy responsibilities.
[Annotation 3] Line: “Philosophy is the antidote to the uncritical acceptance of the world and ourselves as we are.” → The metaphor of antidote frames philosophy as medicine for the mind. Morton presents philosophy not as abstract luxury, but as a tool that helps people resist passivity and question harmful assumptions.
[Annotation 4] Line: “Consider the questions our protagonist could ask herself: Why should she have to take out student loans…?” → Morton uses a series of rapid questions to model critical inquiry. The barrage challenges the reader to reflect and destabilizes passive acceptance, encouraging independent thought.
[Annotation 5] Line: “Imagine that an evil demon is controlling your every thought.” → Descartes’ “evil demon” thought experiment pushes doubt to its extreme. By imagining complete deception, students are forced to question how much of their knowledge can truly be trusted.
[Annotation 6] Line: “Philosophers…are failing as citizens if they turn their back on those in the cave who are less fortunate.” → By invoking Plato’s Allegory of the Cave, Morton reframes philosophy as a moral duty. Knowledge is shown to carry responsibility: those who understand more are obligated to help others see beyond ignorance.
[Annotation 7] Line: “If the groups over-fish, famine ensues and all of the families will die.” → The classroom exercise dramatizes the challenge of limited resources. It highlights how individual greed can harm the whole community, teaching lessons about foresight, cooperation, and restraint.
[Annotation 8] Line: “Rawls asks his readers to imagine a hypothetical social contract…” → Rawls’ “veil of ignorance” is a thought experiment designed to remove bias. It forces people to think about justice from a neutral position, encouraging fairness and equality.
[Annotation 9] Line: “She will be indignant because many of them are the product of unjust political institutions…” → Morton frames indignation as a productive moral response. Philosophy helps people recognize injustice clearly and articulate why unfair systems provoke anger.
[Annotation 10] Line: “The way injustice often undermines our agency is by shrinking the horizons of what we think is possible.” → Morton explains how injustice limits imagination and hope. Philosophy works to expand possibilities, giving people vision and confidence to seek change.
[Annotation 11] Line: “Dogmatism is an enemy to peace, and an insuperable barrier to democracy.” → Russell critiques rigid certainty as harmful to progress. Morton uses this to emphasize how philosophy promotes open-mindedness, tolerance, and democratic dialogue.
[Annotation 12] Line: “Martin Luther King Jr…held up hope in the form of a dream.” → Morton concludes by connecting philosophy with imagination and justice. King’s dream is used as an example of how ideas can inspire hope and collective action against injustice.
Today, the New Zealand government estimates that invasive predators cost the country over 3 Billion NZ dollars (about 2 billion Euros) a year due
Yearly damage is roughly 1.2% of New Zealand's GDP.
Aplicaciones recientes de la ecuación del bio-calor
Creo que no tiene caso dividir el texto con esta subsección si solo hay una subsección. Puede quedar todo el texto (4 párrafos o 3 si quitas el anterior) de la sección 10 sin divisiones.
Por otro lado, no son aplicaciones tan recientes, la más nueva es de hace 7 años, échale un ojo a estas referencias: https://www.scielo.org.mx/scielo.php?pid=S1405-55462018000401573&script=sci_arttext https://www.cell.com/heliyon/fulltext/S2405-8440(24)16140-3 https://www.sciencedirect.com/science/article/pii/S0017931023008438?casa_token=wbiNgqg10poAAAAA:DrtwAK4QYSucDxmgD9UILQje9Jz_0sTD3WMmK3_ycliMbSg94LD3cQi_FQ0T34AplbJWmNNF0cs
o busca bioheat equation en scholar google
Avg inventory turnover = hoe vaak de hele voorraad gebruikt wordt.
Hoevaak je de voorraad gebruikt. Dus stel --> voorraad = 100 --> verkoop = 20 per week. Eens in d)e 5 dagen draai je je voorraad om. Avg inventory turnover.
!: flow time = throughput time, flow rate = throughput rate !: WIP (work in progress) = gemiddelde inventory
WIP = hoeveel flow units gemiddeld in proces. Throughput rate = Hoeveel flow units klaar met proces in tijd. Flow time = gemiddelde tijd flow unit door het proces gaat. Je hebt er 2 nodig om de 3e uit te rekenen. Bijvoorbeeld 9=3x3
Als een bedrijf een desired cycle time heeft, kan het minimale aantal werkstations berekend worden:
Bovenkant formule = tijd van alle taken (1 product/persoon) Onderkant formule = gewenste cycle time (max tijd per station) Hoeveel werk er gedaan moet worden / tijd station mag. Tijd van alle taken --> 60 min Gewenste cycle time --> 15 min 60 / 15 = 4 stations nodig = Minimale aantal werkstations
De efficiëntie kan berekend worden met de volgende formule:
Deze oefenen / doornemen. Vraag 1.
De balancing loss kan ook berekend worden door 1 - efficiency te doen.
Het verlies door een verkeerde verdeling. 1 - Efficiency (stel efficiency is 0,8) 1 - 0,8 = 20% verlies = Balancing loss.
De desired cycle time: dit is de maximale tijd die besteed kan worden per station om een productietarget te halen. De desired cycle time (Cd) wordt op de volgende manier berekend:
Je hebt 120 minuten om 30 producten te maken. 120 / 30 = 4 minuten per product. = Desired cycle time (Cd)
De throughput rate (aantal afgeronde units per tijdseenheid) kan berekend worden via Little's Law maar ook door 1 te delen door de cycle time.
Altijd de tijdseenheid erbij vermelden. Cycletime --> 2 producten per minuut --> 1/2 = 0,5 producten !per minuut! = Throughput rate
Bij line balancing is het belangrijk om het verschil tussen cycle time en throughput time goed te weten. De throughput rate (aantal afgeronde units per tijdseenheid) kan berekend worden via Little's Law maar ook door 1 te delen door de cycle time.
Cycle time kan alleen korter zijn dan de throughput time als er meerdere flow units tegelijk in het proces zitten (parallelle verwerking). Cycle time = hoelang zit er tussen twee flow units die afronden Through put time = hoelang doet een flow unit over afronden
1. a >L b: if and only if there is a trace σ =⟨t1,t2,t3,...,tn⟩ and i ∈{1,...,n−1} such that σ ∈ L and ti = a and ti+1 = b; 2. a →L b if and only if a > L and b >/ L a; 3. a #L b if and only if a >/ b and b >/ a; and 4. a ∥L b if and only if a > L b and b > L a.
Synthèse sur la Sexualité et la Contraception
Résumé
Ce document de synthèse résume les points clés d'une discussion sur la sexualité et la contraception, menée par des intervenantes du Planning Familial d'Indre-et-Loire.
Les thèmes centraux incluent l'importance d'une éducation sexuelle et affective précoce et progressive, fondée sur les connaissances des jeunes pour déconstruire les idées reçues.
Un accent particulier est mis sur la notion de consentement, détaillée à travers l'acronyme "REELS" (Réversible, Éclairé, Enthousiaste, Libre, Spécifique), qui sert de guide pratique pour des relations respectueuses.
Le document présente un panorama exhaustif des méthodes de contraception existantes, classées en cinq catégories (hormonales, mécaniques, naturelles, définitives, d'urgence), en soulignant la responsabilité partagée et la possibilité de changer de méthode au cours de la vie.
Enfin, il aborde la prévention des Infections Sexuellement Transmissibles (IST), l'importance cruciale du dépistage, et les modalités de l'Interruption Volontaire de Grossesse (IVG) en France, tout en listant les structures ressources locales pour les jeunes et les parents.
1. L'Approche du Planning Familial en Matière d'Éducation Sexuelle
Le Planning Familial d'Indre-et-Loire, une association loi 1901 fondée en 1967 localement, a pour mission la prévention, l'information et l'orientation sur la vie affective, relationnelle et la santé sexuelle. Ses interventions sont gratuites, anonymes, confidentielles et basées sur un accueil inconditionnel.
1.1. Méthodologie : L'Éducation Populaire
L'approche de l'association repose sur le principe de l'éducation populaire, qui considère que chaque individu possède des savoirs. Les interventions partent des représentations et des connaissances du public pour ensuite apporter des informations complémentaires.
• Outils utilisés : Le "brainstorming" est un outil central pour recueillir les mots et les idées des participants sur un thème donné (ex: la puberté).
• Objectifs :
◦ Déconstruire les idées reçues et les fausses informations, notamment celles issues d'internet.
◦ Rassurer les jeunes sur les changements corporels et les différences de développement, luttant ainsi contre les complexes. ◦ Adapter le contenu à l'âge des participants.
1.2. Le Programme National d'Éducation à la Sexualité
Un programme gouvernemental, en accord avec une loi de 2002 prévoyant trois séances annuelles de la maternelle au lycée (bien que peu appliquée), structure l'éducation à la vie affective et sexuelle. Ce programme, jugé pertinent par le Planning Familial, propose une approche progressive :
• Maternelle et Primaire : Aborde les émotions, le consentement non-sexuel ("je ne veux pas jouer avec toi"), les stéréotypes de genre et les relations aux autres.
• Collège (à partir de la 4ème) et Lycée : Introduction de la thématique de la sexualité au sens large. L'école est considérée comme le lieu idéal pour une information égalitaire.
2. Puberté et Connaissance du Corps
La puberté est une période de transition majeure, marquée par une "tempête hormonale" qui influence à la fois le corps et les émotions.
2.1. Changements Physiques et Émotionnels
• Émotionnels : Humeur changeante (colère, tristesse), fatigue, susceptibilité. C'est une période de découverte de soi, de ses goûts et de ses limites.
• Physiques : Pilosité, développement des seins, mue de la voix, acné, croissance. Les intervenantes soulignent l'importance de rassurer sur le fait que chaque corps se développe à son propre rythme. La puberté survient en moyenne entre 11 et 16 ans, mais peut être plus précoce (dès 9-10 ans) ou plus tardive.
2.2. Le Cycle Menstruel
Le cycle menstruel est un sujet essentiel abordé avec tous les jeunes, garçons et filles.
• Fonctionnement : Un cycle moyen dure 28 jours, avec une ovulation survenant 14 jours avant le début du cycle suivant.
• Période de Fertilité : Un ovule est viable 24 à 48 heures, tandis qu'un spermatozoïde peut survivre jusqu'à 5 jours dans l'utérus, ce qui définit une fenêtre de fertilité de plusieurs jours.
• Idées Reçues : Il est possible de tomber enceinte dès les premières règles. Des ovulations spontanées peuvent survenir en dehors de la période théorique de fertilité.
• Douleurs : Il est rappelé que des douleurs de règles invalidantes ne sont pas normales et doivent motiver une consultation médicale.
2.3. Ressources Pédagogiques pour les Parents et les Jeunes
Une sélection d'ouvrages est présentée comme support pour aborder ces sujets en famille.
Titre de l'Ouvrage
Public Cible
Thèmes Abordés
Le petit illustré de l'intimité (4 tomes)
Enfants, Adolescents, Parents
Anatomie (pénis, vulve), famille, contraception, genre, orientation sexuelle.
Le petit guide de la foufoune sexuelle
Tous publics
Sexualité, consentement, anatomie, puberté, fausses idées.
Le parcours de Polo Jeunes enfants
Reproduction (comment on fait les bébés) sous forme d'album jeunesse.
Comment on fait les bébés
Jeunes enfants
Reproduction, avec un point de vue humoristique sur les non-dits des parents.
Les règles, Quelle aventure
Adolescents
Les menstruations, sous forme d'histoire.
Rouge
Jeunes enfants
Les émotions (la timidité, le fait de rougir).
Corps, amour et sexualité Parents (Guide)
Encyclopédie par tranche d'âge (5-12 ans) pour répondre aux questions des enfants.
3. Le Consentement : Un Pilier des Relations
Le consentement est un sujet de plus en plus évoqué par les jeunes. Le Planning Familial utilise un moyen mnémotechnique pour en définir les composantes essentielles.
• R - Réversible : Un "oui" peut être retiré à tout moment. L'absence de participation active équivaut à un retrait du consentement.
• E - Éclairé : La personne doit être en pleine capacité de comprendre ce qui est proposé. Le consentement n'est pas valide si la personne est endormie, sous l'influence de l'alcool ou de drogues.
• E - Enthousiaste : Le consentement doit être actif et joyeux, exprimé verbalement et non-verbalement. Une participation passive ou hésitante n'est pas un consentement enthousiaste.
• L - Libre : Le "oui" doit être donné sans pression, chantage, menace ou rapport de hiérarchie.
• S - Spécifique : Le consentement est donné pour un acte précis, à un moment précis. Il n'est pas valable pour d'autres actes ou pour d'autres moments.
4. Panorama des Moyens de Contraception La contraception est présentée comme une responsabilité partagée, les hommes étant fertiles toute l'année. Aucune méthode n'est efficace à 100% et il est possible d'en changer tout au long de sa vie.
4.1. Contraceptions Hormonales
Elles agissent en faisant croire au corps qu'une grossesse est en cours, bloquant ainsi l'ovulation.
• La Pilule : Comprimé quotidien à heure fixe. Efficace à 99%. Peut ne pas convenir à un mode de vie irrégulier.
• L'Implant : Bâtonnet inséré sous la peau du bras, efficace 3 ans.
• Le Patch : À coller sur la peau et à changer chaque semaine. Non remboursé.
• Le Dispositif Intra-Utérin (DIU) Hormonal : Placé dans l'utérus, diffuse des hormones localement. Efficace 5 ans.
• L'Anneau Vaginal : Anneau flexible placé dans le vagin pour 3 semaines. Non remboursé.
• Les Injections d'Hormones : Tous les 3 mois pour les femmes, toutes les semaines pour les hommes.
4.2. Contraceptions Mécaniques
Elles créent une barrière physique pour empêcher la rencontre des spermatozoïdes et de l'ovule.
• Préservatifs (Externe et Interne) : Seul moyen de contraception protégeant également des IST. Le préservatif interne peut être mis jusqu'à 8 heures avant le rapport.
• Le Diaphragme / La Cape Cervicale : Dômes en silicone placés au fond du vagin. Efficacité renforcée avec des spermicides.
• Le DIU au Cuivre : Le cuivre crée une inflammation qui rend l'utérus "hostile" à la nidation. Efficace 5 ans, sans hormones.
• L'Andro-Switch (Anneau) / Le Slip Thermique : Méthodes masculines qui remontent les testicules contre le corps.
La chaleur annule le pouvoir fécondant des spermatozoïdes. Nécessite d'être porté 15h/jour et un suivi médical (spermogrammes).
4.3. Méthodes Naturelles (Contrôle des Naissances)
Leur efficacité est jugée relative et moins fiable.
• Le Retrait : Efficacité limitée car le liquide pré-séminal peut contenir des spermatozoïdes.
• Méthode des Températures : Suivi de la température corporelle pour détecter l'ovulation.
• Méthode de l'Allaitement : L'allaitement peut bloquer le retour de couches, mais reste très peu fiable.
• Méthode Ogino (Calendrier) : Calcul de la période de fertilité, peu fiable en raison des ovulations spontanées.
4.4. Contraceptions Définitives
Elles sont accessibles dès 18 ans, après un délai de réflexion légal.
• La Vasectomie (Homme) : Opération sous anesthésie locale pour sectionner les canaux déférents. L'éjaculation persiste mais sans spermatozoïdes. Considérée comme irréversible.
• La Ligature des Trompes (Femme) : Opération plus lourde, sous anesthésie générale, pour bloquer le passage des ovules.
4.5. Contraception d'Urgence • "Pilule du lendemain" : Comprimé à prendre le plus tôt possible après un rapport à risque (efficace jusqu'à 3-5 jours). Gratuite pour les mineurs et accessible en pharmacie sans avancer de frais pour les majeurs. Un homme peut aller la chercher pour sa partenaire.
5. Infections Sexuellement Transmissibles (IST)
• Protection : Le préservatif est le seul moyen de s'en protéger.
• Dépistage : C'est un acte de responsabilité essentiel pour se protéger soi-même et ses partenaires. La plupart des IST sont asymptomatiques.
◦ Il est possible de se faire dépister sans ordonnance en laboratoire.
◦ Un délai (généralement 6 semaines à 3 mois après le rapport à risque) est nécessaire pour que le test soit fiable.
• Traitement : La majorité des IST se soignent bien si elles sont prises à temps. Le VIH, bien qu'incurable, se traite efficacement, permettant une vie normale et une charge virale indétectable (et donc intransmissible).
6. L'Interruption Volontaire de Grossesse (IVG)
L'IVG est un droit en France, encadré par la loi. • Deux méthodes : 1. IVG médicamenteuse : Jusqu'à 7 semaines de grossesse (9 semaines d'aménorrhée). 2. IVG chirurgicale : Jusqu'à 14 semaines de grossesse (16 semaines d'aménorrhée). • Accompagnement : ◦ Pour une personne mineure, l'accompagnement par une personne majeure de confiance (pas obligatoirement un parent) est obligatoire. ◦ La démarche inclut des entretiens d'information pour permettre un choix éclairé. ◦ Les centres spécialisés (centres d'orthogénie) disposent d'équipes pluridisciplinaires (médecins, psychologues) pour accompagner les personnes.
7. Lieux et Structures Ressources
Plusieurs structures offrent information, accompagnement et soins de manière gratuite et confidentielle en Indre-et-Loire. • Le Planning Familial : Pour l'information, l'écoute et l'orientation. • Le CEGIDD (Centre Gratuit d'Information, de Dépistage et de Diagnostic) : Pour le dépistage des IST. • Les Centres de Santé Sexuelle : Pour le suivi gynécologique, la contraception, le dépistage et l'accompagnement global. • L'Espace Santé Jeunes (ESJ) : Pour les jeunes et les parents sur des questions de santé globale, y compris les addictions. • La Maison des Adolescents : Lieu ressource avec des professionnels variés (psychologues, pédiatres, etc.).
La Cohérence Éducative : Synthèse et Analyse
Résumé
La cohérence éducative constitue le socle d'un développement sain pour l'enfant, en lui offrant un cadre stable, prévisible et sécurisant.
Elle se définit par l'harmonisation des discours, des pratiques et des valeurs entre les différents adultes qui entourent l'enfant.
Cette cohérence ne se limite pas au couple parental, mais s'étend sur cinq sphères interdépendantes : la cohérence interne de chaque adulte avec ses propres valeurs, le couple parental, la famille élargie et l'entourage, les professionnels (école, crèche), et la société dans son ensemble.
Le principal risque de l'incohérence chronique est l'insécurité affective, qui peut entraver la construction de la confiance en soi et pousser l'enfant à tester constamment les limites.
Il est crucial de distinguer la simple différence, qui peut être une source de richesse et d'ouverture d'esprit lorsqu'elle est expliquée, de l'incohérence conflictuelle et de la disqualification d'un adulte par un autre devant l'enfant, qui sont particulièrement préjudiciables.
Les stratégies fondamentales pour cultiver cette cohérence reposent sur la communication, la souplesse et le respect mutuel.
Il s'agit de dialoguer sur les désaccords, de comprendre les besoins sous-jacents aux positions de chacun, de chercher des compromis et d'expliquer les règles à l'enfant.
L'objectif n'est pas une rigidité absolue, mais la construction d'un cadre général fiable qui prépare l'enfant à naviguer sereinement dans un monde où les incohérences font partie de la vie.
1. Le Concept de Cohérence et ses Fondements
1.1. Définition de la Cohérence
La cohérence est définie comme l'adéquation et la logique entre les pensées, les paroles et les actes d'un individu.
Cette harmonie interne est un besoin fondamental ; son absence génère un inconfort psychologique.
1.2. La Dissonance Cognitive
Théorisée par le psychologue Leon Festinger, la dissonance cognitive décrit l'état d'inconfort ressenti lorsqu'il y a un décalage entre les croyances et les comportements.
Pour réduire cette tension, les individus ont tendance à modifier leurs pensées ou à trouver des justifications plutôt que de changer un comportement ancré.
• Exemples concrets :
◦ Un parent qui passe beaucoup de temps sur son écran tout en limitant celui de son enfant.
◦ Un parent qui fume tout en interdisant à son adolescent de le faire.
• Perception par l'enfant : Les enfants sont particulièrement habiles à détecter ces incohérences, ce qui peut miner la crédibilité des règles et des adultes qui les édictent.
2. Les Cinq Sphères de la Cohérence Éducative Le document source structure l'analyse de la cohérence éducative autour d'un schéma central représentant cinq sphères d'influence concentriques, allant de l'individu à la société.
2.1. La Cohérence Interne : La Base Individuelle
Avant de pouvoir être cohérent avec d'autres adultes, il est nécessaire de l'être avec soi-même.
Cette cohérence personnelle est un alignement entre les comportements visibles et les niveaux plus profonds que sont les règles, les croyances et les valeurs fondamentales, souvent héritées de sa propre éducation, de sa culture et de son histoire familiale.
• Questionnement personnel : Il est essentiel pour un adulte de s'interroger sur l'origine de ses valeurs ("Est-ce que finir son assiette est une valeur qui me correspond vraiment ou est-ce un héritage familial que je transmets sans y réfléchir ?").
• Gestion de ses propres incohérences : Nul n'est parfait.
Lorsqu'un parent agit de manière incohérente (par exemple, crier alors que la règle est de ne pas crier), il est bénéfique de le reconnaître devant l'enfant.
Nommer l'incohérence ("Je suis désolé, je me suis emporté") permet de donner du sens, de valider le ressenti de l'enfant et de renforcer la sécurité affective en montrant que les adultes peuvent admettre leurs erreurs.
2.2. Le Couple Parental : Le Premier Cercle de Cohérence
Qu'ils soient ensemble ou séparés, les parents forment le premier duo d'adultes dont la cohérence est primordiale.
L'objectif commun est de protéger l'enfant et de lui offrir un cadre propice à son développement.
• Métaphore de "L'oiseau à deux becs" : Cet album jeunesse illustre deux parents (les deux becs) qui nourrissent un même enfant (le ventre).
Si les becs se disputent, le ventre en pâtit et peut même être "empoisonné". La coopération est donc vitale.
• Sources de conflit : Les désaccords peuvent provenir de modèles éducatifs différents, de tensions conjugales ou de simples divergences de points de vue.
• Stratégies de résolution :
◦ Identifier les besoins : Derrière une émotion comme la colère ou une position rigide se cache souvent un besoin non satisfait (besoin de respect, de sécurité, d'être écouté).
Comprendre le besoin de l'autre permet de dépasser le conflit de surface.
◦ La disqualification : Le risque majeur est de disqualifier l'autre parent devant l'enfant ("De toute façon, toi, tu laisses tout faire").
Cela consiste à nier sa compétence et place l'enfant dans une position intenable de loyauté.
2.3. La Famille et l'Entourage : L'Élargissement du Cadre
Cette sphère inclut les grands-parents, oncles, tantes, amis et voisins. L'enfant évolue dans différents lieux avec des règles potentiellement différentes.
• Différence vs. Incohérence : Avoir des règles différentes chez les grands-parents (plus de bonbons, par exemple) n'est pas une incohérence en soi et peut enrichir l'enfant.
Cela devient problématique lorsque ces différences vont à l'encontre de valeurs fondamentales ou de règles de sécurité non négociables pour les parents.
• Conflit et Clivage de Loyauté :
◦ Conflit de loyauté : L'enfant est partagé entre deux attentes différentes. C'est une situation courante et gérable.
◦ Clivage de loyauté : Situation plus grave où choisir un camp signifie être contre l'autre.
Cela se produit lorsque des adultes dénigrent les autres, forçant l'enfant à prendre parti.
Exemple : un enfant n'ose pas dire qu'il a passé un bon week-end chez un parent par peur de blesser l'autre.
2.4. Les Professionnels : La Collaboration Éducative
Cette sphère concerne les acteurs de la crèche, de l'école, des loisirs ou du secteur médical.
• Légitimité parentale : Les parents sont les premiers éducateurs de leur enfant.
Même face à des experts, ils ont la légitimité de questionner les pratiques qui ne leur semblent pas cohérentes avec leurs valeurs.
• Importance du dialogue : Il est essentiel d'oser interroger les professionnels pour comprendre le sens de leurs pratiques (ex: la "motricité libre" en crèche qui autorise les enfants à monter sur des meubles sécurisés).
Cet échange permet de créer une continuité éducative et de rassurer les parents.
L'enfant est capable de comprendre que les règles peuvent varier selon le contexte (maison vs. crèche), à condition que ce contexte soit clair.
2.5. La Société : Le Contexte Global
C'est la sphère la plus diffuse et la plus difficile à influencer. Elle englobe les codes sociaux, les injonctions médiatiques, les évolutions culturelles et le cadre législatif.
• Pression sociale : Le regard des autres peut pousser un parent à agir contre ses propres principes (ex: gérer une crise de colère d'un enfant dans un magasin).
• Évolution des normes : Des concepts comme "l'éducation bienveillante" ou les apports des neurosciences modifient les attentes sociales envers les parents.
• Le cadre légal : La loi impose un cadre non négociable, comme l'interdiction de la fessée en France depuis 2019, qui s'impose à toutes les pratiques éducatives.
3. Les Risques de l'Incohérence et le Cadre Sécurisant
3.1. L'Impact sur la Sécurité Affective de l'Enfant
La cohérence est un pilier de la sécurité affective. Le chercheur québécois Georges Tarabulsy identifie trois facteurs clés de la sécurité de l'attachement : la chaleur, la prévisibilité et la cohérence.
• La "jauge à sécurité" : Une métaphore est utilisée pour décrire la sécurité interne de l'enfant comme un réservoir.
Des pratiques cohérentes, stables et harmonieuses remplissent cette jauge. Des incohérences chroniques la vident, ce qui peut affecter durablement le développement de l'enfant.
3.2. Conséquences Comportementales et Psychologiques
Un manque de cohérence peut entraîner :
• Une recherche constante des limites pour comprendre un cadre flou.
• Une perte de sens et une atteinte à la confiance en soi.
• Des difficultés à construire son propre système de valeurs, car il n'y a pas eu de base stable à partir de laquelle se différencier.
4. Stratégies Pratiques pour Cultiver la Cohérence
Stratégie Description Exemple
Adapter le cadre à l'enfant Ajuster les règles et les consignes à l'âge, au rythme de développement, aux compétences et aux besoins de l'enfant.
Ne pas proposer à un enfant de 3 ans de sortir seul de l'école. Offrir un choix limité ("le pantalon bleu ou le rouge ?") plutôt qu'un choix trop vaste et insécurisant.
Préciser les consignes
Donner des instructions claires et s'assurer de leur compréhension. Expliquer le "pourquoi" derrière une règle.
Au lieu de "ne dessine pas sur les murs", expliquer : "Je ne veux pas que tu abîmes la peinture. Si tu veux décorer, utilisons des post-it".
Communiquer entre adultes
Dialoguer ouvertement sur les désaccords, loin de la présence de l'enfant si le ton monte. Viser la compréhension mutuelle et le compromis.
Un parent veut que l'enfant marche pieds nus pour sa motricité, l'autre s'inquiète de l'hygiène. Le compromis : pieds nus dans la maison, mais chaussons dans le garage.
Faire preuve de souplesse
Accepter que l'autre puisse avoir un point de vue différent sans que cela ne constitue une attaque personnelle.
Métaphore du livre Quatre petits coins de rien du tout : parfois, il ne faut pas chercher à changer l'enfant (le carré) mais à adapter le cadre (la porte ronde).
Pratiquer la "métacommunication"
Communiquer sur la manière de communiquer : "Quand tu me parles sur ce ton, j'ai l'impression que tu penses que je suis une mauvaise mère."
Permet de clarifier les malentendus et de comprendre l'impact de ses paroles sur l'autre.
5. Conclusion : Viser un Équilibre Dynamique
L'incohérence fait partie de la vie ; l'objectif n'est pas de l'éradiquer, mais de la minimiser et de la gérer.
La clé est de construire un cadre éducatif globalement stable et sécurisant.
Comme le disait le pédagogue Célestin Freinet, il s'agit moins d'éviter les incohérences que de "préparer l'enfant à pouvoir les vivre sereinement".
En dialoguant, en s'ajustant et en faisant preuve de souplesse, les adultes peuvent transformer les différences en une richesse et offrir à l'enfant les repères solides dont il a besoin pour grandir et construire sa propre cohérence.
Dossier d'Information : Consommation et Addiction à l'Adolescence
Résumé
Ce document synthétise les perspectives et données clés issues d'une discussion entre expertes sur la consommation de substances et l'addiction durant l'adolescence. Les points essentiels à retenir sont les suivants :
1. Baisse de la Consommation : Contrairement à une perception médiatique souvent anxiogène, les données épidémiologiques (enquêtes ESCAPAD) montrent une baisse constante et significative de l'expérimentation et de la consommation régulière de tabac, d'alcool et de cannabis chez les adolescents en France depuis plus de dix ans.
2. Distinction Cruciale : Il est impératif de ne pas confondre consommation, expérimentation et addiction.
L'expérimentation est un comportement exploratoire fréquent et même jugé "presque nécessaire" au développement de l'adolescent pour tester ses limites.
L'addiction, caractérisée par une perte de contrôle, reste un phénomène rare à cet âge. Le terme "conduite à risque" est souvent plus approprié.
3. Signaux d'Alerte : L'inquiétude doit naître non pas d'une consommation isolée, mais d'un cumul de plusieurs facteurs : précocité de l'usage, cumul de produits, consommation à visée "autothérapeutique" (pour s'apaiser), recherche systématique d'excès et répétition fréquente. L'évaluation doit être globale, incluant le contexte scolaire, social et familial.
4. Prévention Efficace : Les stratégies de prévention modernes ont abandonné l'approche basée sur la peur et l'information brute, jugée contre-productive. L'accent est désormais mis sur le renforcement des compétences psychosociales (CPS) : estime de soi, gestion des émotions, esprit critique et capacité à dire non.
5. Rôle Parental Fondamental : Les parents sont des acteurs de prévention de premier plan. Leur rôle est de maintenir un dialogue ouvert, d'éviter les jugements hâtifs, de poser un cadre clair sans fermer la communication, et d'être une ressource fiable en cas de difficulté. Discuter des sensations et des limites est plus constructif que la seule répression.
6. Ressources Disponibles : Des structures gratuites, confidentielles et accessibles existent pour les jeunes (12-25 ans) et leur entourage. Les Consultations Jeunes Consommateurs (CJC), hébergées par des entités comme le CSAPA 37 ou l'Espace Santé Jeune, offrent un lieu d'écoute et d'évaluation sans jugement.
1. Définitions Clés : De la Substance Psychoactive à l'Addiction
Selon les intervenantes, il est essentiel de clarifier les termes employés pour aborder sereinement le sujet.
• Drogue / Substance Psychoactive : Une drogue est définie comme tout produit, licite ou illicite, qui modifie le fonctionnement du cerveau et du psychisme. Cette définition inclut donc l'alcool et le tabac, qui sont les substances les plus consommées et les principaux enjeux de santé publique.
• Mécanismes Cérébraux : Le corps humain produit naturellement des substances psychoactives (endogènes) comme la dopamine ou les endorphines, qui génèrent du plaisir suite à des actions comme manger ou faire du sport.
Les substances externes (exogènes) stimulent ces mêmes circuits de récompense de manière beaucoup plus rapide et intense, ce qui peut inciter le cerveau à privilégier cette voie pour obtenir une satisfaction immédiate.
• Consommation vs. Addiction : Toute consommation n'est pas une addiction. L'addiction est un processus qui s'installe dans le temps et se caractérise par une "perte de la liberté de s'abstenir". Pour les adolescents, les professionnelles préfèrent parler de conduites à risque plutôt que d'addiction, cette dernière étant rare dans cette tranche d'âge.
2. La Réalité de la Consommation chez les Adolescents : Données et Tendances
Les intervenantes insistent sur la nécessité de dédramatiser le phénomène en s'appuyant sur des données objectives issues d'enquêtes nationales (ESCAPAD, menée par l'OFDT).
Tendance Observée Détails
Baisse Générale
Une baisse constante des expérimentations et des consommations régulières de tabac, d'alcool et de cannabis est observée chez les collégiens et lycéens depuis plus de 10 ans.
Recul de l'Âge
Contrairement aux idées reçues, l'âge des premières consommations ne baisse pas ; il a même tendance à reculer.
Les premières expérimentations ont souvent lieu vers 16-17 ans.
Exemple du Tabac
Le pourcentage de fumeurs réguliers à 17 ans est passé de 40% en 2002 à moins de 16% en 2022.
Hiérarchie des Produits
Les produits les plus consommés restent, de loin, le tabac et l'alcool, suivis par le cannabis. Les autres substances illicites ne représentent qu'une part mineure des consommations (environ 5%).
Première Initiation
L'initiation à l'alcool, produit très normalisé et culturellement ancré en France, se fait très souvent dans le cadre familial lors d'événements festifs.
Cette perspective factuelle est cruciale pour éviter deux écueils : l'inquiétude parentale excessive et l'incitation paradoxale des jeunes qui, se croyant en décalage, pourraient être tentés d'expérimenter pour se conformer à une norme perçue.
3. Identifier les Signes d'Alerte : Quand Faut-il s'Inquiéter ?
Une consommation devient une conduite à risque préoccupante non pas à cause d'un seul acte, mais lorsque plusieurs signaux s'accumulent et indiquent un changement de comportement global. L'évaluation se base sur un faisceau d'indices.
Les cinq indicateurs principaux à surveiller :
1. La Précocité des Consommations : Un usage commencé très jeune (collège) est un facteur de risque majeur en raison de l'immaturité du cerveau.
2. Le Cumul des Consommations : L'utilisation simultanée ou alternée de plusieurs produits (alcool, tabac, cannabis...).
3. Les Consommations Autothérapeutiques : L'usage d'un produit pour s'apaiser, gérer une angoisse ou fuir une difficulté. Le produit devient alors une sorte de "médicament".
4. La Recherche d'Excès : La volonté systématique d'atteindre des états extrêmes.
5. La Répétition des Consommations : Une fréquence élevée qui peut entraîner une accoutumance et un besoin d'augmenter les doses.
En parallèle, il faut observer le contexte général de l'adolescent :
• Scolarité : Y a-t-il un désintérêt, un décrochage ?
• Vie Sociale : L'adolescent s'isole-t-il ? Change-t-il de cercle d'amis ?
• Centres d'Intérêt : Abandonne-t-il des activités qui lui procuraient du plaisir auparavant ?
• Communication Familiale : Le dialogue est-il rompu ? Le comportement de consommation est souvent le symptôme d'un mal-être sous-jacent. L'analyse doit donc être systémique, en prenant en compte l'individu, la substance et son environnement.
4. Le Processus Addictif Illustré : L'Allégorie "Nuggets" Le court-métrage d'animation "Nuggets" (Andreas Hykade, 2014) est utilisé pour décomposer le cheminement qui peut mener d'une expérimentation à une addiction.
1. La Lune de Miel : La première rencontre avec le produit est une expérience positive, une recherche de plaisir pur. Il n'y a pas encore d'envie irrépressible de consommer (craving).
2. L'Apparition des Dommages : La consommation apporte toujours du plaisir, mais les premières conséquences négatives apparaissent (physiques, sociales, financières). La tonalité devient plus sombre.
3. La Compulsion et la Tolérance : L'envie devient pressante, compulsive. Les effets positifs durent moins longtemps et sont moins intenses. Une tolérance s'installe, obligeant à augmenter les doses pour retrouver les effets initiaux. C'est l'étape de la "perte de la liberté de s'abstenir".
4. La Perte de Contrôle : Le comportement devient obsessionnel. La consommation se poursuit de manière frénétique malgré la dégradation de l'état du sujet et l'absence quasi totale de plaisir. Le but n'est plus de ressentir du plaisir, mais de soulager la souffrance du manque.
Ce processus montre que l'addiction ne s'installe pas du jour au lendemain et que la continuité de la consommation est un facteur déterminant.
5. L'Adolescence : Une Période de Vulnérabilité et d'Expérimentation La consommation de produits doit être comprise dans le contexte unique de l'adolescence, une période de profonds bouleversements.
• Une Quête Nécessaire : L'expérimentation, y compris avec des produits, fait partie du processus normal de construction de l'identité. C'est une manière pour l'adolescent de tester ses limites, de se connaître, de développer son estime de soi et de s'autonomiser (processus d'individuation).
• Fonctions Sociales : La consommation peut être un rite de passage, un moyen d'intégration dans un groupe de pairs, ou un acte d'imitation.
• Vulnérabilité Psychique : Pour les adolescents plus fragiles, la substance peut devenir un "support" pour gérer des difficultés, notamment le détachement des parents ou un mal-être profond.
• Le Danger de l'Identification : Le discours ambiant qui associe systématiquement adolescence et consommation problématique peut créer une identification négative. L'adolescent, en quête de modèles, peut se conformer à cette image et adopter le comportement attendu.
6. Stratégies de Prévention Efficaces L'approche de la prévention a radicalement évolué, s'éloignant des méthodes jugées inefficaces pour se concentrer sur l'outillage de l'adolescent.
Ce qui ne fonctionne pas :
• Le Discours de Peur : Agiter les menaces ("tu vas mourir", "tu iras en prison") génère du stress (cortisol) et peut avoir un effet inverse, incitant l'adolescent à chercher une substance pour apaiser cette angoisse.
• L'Apport de Connaissances Brutes : Le cerveau adolescent est dominé par l'impulsivité et le système émotionnel. La connaissance des risques ne suffit pas à empêcher le passage à l'acte.
Ce qui fonctionne : le Renforcement des Compétences Psychosociales (CPS)
L'objectif est de donner à l'adolescent les outils pour faire des choix éclairés et se protéger.
Cela inclut le travail sur :
Le rôle des parents et de l'entourage est central : • Maintenir la Communication : Laisser la porte du dialogue ouverte est la clé.
L'adolescent doit savoir qu'il peut appeler un parent en cas de difficulté, même après une consommation excessive, sans craindre une punition disproportionnée.
• Accueillir et Discuter : Face à une première ivresse, il est plus constructif d'ouvrir une discussion sur les sensations ressenties (positives et négatives) plutôt que de réprimer uniquement.
• Poser un Cadre : L'échange ne signifie pas la permission. Il est du rôle du parent de rappeler les règles et la loi (ex: le cannabis est illégal).
• Prévention Collective : La prévention est l'affaire de tous (parents, enseignants, éducateurs). Créer un environnement bienveillant et sécurisant, où le jeune se sent bien, est un facteur de protection puissant.
Les adolescents eux-mêmes développent des stratégies de réduction des risques, comme le "capitaine de soirée" (celui qui ne boit pas) ou le fait de dormir sur place après une fête, montrant une prise de conscience des dangers.
7. Ressources et Soutien Disponibles Un réseau de structures professionnelles existe pour accompagner les jeunes et leur entourage.
• Les Consultations Jeunes Consommateurs (CJC) :
◦ Public : Jeunes de 12 à 25 ans, mais aussi leur entourage (parents, amis, grands-parents).
◦ Principes : Accueil gratuit, confidentiel et potentiellement anonyme. Il n'est pas nécessaire d'être en situation d'addiction ; toute question sur la consommation est légitime.
◦ Mission : Offrir un lieu d'écoute pour faire le point, évaluer une situation, obtenir de l'information, apprendre à réduire les risques et, si besoin, être orienté.
◦ Lieux : Elles sont implantées dans des structures spécialisées (comme le CSAPA - Centre de Soins, d'Accompagnement et de Prévention en Addictologie) et des lieux plus généralistes pour éviter la stigmatisation (Espace Santé Jeune, Maison des Adolescents).
• Programmes de Prévention : ◦ En Milieu Scolaire : Des programmes probants comme "Unplugged" sont déployés dans les établissements pour travailler sur les compétences psychosociales en co-animation avec les enseignants. ◦ Soutien à la Parentalité : Des groupes de parole pour parents, comme "Mon Ado et Moi", permettent d'échanger sur les inquiétudes et de dédramatiser les situations.
8. Nouvelles Tendances de Consommation
Bien que la consommation des produits "classiques" soit en baisse, de nouveaux produits émergent et suscitent des inquiétudes :
• La "Puff" : Une cigarette électronique jetable, très attractive pour les jeunes par son design coloré et ses goûts sucrés. Elle constitue une nouvelle porte d'entrée vers la consommation de nicotine pour des jeunes qui n'auraient pas commencé à fumer autrement.
• Le Protoxyde d'Azote : Gaz hilarant contenu dans des cartouches. Sa consommation, perçue à tort comme inoffensive, peut entraîner des dommages neurologiques et physiques importants.
• La Chicha : Narguilé dont l'usage reste populaire et qui est également nocif.
Chapter 4: Common Writing Assignments College writing assignments serve a different purpose than the typical writing assignments you completed in high school. The textbook Successful Writing explains that high school teachers generally focus on teaching you to write in a variety of modes and formats, including personal writing, expository writing, research papers, creative writing, and writing short answers and essays for exams. Over time, these assignments help you build a foundation of writing skills. In college, many instructors will expect you to already have that foundation. Your college composition courses will focus on writing for its own sake, helping you make the transition to college-level writing assignments. However, in most other college courses, writing assignments serve a different purpose. In those courses, you may use writing as one tool among many for learning how to think about a particular academic discipline. Additionally, certain assignments teach you how to meet the expectations for professional writing in a given field. Depending on the class, you might be asked to write a lab report, a case study, a literary analysis, a business plan, or an account of a personal interview. You will need to learn and follow the standard conventions for those types of written products. Finally, personal and creative writing assignments are less common in college than in high school. College courses emphasize expository writing, writing that explains or informs. Often expository writing assignments will incorporate outside research, too. Some classes will also require persuasive writing assignments in which you state and support your position on an issue. College instructors will hold you to a higher standard when it comes to supporting your ideas with reasons and evidence. Common Types of College Writing Assignments Below you will find a list of different types of writing assignments you may write as you pursue your academic goals. Review each assignment and think about the writing you’ve done in high school and how these assignments might look different in your college composition classes. Figure 1 After reviewing Figure 1 and the descriptions of various types of writing assignments, watch the following video about the writing process. No matter what type of assignment you are writing, it will be important for you to follow a writing process: a series of steps a writer takes to complete a writing task. Making use of a writing process ensures that you stay organized and focused while allowing you to break up a larger assignment into several distinct tasks. Not every writer follows the same process, and part of the work you will do in your writing classes is to discover the writing process that works best for you. Even though the writing process is often presented as a linear set of steps that writers follow from beginning to end, composition scholars now recognize the recursive nature of writing. In other words, many writers repeat steps in the process and not all writers invest an equal amount of time in each stage. Instead, writers often loop back to individual stages as needed in order to develop and refine their work. As you watch the video below, consider your current writing process (if you have one) and reflect upon how you might develop your process to support your growth as a writer—and to save yourself time and stress when completing college writing assignments. In the previous chapters, we covered college writing at CNM and reading strateg
The key to this is there are different types of writing assignments that has in the common writing assignments.
I have been out of school for a long time. Going back into schooling again is showing me that I can do it with all the resources that's out there. Focusing on learning new things that we do in everyday life.
As an illustration, we have developed a multiwell format in vitro assay that allows researchers to measure the activity of in-plate expressed and exported VNp-uricase protein (Figure 3), by following changes in 293 nm absorbance to monitor enzyme dependent breakdown of uric acid
I'm guessing that you measured this in your initial paper, but might be worth mentioning here as well. Have you shown that the VNp tag doesn't affect enzyme activity, stability, folding?
What does lichen do, not just in this poem, but outside it? What ways of being does it bring into the poem? How might we regard it as an agent of “enlichenment” (Goward)?
enlichenment <3
Data reflects official member state reporting with typical 2-3 month lags for newest installations.
Replace with: "Data reflects official member state reporting, with typical 2-3 month lags for newest installations."
(~3 decades)
takes species 30b years to adequately adjust
Reviewer #1 (Public review):
Summary:
It is well known that autophagosomes/autolysosomes move along microtubules. However, as these previous studies did not distinguish between autophagosomes and autolysosomes, it remains unknown whether autophagosomes begin to move after fusion with lysosomes or even before fusion. In this manuscript, the authors show using fusion-deficient vps16a RNAi cells that both pre-fusion autophagosomes and lysosomes can move along the microtubules towards the minus end. This was confirmed in snap29 RNAi cells. By screening motor proteins and Rabs, the authors found that autophagosomal traffic is primarily regulated by the dynein-dynactin system and can be counter-regulated by kinesins. They also show that Rab7-Epg5 and Rab39-ema interactions are important for autophagosome trafficking.
Strengths:
This study uses reliable Drosophila genetics and high-quality fluorescence microscopy. The data are properly quantified and statistically analyzed. It is a reasonable hypothesis that gathering pre-fusion autophagosomes and lysosomes in close proximity improves fusion efficiency.
Weaknesses:
(1) This study investigates the behavior of pre-fusion autophagosomes and lysosomes using fusion-incompetent cells (e.g., vps16a RNAi cells). However, the claim that these cells are truly fusion-incompetent relies on citations from previous studies. Since this is a foundational premise of the research, it should be rigorously evaluated before interpreting the data. It's particularly awkward that the crucial data for vps16a RNAi is only presented at the very end of Figure 10-S1; this should be among the first data shown (the same for SNAP29). It would be important to determine the extent to which autophagosomes and lysosomes are fusing (or tethered in close proximity), within each of these cell lines.
(2) In the new Figures 8 and 9, the authors analyze autolysosomes without knocking down Vps16A (i.e., without inhibiting fusion). However, as this reviewer pointed out in the previous round, it is highly likely that both autophagosomes and autolysosomes are present in these cells. This is particularly relevant given that the knockdown of dynein-dynactin, Rab7, and Epg5 only partially inhibits the fusion of autophagosomes and lysosomes (Figure 10H). If the goal is to investigate the effects of fusion, it would be more appropriate to analyze autolysosomes and autophagosomes separately. The authors mention that they can differentiate these two structures based on the size of mCherry-Atg8a structures. If this is the case, they should perform separate analyses for both autophagosomes and autolysosomes.
(3) This is also a continued Issue from the previous review. The authors suggest that autophagosome movement is crucial for fusion, based on the observed decrease in fusion rates in Rab7 and Epg5 knockdown cells (Fig. 10). However, this conclusion is not well supported. It is known that Rab7 and Epg5 are directly involved in the fusion process itself. Therefore, the possibility that the observed decrease is simply due to a direct defect in fusion, rather than an impairment of movement, has not been ruled out.
(4) The term "autolysosome maturation" appears multiple times, yet its meaning remains unclear. Does it refer to autolysosome formation (autophagosome-lysosome fusion), or does it imply a further maturation process occurring after autolysosome formation? This is not a commonly used term in the field, so it requires a clear definition.
(5) In Figure 1-S1D, the authors state that the disappearance of the mCherry-Atg8a signal after atg8a RNAi indicates that the observed structures are not non-autophagic vacuoles. This reasoning is inappropriate. Naturally, knocking down Atg8 will abolish its signal, regardless of the nature of the vacuoles. This does not definitively distinguish autophagic from non-autophagic structures.
Reviewer #3 (Public review):
Summary:
In multicellular organisms, autophagosomes are formed throughout the cytosol, while late endosomes/lysosomes are relatively enriched in the perinuclear region. It is known that autophagosomes gain access to the lysosome-enriched region by microtubule-based trafficking. The mechanism by which autophagosomes move along microtubules remains incompletely understood. In this manuscript, Péter Lőrincz and colleagues investigated the mechanism driving the movement of nascent autophagosomes along microtubule towards non-centrosomal microtubule organizing center (ncMTOC) using fly fat body as a model system. The authors took an approach by examining autophagosome positioning in cells where autophagosome-lysosome fusion was inhibited by knocking down the HOPS subunit Vps16A. Despite being generated at random positions in the cytosol, autophagosomes accumulate around the nucleus when Vps16A is depleted. They then performed an RNA interference screen to identify the factors involved in autophagosome positioning. They found that the dynein-dynactin complex is required for trafficking of autophagosomes toward ncMTOC. Dynein loss leads to the peripheral relocation of autophagosomes. They further revealed that a pair of small GTPases and their effectors, Rab7-Epg5 and Rab39-ema, are required for bidirectional autophagosome transport. Knockdown of these factors in Vps16a RNAi cells causes scattering of autophagosomes throughout the cytosol.
Strengths:
The data presented in this study help us to understand the mechanism underlying the trafficking and positioning of autophagosomes.
Weaknesses:
(1) The experiments were performed in Vps16A RNAi KD cells. Vps16A knockdown blocks fusion of vesicles derived from the endolysosomal compartments such as fusion between lysosomes. The pleiotropic effect of Vps16A RNAi may complicate the interpretation.
(2) In this study, the transport of autophagosomes is investigated in fly fat cells. In fat cells, a large number of large lipid droplets accumulate and the endomembrane systems are distinct from that in other cell types. The knowledge gain from this study may not apply to other cell types.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
(1) To distinguish autophagosomes from autolysosomes, the authors used vps16 RNAi cells, which are supposed to be fusion deficient. However, the extent to which fusion is actually inhibited by knockdown of Vps16A is not shown. The co-localization rate of Atg8 and Lamp1 should be shown (as in Figure 8). Then, after identifying pre-fusion autophagosomes and lysosomes, the localization of each should be analyzed.
Thank you for this insightful comment. We analyzed the colocalization of 3xmCherry-Atg8a and GFP-Lamp1, which label autophagic structures and lysosomes, respectively, in Vps16A RNAi fat body cells. As expected, Vps16A silencing markedly reduced the overlap between these two signals, indicating a strong block in autophagosome–lysosome fusion. Moreover, both 3xmCherry-Atg8a and GFP-Lamp1 became more perinuclearly localized compared to the control (luciferase RNAi) cells.
It is also possible that autophagosomes and lysosomes are tethered by factors other than HOPS (even if they are not fused). If this is the case, autophagosomal trafficking would be affected by the movement of lysosomes.
Thank you for raising this possibility. While we cannot fully exclude that autophagosomes might be indirectly transported via tethering to lysosomes, we consider this unlikely. We believe that in Drosophila fat cells, autophagosomes and lysosomes rapidly fuse once in close proximity. Therefore, even if alternative tethering mechanisms exist, they are unlikely to permit prolonged joint trafficking without fusion.
(2) The authors analyze autolysosomes in Figures 6 and 7. This is based on the assumption that autophagosome-lysosome fusion takes place in cells without vps16A RNAi. However, even in the presence of Vps16A, both pre-fusion autophagosomes and autolysosomes should exist. This is also true in Figure 8H, where the fusion of autophagosomes and lysosomes is partially suppressed in knockdown cells of dynein, dynactin, Rab7, and Epg5. If the effect of fusion is to be examined, it is reasonable to distinguish between autophagosomes and autolysosomes and analyze only autolysosomes.
Thank you for this careful observation. The 3xmCherry-Atg8a reporter is well suited to identify both autophagosomes and autolysosomes, as the mCherry fluorophore is resistant to degradation in the acidic environment of autolysosomes. Notably, mCherry-Atg8a–positive autolysosomes appear larger and brighter than pre-fusion autophagosomes, which are typically smaller and dimmer, especially under fusion-deficient conditions (e.g., Figure 4). Therefore, we use these morphological differences as a proxy to distinguish between the two.
To improve structural assignment, we incorporated endogenous Lamp1 staining (Figure 10) and a Lamp1-GFP reporter (Figure 10—figure supplement 1). Vesicles positive for mCherryAtg8a but negative for Lamp1 are considered pre-fusion autophagosomes. Structures double-positive for mCherry-Atg8a and Lamp1 represent autolysosomes, while Lamp1positive, Atg8a-negative vesicles correspond to non-autophagic lysosomes. To clarify these interpretations, we revised the Results section and explained these reporters in more detail.
(3) In this study, only vps16a RNAi cells were used to inhibit autophagosome-lysosome fusion. However, since HOPS has many roles besides autophagosome-lysosome fusion, it would be better to confirm the conclusion by knockdown of other factors (e.g., Stx17 RNAi).
Thank you for this valuable suggestion. We initially considered using Syntaxin17 RNAi; however, our recent findings indicate that loss of Syx17 results in a HOPS-dependent tethering lock between autophagosomes and lysosomes (DOI: 10.1126/sciadv.adu9605). In this case, tethered vesicles would likely move together, confounding the interpretation of autophagosome-specific trafficking.
Therefore, we turned to other SNAREs such as Vamp7 and Snap29. One Snap29 RNAi was located on the appropriate chromosome needed for our genetic experiments. We generated a transgenic fly line expressing both Snap29 RNAi and the mCherry-Atg8a reporter under a fat body-specific R4 promoter. When we tested our key trafficking hits in this background, we observed similar autophagosome localization phenotypes as in Vps16A RNAi cells. These results, now included in the revised manuscript (see Figure 6), confirm that the observed transport phenotypes are not specific to Vps16A or HOPS complex loss.
(4) Figure 8: Rab7 and Epg5 are also known to be directly involved in autophagosomelysosome tethering/fusion. Even if the fusion rate is reduced in the absence of Rab7 and Epg5, it may not be the result of defective autophagosome movement, but may simply indicate that these molecules are required for fusion itself. How do the authors distinguish between the two possibilities?
Thank you for this important point. While Rab7 and Epg5 indeed participate in autophagosome–lysosome tethering and fusion, our data suggest they also contribute to autophagosome movement. This is evident from the distinct phenotypes observed upon Rab7 or Epg5 RNAi compared to Vps16A or SNARE RNAi. Depletion of Vps16A, Syx17, Vamp7, or Snap29 (factors involved specifically in fusion) results in perinuclear accumulation of autophagosomes. In contrast, Rab7 or Epg5 RNAi leads to a dispersed autophagosome pattern throughout the cytoplasm.
These differences suggest that Rab7 and Epg5 play additional roles in positioning autophagosomes. Supporting this, our co-immunoprecipitation experiments show that Epg5 interacts with dynein motors. Therefore, we propose that Rab7 and Epg5 influence both autophagosome fusion and their microtubule-based transport.
Reviewer #2 (Public review):
One limitation of the study is the genetic background that serves as the basis for the screening. In addition to preventing autophagosome-lysosome fusion, disruption of Vps16A has been shown to inhibit endosomal maturation and block the trafficking of components to the lysosome from both the endosome and Golgi apparatus. Additional effects previously reported by the authors include increased autophagosome production and reduced mTOR signaling. Thus Vps16A-depleted cells have a number of endosome, lysosome, and autophagosome-related defects, with unknown downstream consequences. Additionally, the cause and significance of the perinuclear localization of autophagosomes in this background is unclear. Thus, interpretations of the observed reversal of this phenotype are difficult, and have the caveat that they may apply only to this condition, rather than to normal autophagosomes. Additional experiments to observe autophagosome movement or positioning in a more normal environment would improve the manuscript.
Thank you for highlighting this limitation. We have tried to conduct time-lapse imaging of live fat body cells expressing 3xmCherry-Atg8a and GFP-Lamp1 to visualize the movement and fusion events of pre-fusion autophagosomes (3xmCherry-Atg8a positive and GFP-Lamp1 negative) and lysosomes (GFP-Lamp1 positive). Despite different experimental setups and durations of starvation, no vesicle movement was observed at all, so live imaging of larval Drosophila fat tissue will require time-consuming optimizations of in vitro culture conditions. Consistent with this, we did not find any literature data where organelle motility in fat body cells was successfully observed. Nuclear positioning in fat body cells was investigated in detail in an excellent study, however the authors were able to observe only very little movement of the nuclei by live imaging (Zheng et al. Nat Cell Biol. 2020 Mar;22(3):297-309. doi: 10.1038/s41556-020-0470-7), further highlighting the technical difficulties of live or timelapse imaging in this tissue type.
Specific comments
(1) Several genes have been described that when depleted lead to perinuclear accumulation of Atg8-labeled vesicles. There seems to be a correlation of this phenotype with genes required for autophagosome-lysosome fusion; however, some genes required for lysosomal fusion such as Rab2 and Arl8 apparently did not affect autophagosome positioning as reported here. Thus, it is unclear whether the perinuclear positioning of autophagosomes is truly a general response to disruption of autophagosome-lysosome fusion, or may reflect additional aspects of Vps16A/HOPS function. A few things here would help. One would be an analysis of Atg8a vesicle localization in response to the depletion of a larger set of fusionrelated genes. Another would be to repeat some of the key findings of this study (effects of specific dynein, dynactin, rabs, effectors) on Atg8a localization when Syx17 is depleted, rather than Vps16A. This should generate a more autophagosome-specific fusion defect.
Thank you for this insightful suggestion. We recently discovered that Syx17 depletion induces a HOPS-dependent tethering lock between autophagosomes and lysosomes (DOI: 10.1126/sciadv.adu9605), making it unsuitable for modeling autophagosome-specific fusion defects. In contrast, Vamp7 and Snap29 knockdowns do not appear to cause such tethering lock. We were able to generate a suitable Drosophila line using a Snap29 RNAi transgene located on a compatible chromosome. Upon testing key hits from our screen in this background, we found that autophagosomes redistributed similarly, supporting our conclusions. These new results have been included in the revised manuscript (see Figure 6)
Third, it would greatly strengthen the findings to monitor pre-fusion autophagosome localization without disrupting fusion. Such vesicles could be identified as Atg8a-positive Lamp-negative structures. The effects of dynein and rab depletion on the tracking of these structures in a post-induction time course would serve as an important validation of the authors' findings.
Thank you for this helpful suggestion. As described above, we attempted time-lapse imaging of 3xmCherry-Atg8a and GFP-Lamp1-expressing fat body cells under various conditions to identify motile pre-fusion autophagosomes. However, we did not observe any vesicle movement, regardless of the starvation duration or experimental setup. As this likely reflects technical limitations of ex vivo fat body imaging, we were unable to achieve live tracking of autophagosome dynamics without introducing perturbations. This limitation is now discussed in the revised manuscript.
(2) The authors nicely show that depletion of Shot leads to relocalization of Atg8a to ectopic foci in Vps16A-depleted cells; they should confirm that this is a mislocalized ncMTOC by colabeling Atg8a with an MTOC component such as MSP300. The effect of Shot depletion on Atg8a localization should also be analyzed in the absence of Vps16A depletion.
Thank you for this positive comment. We co-labeled Atg8a with the minus-end microtubule marker Khc-nod-LacZ in both shot single knockdown and shot; vps16A double knockdown cells. Ectopic Khc-nod-LacZ-positive MTOC foci were clearly visible in both conditions, and Atg8a-positive autophagosomes accumulated around these structures. These findings confirm that Shot depletion induces ectopic MTOC formation, which correlates with autophagosome relocalization. The new data have been incorporated into the revised manuscript (see Figure 1O-S).
(3) The authors report that depletion of Dynein subunits, either alone (Figure 6) or codepleted with Vps16A (Figure 2), leads to redistribution of mCherry-Atg8a punctae to the "cell periphery". However, only cell clones that contact an edge of the fat body tissue are shown in these figures. Furthermore, in these cells, mCherry-Atg8a punctae appear to localize only to contact-free regions of these cells, and not to internal regions of clones that share a border with adjacent cells. Thus, these vesicles would seem to be redistributed to the periphery of the fat body itself, not to the periphery of individual cells. Microtubules emanating from the perinuclear ncMTOC have been described as having a radial organization, and thus it is unclear that this redistribution of mCherry-Atg8a punctae to the fat body edge would reflect a kinesin-dependent process as suggested by the authors.
Thank you for this detailed observation. We frequently observe autophagosomes accumulating in contact-free peripheral regions of dynein-depleted cells, resulting in an asymmetric distribution. While previous studies describe a radial microtubule organization in fat body cells, none of them directly label MT plus ends, the direction of kinesin-based transport.
To further explore this, we overexpressed a HA-tagged kinesin, Klp98A-3xHA, in both control and Vps16A RNAi backgrounds. Immunolabeling revealed that Klp98A localizes to the contact-free peripheral regions in both conditions, consistent with the distribution of autophagosomes in dynein knockdown cells. This supports our interpretation that kinesindependent transport drives autophagosome redistribution in the absence of dynein, and that fat body cells exhibit subtle asymmetries in MT polarity that influence this transport. These new results have been included in the revised manuscript (see Figure 3G, H).
(4) To validate whether the mCherry-Atg8a structures in Vps16A-depleted cells were of autophagic origin, the authors depleted Atg8a and observed a loss of mCherry- Atg8a signal from the mosaic cells (Figure S1D, J). A more rigorous experiment would be to deplete other Atg genes (not Atg8a) and examine whether these structures persist.
Thank you for the suggestion to further validate our reporter. We depleted Atg1, a key kinase required for phagophore initiation, in the Vps16A RNAi background. This completely abolished the punctate mCherry-Atg8a distribution in knockdown cells (see Figure 1—figure supplement 1E, K), confirming that the labeled structures are indeed of autophagic origin.
(5) The authors found that only a subset of dynein, dynactin, rab, and rab effector depletions affected mCherry-Atg8a localization, leading to their suggestion that the most important factors involved in autophagosome motility have been identified here. However, this conclusion has the caveat that depletion efficiency was not examined in this study, and thus any conclusions about negative results should be more conservative.
Thank you for this constructive feedback. We agree that negative results must be interpreted conservatively due to potential differences in knockdown efficiency. We have revised our conclusions accordingly, clarifying that the factors identified are key for autophagosome motility, while acknowledging the possibility of false negatives.
Reviewer #3 (Public review):
Major concerns:
(1) The localization of EPG5 should be determined. The authors showed that EPG5 colocalizes with endogenous Rab7. Rab7 labels late endosomes and lysosomes. Previous studies in mammalian cells have shown that EPG5 is targeted to late endosomes/lysosomes by interacting with Rab7. EPG5 promotes the fusion of autophagosomes with late endosomes/lysosomes by directly recognizing LC3 on autophagosomes and also by facilitating the assembly of the SNARE complex for fusion. In Figure 5I, the EPG5/Rab7colocalized vesicles are large and they are likely to be lysosomes/autolysosomes.
Thank you for suggesting to improve our Epg5 localization data. We performed triple immunostaining for Atg8a, Lamp1-3xmCherry, and Epg5-9xHA in S2R+ cells. In addition to triple-positive structures—likely representing autolysosomes—we observed Atg8a and Epg59xHA double-positive vesicles that lacked Lamp1-3xmCherry signal, which likely correspond to pre-fusion autophagosomes. Based on these results, we propose that in addition to arriving via the endocytic route, Epg5 may also reach lysosomes through autophagosomes. These findings have been included in the revised manuscript (see Figure 5K).
(2) The experiments were performed in Vps16A RNAi KD cells. Vps16A knockdown blocks fusion of vesicles derived from the endolysosomal compartments such as fusion between lysosomes. The pleiotropic effect of Vps16A RNAi may complicate the interpretation. The authors need to verify their findings in Stx17 KO cells, as it has a relatively specific effect on the fusion of autophagosomes with late endosomes/lysosomes.
Thank you for this valuable suggestion. We initially considered Syntaxin17 for validation; however, we recently found that loss of Syx17 leads to a HOPS-dependent tethering lock between autophagosomes and lysosomes, which would confound interpretation, as autophagosomes remain tethered to lysosomes (DOI: 10.1126/sciadv.adu9605). Therefore, Syntaxin17 loss is not suitable for our purpose. Among the remaining fusion SNAREs, one RNAi line targeting Snap29 was available on a compatible chromosome for generating Drosophila lines equivalent to those used in the screen. We established this Snap29 RNAicontaining tester line and crossed it with our top hits. We observed that autophagosome motility was comparable to that in the Vps16A RNAi background, further supporting our conclusions. These results have been incorporated into the revised manuscript (see Figure 6)
(3) Quantification should be performed in many places such as in Figure S4D for the number of FYVE-GFP labeled endosomes and in Figures S4H and S4I for the number and size of lysosomes.
Thank you for pointing this out. We performed the suggested quantifications and statistical analyses for FYVE-GFP labeled endosomes, as well as for the number and size of lysosomes. The updated data are now presented in the revised Figure 5—figure supplement 1.
(4) In this study, the transport of autophagosomes is investigated in fly fat cells. In fat cells, a large number of large lipid droplets accumulate and the endomembrane systems are distinct from that in other cell types. The knowledge gained from this study may not apply to other cell types. This needs to be discussed.
Thank you for raising this important point. We agree that our findings may not be fully generalizable to all cell types. Given that the organization of the microtubule network depends on both cell function and developmental stage, it is plausible that the molecular machinery described here operates differently elsewhere. We now mention this limitation in the Discussion.
Minor concerns:
(5) Data in some panels are of low quality. For example, the mCherry-Atg8a signal in Figure 5C is hard to see; the input bands of Dhc64c in Figure 5L are smeared.
Thank you for pointing this out. We repeated the experiment shown in Figure 5C and replaced the panel with a clearer image. The smeared Dhc64C input bands in Figure 5L result from the unusually large size of this protein, which affects its electrophoretic migration. We mentioned this point in the corresponding figure legend.
(6) In this study, both 3xmCherry-Atg8a and mCherry-Atg8a were used. Different reporters make it difficult to compare the results presented in different figures.
Thank you for this comment. Both 3xmCherry-Atg8a and mCherry-Atg8a are well-established reporters that behave similarly as autophagic markers. Nevertheless, to avoid confusion, we ensured that each figure uses only one type of reporter consistently, which is now clearly indicated in the revised manuscript.
(7) The small autophagosomes presented in Figures such as in Figure 1D and 1E are not clear. Enlarged images should be presented.
Thank you for your suggestion. We repeated these experiments and replaced the relevant panels with higher-quality images, including enlarged insets to better visualize small autophagosomes. These updated figures are now included in the revised manuscript.
(8) The authors showed that Epg5-9xHA coprecipitates with the endogenous dynein motor Dhc64C. Is Rab7 required for the interaction?
Thank you for this insightful question. We tested this by co-transfecting S2R+ cells with Epg5-9xHA and different forms of Rab7: wild-type, GTP-locked (constitutively active), and GDP-locked (dominant-negative). Our results indicate that the strength of Epg5-Dhc interaction does not change in the presence of either GTP-locked or GDP-locked Rab7. However, we believe that Epg5 and dynein are recruited to the vesicle membranes via Rab7 in vivo, so we did not include these results in the revised manuscript.
(9) The perinuclear lysosome localization in Epg5 KD cells has no indication that Epg5 is an autophagosome-specific adaptor.
Thank you for this important comment. Accordingly, we have toned down our statements about Epg5 functions throughout the revised manuscript.
Reviewer #1 (Recommendations for the authors):
(1) Figure 6: What do "autolysosome maturation" and "small autolysosomes" mean? Do different numbers of lysosomes fuse to a single autophagosome?
Thank you for highlighting this point. We concluded that the formation of smaller autolysosomes—compared to controls—is likely due to a defect in autolysosome maturation, as is often the case. We had not explicitly considered whether a different number of lysosomes fuse with each autophagosome during this process. We clarified this issue in the revised manuscript.
(2) Figure 5A shows that the localization of endogenous Atg8 requires Epg5, but the data is not as clear as for mCherry-Atg8 (Figure 4B). Why the difference?
Thank you for this question. The difference arises because the mCherry-Atg8a reporter strongly labels autolysosomes, as the mCherry fluorophore remains stable in acidic compartments. As a result, mCherry-Atg8a labels both autophagosomes and autolysosomes, but the strong autolysosomal signal originating from the surrounding GFP negative, nonRNAi cells can make accumulated autophagosomes appear fainter in fusion-defective cells (as in Figure 4). In contrast, endogenous Atg8a is degraded in lysosomes, and therefore labels only autophagosomes. This means that the appearance of these two experiments can be slightly different, but since in both cases autophagosomes no longer accumulate in the perinuclear region of Vps16A,Epg5 double RNAi cells we can conclude that Epg5 is required for autophagosome positioning. We explained this difference of the two methods in the revised manuscript where it first appears (Figure 1B and Figure 1—figure supplement 1A).
(3) Blue letters on the black micrographs are hard to see. Some of the other letters are also small and hard to read.
Thank you for this suggestion. We improved the visibility and readability of the labels in the revised figures.
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Reviewer #3 (Public review):
Summary:
In their revised manuscript, the authors addressed most of the reviewers' concerns. One concern was the emphasis on increased MLEC-OST interactions during infection, which the authors toned down in the revision. They clarified that MLEC interaction with OST is maintained-rather than increased-during infection, while its interaction with other QC factors decreases. They also added context and discussion of the co-localization of viral proteins with ER and mitochondrial proteins, noting that both nsp2 and MLEC localize to mitochondria-associated membranes (MAMs), providing a plausible explanation for these interactions.
Another concern involved the effects of MLEC KD on the cellular environment. To address this, the authors analyzed stress pathway activation and glycosylation of endogenous proteins in MLEC KD cells. They found only modest upregulation of the HSF1 pathway and no changes in the UPR or other stress responses, suggesting MLEC KD does not broadly disrupt ER proteostasis. Additionally, glycopeptide profiling showed only minor changes in host protein glycosylation, supporting a more direct role for MLEC in viral replication rather than general host glycoprotein disruption.
However, some weaknesses remain. Direct interaction between MLEC and nsp2 during infection was not detected, and the identified viral glycopeptides were limited to only five Spike sites. Furthermore, the mechanism by which MLEC promotes viral replication is still unclear.
In summary, the authors strengthened the manuscript by addressing reviewers' concerns through additional data, clarified language, and expanded discussion. While the overall support for MLEC's pro-viral role is solid, its precise mechanism of action remains speculative. Future work will be needed to directly link MLEC's activity to specific steps in viral protein biogenesis and replication.
Original summary: In this study, Davies and Plate set out to discover conserved host interactors of coronavirus non-structural proteins (Nsp). They used 293T cells to ectopically express flag-tagged Nsp2 and Nsp4 from five human and mouse coronaviruses, including SARS-CoV-1 and 2, and analyzed their interaction with host proteins by affinity purification mass-spectrometry (AP-MS). To confirm whether such interactors play a role in coronavirus infection, the authors measured the effects of individual knockdowns on replication of murine hepatitis virus (MHV) in mouse Delayed Brain Tumor cells. Using this approach, they identified a previously undescribed interactor of Nsp2, Malectin (Mlec), which is involved in glycoprotein processing and shows a potent pro-viral function in both MHV and SARS-CoV-2. Although the authors were unable to confirm this interaction in MHV-infected cells, they show that infection remodels many other Mlec interactions, recruiting it to the ER complex that catalyzes protein glycosylation (OST). Mlec knockdown reduced viral RNA and protein levels during MHV infection, although such effects were not limited to specific viral proteins. However, knockdown reduced the levels of five viral glycopeptides that map to Spike protein, suggesting it may be affected by Mlec.
Strengths:
This is an elegant study that uses a state-of-the-art quantitative proteomic approach to identify host proteins that play critical roles in viral infection. Instead of focusing on a single protein from a single virus, it compares the interactomes of two viral proteins from five related viruses, generating a high confidence dataset. The functional follow-ups using multiple live and reporter viruses, including MHV and CoV2 variants, convincingly depict a pro-viral role for Mlec, a protein not previously implicated in coronavirus biology.
Weaknesses:
Although a commonly used approach, AP-MS of ectopically expressed viral proteins may not accurately capture infection-related interactions. The authors observed Mlec-Nsp2 interactions in transfected 293T cells (1C) but were unable to reproduce those in mouse cells infected with MHV (3C). EIF4E2/GIGYF2, two bonafide interactors of CoV2 Nsp2 from previous studies, are listed as depleted compared to negative controls (S1D). Most other CoV2 Nsp2 interactors are also depleted by the same analysis (S1D). Previously reported MERS Nsp2 interactors, including ASCC1 and TCF25, are also not detected (S1D). Furthermore, although GIGYF2 was not identified as an interactor of MHV Nsp2/4 in human cells (S1D), its knockdown in mouse cells reduced MHV titers about 1000 fold (S4). The authors should attempt to explain these discrepancies.
More importantly, the authors were unable to establish a direct link between Mlec and the biogenesis of any viral or host proteins, by mass-spectrometry or otherwise. Although it is clear that Mlec promotes coronavirus infection, the mechanism remains unclear. Its knockdown does not affect the proteome composition of uninfected cells (S15B), suggesting it is not required for proteome maintenance under normal conditions. The only viral glycopeptides detected during MHV infection originated from Spike (5D), although other viral proteins are also known to be glycosylated. Cells depleted for Mlec produce ~4-fold less Spike protein (4E) but no more than 2-fold less glycosylated spike peptides (5D), compounding the interpretation of Mlec effects on viral protein biogenesis. Furthermore, Spike is not essential for the pro-viral role of Mlec, given that Mlec knockdown reduces replication of SARS-CoV-2 replicons that express all viral proteins except for Spike (6A/B).
Any of the observed effects on viral protein levels could be secondary to multiple other processes. Interventions that delay infection for any reason could lead to imbalance of viral protein levels, because Spike and other structural proteins are produced at a much higher rate than non-structural proteins due to the higher abundance of their cognate subgenomic RNAs. Similarly, the observation that Mlec depletion attenuates MHV-mediated changes to the host proteome (S15C/D) can also be attributed to indirect effects on viral replication, regardless of glycoprotein processing. In the discussion, the authors acknowledge that Mlec may indirectly affect infection through modulation of replication complex formation or ER stress, but do not offer any supporting evidence. Interestingly, plant homologs of Mlec are implicated in innate immunity, favoring a more global role for Mlec in mammalian coronavirus infections.
Finally, the observation that both Nsp2 (3C) and Mlec (3E/F) are recruited to the OST complex during MHV infection neither support nor refute any of these alternate hypotheses, given that Mlec is known to interact with OST in uninfected cells and that Nsp2 may interact with OST as part of the full length unprocessed Orf1a, as it co-translationally translocates into the ER.
Therefore, the main claims about the role of Mlec in coronavirus protein biogenesis are only partially supported.
Comments on revisions:
Figure 7B should be revised to show that MLEC maintains interactions with rather than recruited to the OST.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
In this manuscript, the authors employ a combined proteomic and genetic approach to identify the glycoprotein QC factor malectin as an important protein involved in promoting coronavirus infection. Using proteomic approaches, they show that the non-structural protein NSP2 and malectin interact in the absence of viral infection, but not in the presence of viral infection. However, both NSP2 and malectin engage the OST complex during viral infection, with malectin also showing reduced interactions with other glycoprotein QC proteins. Malectin KD reduce replication of coronaviruses, including SARS-COV2. Collectively, these results identify Malectin as a glycoprotein QC protein involved in regulating coronavirus replication that could potentially be targeted to mitigate coronavirus replication.
Overall, the experiments described appear well performed and the interpretations generally reflect the results. Moreover, this work identifies Malectin as an important pro-viral protein whose activity could potentially be therapeutically targeted for the broad treatment of coronavirus infection. However, there are some weaknesses in the work that, if addressed, would improve the impact of the manuscript.
Notably, the mechanism by which malectin regulates viral replication is not well described. It is clear from the work that malectin is a pro-viral protein in the work presented, but the mechanistic basis of this activity is not pursued. Some potential mechanisms are proposed in the discussion, but the manuscript would be strengthened if additional insight was included. For example, does the UPR activated to higher levels in infected cells depleted of malectin? Do glycosylation patterns of viral (or non-viral) proteins change in malectindepleted cells? Additional insight into this specific question would significantly improve the manuscript.
We concur with the reviewer that the mechanism by which Malectin regulates viral replication is an important point to elucidate further. Our proteomics data were able to offer additional insight into the questions posed here. We examined the upregulation of protein markers of the UPR and other stress response pathways in cells depleted of MLEC (Fig. S15D). We find that the UPR pathways are moderately but insignificantly upregulated, while the Heat Shock Factor 1 (HSF1) pathway is moderately and significantly upregulated. The fold change increase of these marker proteins are relatively small, so while upregulation of this pathway may contribute to the suppression of CoV replication, it may not fully explain the phenotype.
In addition, to address the second question, we compared the glycosylation patterns of endogenous proteins in MLEC-KD cells (Fig. S15E-G). We found that there is a small increase in abundance of glycopeptides associated with LAMP2, SERPHINH1, RDX, RPL3/5, CADM4, and ITGB1, however these fold changes are small and tested to be insignificant. These results indicate there is relatively little modification of endogenous glycoproteins upon MLEC-depletion. These findings support a more direct role for MLEC in regulating viral replication.
We added the following section to the manuscript text to discuss these results:
“In uninfected cells, MLEC KD leads to relatively little proteome-wide changes, with MLEC being the only protein significantly downregulated and no other proteins significantly upregulated, supporting the specificity of MLEC KD in MHV suppression (Fig. S15C). To determine whether MLEC KD alters general host proteostasis, we further examined the levels of protein markers of stress pathways based on previous gene pathway definitions(Davies et al., 2023; Grandjean et al., 2019; Shoulders et al., 2013) (Fig. S15D). We find that there are modest but significant increases in protein levels associated with the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response (UPR) pathways are largely unmodified.
We also probed the effect of MLEC KD on endogenous protein glycosylation. We find that there is only a small increase in abundance of glycopeptides, including those associated with the ribosome (Rpl3, Rpl5), a cytoskeletal protein (Rdx), the integrin Itgb1, and the ER-resident chaperone Serphinh1 (Fig. S15E-G).”
“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). In addition, there are only minor increases in endogenous glycopeptide levels (Fig. S15E-G). Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”
Further, the evidence for increased interactions between OST and malectin during viral infection is fairly weak, despite being a major talking point throughout the manuscript. The reduced interactions between malectin and other glycoproteostasis QC factors is evident, but the increased interactions with OST are not well supported. I'd recommend backing off on this point throughout the text, instead, continuing to highlight the reduced interactions.
We agree that the fold change increase of OST interactions with malectin are small compared to the fold change decrease of other glycoproteostasis factors We have modified the text to less emphasize this point and instead highlight the reduced interactions:
“Further, MHV infection retains the association of MLEC with the OST complex while titrating off other interactors, potentially leading to more efficient glycoprotein biogenesis.”
I was also curious as to why non-structural proteins, nsp2 and nsp4, showed robust interactions with host proteins localized to both the ER and mitochondria? Do these proteins localize to different organelles or do these interactions reflect some other type of dysregulation? It would be useful to provide a bit of speculation on this point.
We also find these ER and mitochondrial protein interactions curious, which we initially reported on (Davies, Almasy et al. 2020 ACS Infectious Diseases). In this prior report, we found that when expressed in HEK293T cells, SARS-CoV-2 nsp2 and nsp4 have partial localization to mitochondrial-associated ER membranes (MAMs), as determined by subcellular fractionation. Given that malectin has also been shown to have MAMs localization (Carreras-Sureda, et al. 2019 Nature Cell Biology), we have added additional text in the Discussion to speculate on this point:
“Additionally, MLEC has also been shown to localize to ER-mitochondria contact sites (MAMs)(Carreras-Sureda et al., 2019), which regulate mitochondrial bioenergetics. We have previously shown that SARS-CoV-2 nsp2 and nsp4 can partially localize to MAMs(Davies et al., 2020), so these viral proteins may also dysregulate MLEC and MAMs activity to promote infection.”
Again, the overall identification of malectin as a pro-viral protein involved in the replication of multiple different coronaviruses is interesting and important, but additional insights into the mechanism of this activity would strengthen the overall impact of this work.
Thank you for this endorsement. We hope the additional analyses and discussion points in the revised manuscript further homed in on a direct mechanistic function for MLEC in modulating viral replication.
Reviewer #2 (Public Review):
Summary:
A strong case is presented to establish that the endoplasmic reticulum carbohydrate binding protein malectin is an important factor for coronavirus propagation. Malectin was identified as a coronavirus nsp2 protein interactor using quantitative proteomics and its importance in the viral life cycle was supported by using a functional genetic screen and viral assays. Malectin binds diglucosylated proteins, an early glycoform thought to transiently exist on nascent chains shortly after translation and translocation; yet a role for malectin has previously been proposed in later quality control decisions and degradation targeting. These two observations have been difficult to reconcile temporally. In agreement with results from the Locher lab, the malectininteractome shown here includes a number of subunits of the oligosaccharyltransferase complex (OST). These results place malectin in close proximity to both the co-translational (STT3A or OST-A) and post-translational (STT3B or OST-B) complexes. It follows that malectin knockdown was associated with coronavirus Spike protein hypoglycosylation.
Strengths:
Strengths include using multiple viruses to identify interactors of nsp2 and quantitative proteomics along with multiple viral assays to monitor the viral life cycle.
Weaknesses:
Malectin knockdown was shown to be associated with Spike protein hypoglycosylation. This was further supported by malectin interactions with the OSTs. However, no specific role of malectin in glycosylation was discussed or proposed.
We have emphasized our hypotheses on this point in the discussion and added a summary figure to highlight the specific role of malectin.
Given the likelihood that malectin plays a role in the glycosylation of heavily glycosylated proteins like Spike, it is unfortunate that only 5 glycosites on Spike were identified using the MS deamidation assay when Spike has a large number of glycans (~22 sites). The mass spec data set would also include endogenous proteins. Were any heavily glycosylated endogenous proteins hypoglycosylated in the MS analysis in Fig 5D?
Thank you for this suggestion. We compared the glycosylation patterns of endogenous proteins in MLEC-KD cells (Fig. S15E-G). We found that there is a small increase in abundance of glycopeptides associated with LAMP2, SERPHINH1, RDX, RPL3/5, CADM4, and ITGB1, however these fold changes are small and tested insignificant. These results indicate there is relatively little modification of endogenous glycoproteins upon MLEC-depletion. We added the following sections:
“We also probed the effect of MLEC KD on endogenous protein glycosylation. We find that there is only a small increase in abundance of glycopeptides, including those associated with the ribosome (Rpl3, Rpl5), a cytoskeletal protein (Rdx), the integrin Itgb1, and the ER-resident chaperone Serphinh1 (Fig. S15E-G).”
“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). In addition, there are only minor increases in endogenous glycopeptide levels (Fig. S15E-G). Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”
The inclusion of the nsp4 interactome and its partial characterization is a distraction from the storyline that focuses on malectin and nsp2.
We believe the nsp4 comparative interactome and functional genomics data offers a rich resource for further functional investigation by others, if made public. While we found the malectin and nsp2 storyline the most compelling to pursue, we believe the inclusion of the nsp4 data strengthens the overall approach, in agreement with Reviewer #3’s comments.
Reviewer #3 (Public Review):
Summary:
In this study, Davies and Plate set out to discover conserved host interactors of coronavirus non-structural proteins (Nsp). They used 293T cells to ectopically express flag-tagged Nsp2 and Nsp4 from five human and mouse coronaviruses, including SARS-CoV-1 and 2, and analyzed their interaction with host proteins by affinity purification mass-spectrometry (AP-MS). To confirm whether such interactors play a role in coronavirus infection, the authors measured the effects of individual knockdowns on replication of murine hepatitis virus (MHV) in mouse Delayed Brain Tumor cells. Using this approach, they identified a previously undescribed interactor of Nsp2, Malectin (Mlec), which is involved in glycoprotein processing and shows a potent pro-viral function in both MHV and SARS-CoV-2. Although the authors were unable to confirm this interaction in MHVinfected cells, they show that infection remodels many other Mlec interactions, recruiting it to the ER complex that catalyzes protein glycosylation (OST). Mlec knockdown reduced viral RNA and protein levels during MHV infection, although such effects were not limited to specific viral proteins. However, knockdown reduced the levels of five viral glycopeptides that map to Spike protein, suggesting it may be affected by Mlec.
Strengths:
This is an elegant study that uses a state-of-the-art quantitative proteomic approach to identify host proteins that play critical roles in viral infection. Instead of focusing on a single protein from a single virus, it compares the interactomes of two viral proteins from five related viruses, generating a high confidence dataset. The functional follow-ups using multiple live and reporter viruses, including MHV and CoV2 variants, convincingly depict a pro-viral role for Mlec, a protein not previously implicated in coronavirus biology.
Weaknesses:
Although a commonly used approach, AP-MS of ectopically expressed viral proteins may not accurately capture infection-related interactions. The authors observed Mlec-Nsp2 interactions in transfected 293T cells (1C) but were unable to reproduce those in mouse cells infected with MHV (3C). EIF4E2/GIGYF2, two bonafide interactors of CoV2 Nsp2 from previous studies, are listed as depleted compared to negative controls (S1D). Most other CoV2 Nsp2 interactors are also depleted by the same analysis (S1D). Previously reported MERS Nsp2 interactors, including ASCC1 and TCF25, are also not detected (S1D). Furthermore, although GIGYF2 was not identified as an interactor of MHV Nsp2/4 in human cells (S1D), its knockdown in mouse cells reduced MHV titers about 1000 fold (S4). The authors should attempt to explain these discrepancies.
We acknowledge these limitations in AP-MS from ectopically expressed viral proteins and have addressed these discrepancies with further elaboration in the text:
“A limitation of our study is the initial overexpression of individual proteins for AP-MS, in which we find some variation between our data with other AP-MS studies. We sought to overcome these variations by focusing on conserved interactors and testing interactions in a live infection context.”
“We also found GIGYF2-KD strongly suppressed MHV infection, despite GIGYF2 not interacting with MHV nsp2 (Fig. S1D), highlighting the importance of proteostasis factors in infection regardless of direct PPIs.”
More importantly, the authors were unable to establish a direct link between Mlec and the biogenesis of any viral or host proteins, by mass-spectrometry or otherwise. Although it is clear that Mlec promotes coronavirus infection, the mechanism remains unclear. Its knockdown does not affect the proteome composition of uninfected cells (S15B), suggesting it is not required for proteome maintenance under normal conditions. The only viral glycopeptides detected during MHV infection originated from Spike (5D), although other viral proteins are also known to be glycosylated. Cells depleted for Mlec produce ~4-fold less Spike protein (4E) but no more than 2-fold less glycosylated spike peptides (5D), compounding the interpretation of Mlec effects on viral protein biogenesis. Furthermore, Spike is not essential for the pro-viral role of Mlec, given that Mlec knockdown reduces replication of SARS-CoV-2 replicons that express all viral proteins except for Spike (6A/B).
Thank you, these are all important points. We have acknowledged these compounding factors in the Discussion:
“Concurrently, knockdown of MLEC leads to impediment of nsp production and aberrant glycosylation of other viral proteins like Spike, though it should be noted that the decrease in Spike glycopeptides is compounded by the overall decrease in Spike protein. Given that MLEC is pro-viral in a SARS-CoV-2 replicon model lacking Spike (Fig. 6), MLEC can promote CoV replication independent of Spike production.”
Any of the observed effects on viral protein levels could be secondary to multiple other processes.Interventions that delay infection for any reason could lead to an imbalance of viral protein levels because Spike and other structural proteins are produced at a much higher rate than non-structural proteins due to the higher abundance of their cognate subgenomic RNAs. Similarly, the observation that Mlec depletion attenuates MHV-mediated changes to the host proteome (S15C/D) can also be attributed to indirect effects on viral replication, regardless of glycoprotein processing. In the discussion, the authors acknowledge that Mlec may indirectly affect infection through modulation of replication complex formation or ER stress, but do not offer any supporting evidence. Interestingly, plant homologs of Mlec are implicated in innate immunity, favoring a more global role for Mlec in mammalian coronavirus infections.
We examined the upregulation of protein markers of the UPR and other stress response pathways in cells depleted of MLEC (Fig. S15D). We find that the UPR pathways are moderately but insignificantly upregulated, while the Heat Shock Factor 1 (HSF1) pathway is moderately and significantly upregulated. The fold change increase of these marker proteins are relatively small, so while upregulation of this pathway may contribute to the suppression of CoV replication, it may not fully explain the phenotype. Please all see similar points brought up by reviewer 1 (comment 1). We added the following section to the manuscript text to discuss these results:
“In uninfected cells, MLEC KD leads to relatively little proteome-wide changes, with MLEC being the only protein significantly downregulated and no other proteins significantly upregulated, supporting the specificity of MLEC KD in MHV suppression (Fig. S15C). To determine whether MLEC KD alters general host proteostasis, we further examined the levels of protein markers of stress pathways based on previous gene pathway definitions(Davies et al., 2023; Grandjean et al., 2019; Shoulders et al., 2013) (Fig. S15D). We find that there are modest but significant increases in protein levels associated with the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response (UPR) pathways are largely unmodified.
“Our proteomics data reveals that there is only a modest increase in the Heat Shock Factor 1 (HSF1) pathway, while the Unfolded Protein Response is relatively unchanged (Fig. S15D). […] Together, these results indicate that while MLEC KD leads to some alterations in ER proteostasis and host glycosylation, these changes are modest and may not be the primary mechanism by which MLEC KD hinders CoV replication.”
Finally, the observation that both Nsp2 (3C) and Mlec (3E/F) are recruited to the OST complex during MHV infection neither support nor refute any of these alternate hypotheses, given that Mlec is known to interact with OST in uninfected cells and that Nsp2 may interact with OST as part of the full length unprocessed Orf1a, as it co-translationally translocates into the ER. Therefore, the main claims about the role of Mlec in coronavirus protein biogenesis are only partially supported.
We have acknowledged this point in the Discussion.
“We find that nsp2 interacts with several OST complex members, including DDOST, STT3A, and RPN1, though whether this is as part of the uncleaved Orf1a polyprotein during co-translational ER translocation or as an individual protein is unclear.”
Reviewer #2 (Recommendations For The Authors):
What is the proof that MLEC is a type I membrane protein? If it is strictly sequence analysis, this conclusion should be tapered in the text.
Our response: We have added appropriate evidence on the biochemical characterization of MLEC topology from Galli et al., 2011, and cryo-EM structural characterization by Ramírez et al., 2019.
“As it was surprising that nsp2, a non-glycosylated, cytoplasmic protein, would interact with MLEC, an integral ER membrane protein with a short two amino acid cytoplasmic tail(Galli et al., 2011; Ramírez et al., 2019), we assessed a broader genetic interaction between nsp2 and MLEC.”
Validation of some of the nsp2 and malectin interactome components by pulldowns should be included.
Our response: The interactions between nsp2 and Ddost, Stt3A, and Rpn1 passed a stringent confidence filter in our AP-MS experiment (Fig. 3C) based on several replication. For this reason, we do not believe additional validation by Western blotting will offer much useful information.
NGI-1 inhibition of glycosylation looks to be very weak in Fig. 5B and Fig. S14B.
Our response: It is important to note that the NGI-1 inhibition assay used a suboptimal NGI-1 concentration to prevent full suppression of MHV infection, which we have found previously. We have added this justification in the Methods section and associated figure legend (Fig. S14A).
“The 5 uM NGI-1 dosage was chosen as it resulted in partial inhibition of glycosylation while not completely blocking MHV infection.”
“This dosage and timing were chosen to partially inhibit the OST complex without fully ablating viral infection, as NGI-1 has been shown previously to be a potent positive-sense RNA virus inhibitor(Puschnik et al., 2017) (Fig. S14)”
Summary model figure at the end would help to communicate the conclusions.
Our response: Thank you for this suggestion. We agree and have added a summary model figure at the end as suggested.
Reviewer #3 (Public review):
Significance:
About 5% of metastatic castration-resistant prostate cancers (mCRPC) display genomic alterations in the transcriptional kinase CDK12. The mechanisms by which CDK12 alterations drive tumorigenesis in this molecularly-defined subset of mCRPC have remained elusive. In particular, some studies have suggested that CDK12 loss confers a homologous recombination deficiency (HRd) phenotype, However, clinical studies have not borne out the benefit to PARP inhibitors in patients with CDK12 alterations, despite the fact that these agents are typically active against tumors with HRd.
In this study, Frank et al. reconcile these findings by showing that: (1) tumors with biallelic CDK12 alterations do not have genomic features of HRd; (2) in vitro, HR gene downregulation occurs with acute depletion of CDK12 but is far less pronounced with chronic CDK12 loss; (3) CDK12-altered cells are uniquely sensitive to genetic or pharmacologic inhibition of CDK13.
Strengths:
Overall, this is an important study that reconciles disparate experimental and clinical observations. The genomic analyses are comprehensive and conducted with a high degree of rigor and represent an important resource to the community regarding the features of this molecular subtype of mCRPC.
Weaknesses:
(1) It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.
(2) It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
Given that there are different mutations identified at different CDK12 sites as illustrated in Figure 1B it would be nice to know which ones have been functionally classified as pathogenic and for which ones that the pathogenicity has not been determined. This would be especially interesting to perform in light of the differences in the LOH scores and WES data presented - specifically, are the pathogenic mutations vs the mutations for which true pathogenicity is unknown more likely to display LOH or TD?
Alterations were classified as pathogenic when resulting in frameshift, nonsense, or cause an aminoacid change likely to alter function (according to ANNOVAR). Four patients were called CDK12<sup>BAL</sup> but were negative for TDP signatures. Three of these had CDK12 mutations downstream of the kinase domain, which may be less likely to ablate protein activity. Most functionally validated pathogenic mutations include disruption of the kinase domain (PMID: 25712099). We added a sentence to the Results section (under “Identification of genomic characteristics that associate with CDK12 loss in prostate cancer”) to highlight this caveat on pathogenic mutation calls.
For the cell inhibition studies with the CDK12/13 inhibitor, more details characterizing the specificity of this molecule to these targets would be useful. Additionally, could the authors perform short-term depletion studies with a PROTAC to the target or short shRNA or non-selected pool CRISPR deletion studies of CDK12 in these same cell lines to complement their pharmacological studies with genetic depletion studies? Also perhaps performing these same inhibitor studies in CDK12/13 deleted cells to test the specificity of the molecule would be useful.
We are not aware of a CDK12-specific PROTAC, and generate such as reagent is beyond the scope of the present study. Regarding the specificity of the CDK12/13 inhibitor molecules, additional information on the specificity and in vivo dose selection were added to the Results section (under “CDK13 is synthetic lethal in cells with biallelic CDK12 loss”). Cells with CDK12-KO did not tolerate CDK13-KO, so we were unable to generate double knockouts to test for CDK12/13 inhibitor non-specific effects.
Additionally, expanding these studies to additional prostate cancer cell lines or organdies models would strengthen the conclusions being made. More information should be provided about the dose and schedule chosen and the rationale for choosing those doses and schedules for the in vivo studies proposed should be presented and discussed. Was there evidence for maximal evidence of inhibition of the target CDK12/13 at the dose tested given the very modest tumor growth inhibition noted in these studies.
With respect to additional acute CDK12 loss models, our Tet-inducible shCDK12 models show only minor growth slowdown and do not appear to phenocopy the strong arrest or apoptosis seen with CDK12 KO or inhibition, respectively. Future work is ongoing to generate CDK12-degron regulated cell lines. We added a new immunoblot panel showing that acute CRISPR/sgRNA targeting of CDK12 does indeed lead to BRCA2 and ATM protein decrease (Fig. S4g), providing some orthogonal genomic targeting evidence of the acute HR gene effect. We are continuing efforts to collect and generate additional CDK12<sup>BAL</sup> cell models, in both 2D and 3D culture systems, but none are presently available. We added a 3D culture drug dose curve with LuCaP189.4 exposed to THZ531 (Fig. S7m), which confirms heightened sensitivity vs two CDK12-intact lines.
Regarding assessments of CDK12 targets; as we are not aware of any unique CDK12 substrates, it is fair to ask but difficult to measure precise CDK12 inhibition by the compounds in tumors. We dosed mice using the same protocol as detailed in the original report testing SR4835 in mice (PMID: 31668947). We performed immunoblots on lysates from 3 and 28 day treated PDX tumors and did not see any consistent decreases in pRBP1(Ser2) or ATM or increases in γH2A.X (data not shown). However, we did see increases in APA usage and downregulation of DNA repair transcripts with three day treatment (Fig. 6k-l), as would be expected from on target acute effects.
Reviewer #2 (Public review)
One caveat that continues to be unclear as presented, is the uncoupling of cell cycle/essentiality of CDK12/13 from HR-directed mechanisms. Is this purely a cell cycle arrest phenotype acutely with associated down-regulation of many genes?
In regard to untangling the effects of cell arrest on HR gene expression, this is a difficult question given that many HR genes, including BRCA2, are S/G2 linked. We attempted to account for those effects in the acute CDK12 inhibition experiment by including a palbociclib (CDK4/6i) control, which caused cell arrest and decreased BRCA1/2 RNA expression with no apparent 5/3’ transcript imbalance determined by qPCR (Fig. 4e,g). Though overall BRCA1 and BRCA2 mRNA levels are lower in the stable 22Rv1-CDK12-KO2 and KO5 lines, they do not show selective 3’ loss (Fig. 5c), suggesting the downregulation in these lines is mostly due to their slower growth (Fig. S4k) and not intronic polyA usage.
While the RAD51 loading ssRNA experiments are informative, the Tet-inducible knockdown of BRCA2 and CDK12 is confusing as presented in Figure 5, shBRCA2 + and -dox are clearly shown. However, were the CDK12_K02 and K05 also knocked down using inducible shRNA or a stable knockout? The importance of this statement is the difference between acute and chronic deletion of CDK12. Previously, the authors showed that acute knockdown of CDK12 led to an HR phenotype, but here it is unclear whether CDK12K02/05 are acute knockdowns of CDK12 or have been chronically adapted after single cell cloning from CRISPR-knockout.
As a clarification, the 22Rv1-CDK12-KO2 and 22Rv1-CDK12-KO5 are stable CRISPR knockout clonal lines that were expanded from single cells. We added a new figure to include more validation of these lines (Fig. S5). We tried multiple times to reproduce the HRd phenotype and PARPi sensitivity with siRNA and inducible shRNA lines but were unable to see clear sensitivity differences, despite seeing the expected shifts with shBRCA2 controls (data not shown). It is possible the degree of knockdown (~80%), timing (8 days), or specific cell lines used in our experiments were not sufficient to expose the acute phenotype by this method.
However, we were able to see acute HR gene decreases by inhibitor treatment (Fig. 4) or acute CRISPR (Fig. S4g).
Given the multitude of lines, including some single-cell clones with growth inhibitory phenotypes and ex-vivo derived xenografts, the variability of effects with SR4835, ATM, ATR, and WEE1 inhibitors in different models can be confusing to follow. Overall, the authors suggest that the cell lines differ in therapeutic susceptibility as they may have alternate and diverse susceptibilities. It may be possible that the team could present this more succinctly and move extraneous data to the supplement.
We appreciate the complexity of the data and attempted to use multiple models to report consistency and variability. We are not able to ascertain what data would be extraneous, and elected to present data we view as relevant in the main figures while moving supporting data in the supplement.
The in-vitro data suggests that SR4835 causes growth inhibition acutely in parental lines such as 22RV1. However, in vivo, tumor attenuation appears to be observed in both CDK12 intact and deficient xenografts, LuCAP136 and LuCaP 189.4 (albeit the latter is only nominally significant). Is there an effect of PARPi inhibition specifically in either model? What about the 22RV1-K02/05? Do these engraft? Given the role of CDK12/13 in RNAP II, these data might suggest that the window of susceptibility in CDK12 (mutant) tumors may not be that different from CDK12 intact tumors (or intact tissue) when using dual CDK12/13 inhibitors but rather represent more general canonical essential functions of CDK12 and CDK13 in transcription. From a therapeutic development strategy, the authors may want to comment in the discussion on the ability to target CDK13 specifically.
Though the response of the CDK12<sup>BAL</sup> models to some compounds is variable, we believe those mixed results are important and future studies may be able to better explain why some show shifts in sensitivity while others do not. We hope future studies with additional models will help determine which sensitivities are more consistently true, and perhaps provide explanations for differences between models.
Regarding SR4835, we find, and others have reported, a toxic (i.e. apoptotic) effect for in vitro treatment with dual CDK12/13 inhibitors (Fig. 4f, S4e,f); in fact, that may be why previous studies have used short timepoints in cell culture assays with these dual inhibitors. In mice, SR4835 was tolerated well but only LuCaP 189.4 showed statistically significant decreases in tumor volume and weight (Fig. 6j). We did not test PARPi responses in the PDX models, nor did we attempt engrafting the 22Rv1-CDK12-KO cell lines, but both would be worthwhile experiments in the future. Beyond CDK12<sup>BAL</sup> tumors, we agree that CDK12/13 inhibitors could be effective in cancer therapies more generally (e.g. triggering acute HRd, loss of RNAP2 phosphorylation). We added a line to the discussion section about ongoing efforts to combine PARPi and CDK12/13i, which we expect to be synergistic in CDK12-intact tumors due to the acute loss phenotype. We certainly agree that development of a specific CDK13 inhibitor would be the ideal therapeutic option for CDK12<sup>BAL</sup> tumors. However, CDK12 and CDK13 are 43% conserved at the protein level (PMID: 26748711), with 92% conservation in the active site (PMID: 30319007), and there are no available pharmacologic inhibitors that discriminate between CDK12 and CDK13.
Reviewer #3 (Public review):
It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.
Thank you for the feedback. It is a keen observation that homozygous deletions of CDK12 are not typical, though many mutations are upstream frameshifts that are expected to lead to loss of functional protein and mRNA via nonsense mediated decay. LuCaP189.4, our only natural mutant model, has two upstream frameshifts leading to complete protein loss (Fig 5b, S4h-i). We also added a caveat previously mentioned (in response to Reviewer 1) that mutations downstream of the kinase domain may be less likely to be fully pathogenic. For upstream missense mutations, the possibility of neuromorphic function remains an intriguing possibility that cannot be ruled out and would not be captured in our current models. Hopefully additional models can be developed, both natural and engineered, to help dissect that question in future studies.
It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).
We concur. Another caveat with the CRISPR models, beyond reliance on upstream frameshift mutations, is the simultaneous loss of alleles. In human tumors, there may be a period of single copy loss before the second hit that may provide a window for adaptation. It is possible that sequential loss is far easier for a cell to tolerate than acute bi-allelic inactivation. We agree that the question of what (if any) cooperating genetic alterations are required to tolerate CDK12 loss is an important one that we plant to further explore in future work.
Recommendations for Authors:
Reviewer #1 (Recommendations for Authors):
The authors have thoroughly addressed all issues of data availability, reagents, in vivo protocols, and animal approvals associated with the studies presented in this manuscript. Specific comments and experimental suggestions that in my opinion would strengthen the conclusions of this interesting and compelling manuscript are included above
Reviewer #2 (Recommendations for the authors):
The authors were thorough in their studies. As a general note, switching between the cell lines is often overwhelming in interpreting the data given cell-to-cell variability in response. If possible, consolidating the text/conclusions in results would improve the readability of the manuscript.
The variety of cell lines and models is perhaps expansive at times, but we hope the inclusion of these different models helps support the conclusions.
Is it possible to knockout CDK12 acutely using a degron-based approach, instead of utilizing an inhibitor that targets both CDK12/13?
There is a HeLa cell line made with analog-sensitive CDK12 (Bartkowiak, Yan, and Greenleaf 2016) but we were unaware of any such prostate lines at the time of this work. We are attempting to develop engineered prostate lines with specific CDK12 degradation but do not yet have them available.
How do the authors address a lower BRCA1/2 level in for example 22RV1-K05, does this cell line have increased sensitivity to PARPi over its parental 22RV1 line? Could this be added to Figure 5h/i?
The lower BRCA2 levels in 22Rv1CDK12-KO5 is likely due to the slower growth rate (Fig. S4k), as BRCA2 expression is S/G2 linked. While the mRNA level of BRCA2 overall is lower in the KO5 line, we do not observe the 5’/3’ transcript imbalance (Fig. 5c). The 22Rv1-CDK12-KO lines did not show increased sensitivity to carboplatin, while inducible shBRCA2 did (Fig. S7a), so we do not believe this lower BRCA2 confers functional HRd. We did test the KO lines with olaparib (Fig. S7d) and saw a modest increased sensitivity compared to parental 22Rv1, but not to the extent measured in the BRCA1 mutant line UWB1.289.
What is the clonality of the LuCAP 189.4 lines upon derivation? Is biallelic CDK12 loss seen in all cells?
We do not know the exact clonality of the LuCAP 189.4 PDX or CL model, but we do see highly uniform CDK12 protein loss in these cells (quantified by IHC staining, data not shown).
The authors state that 22RV1-K02/05 has an increased growth arrest to CDK13 inhibition. However, in Figure 6h, it appears the viability is not significantly different compared to the parental 22RV1 line. Similar aspects noted in 189.4-vec/CDK12?
We found that 22Rv1 KO2/KO5 have growth arrest with sgCDK13 and cell death with CDK12/13 inhibitor. We did notice that SR4835 did not show the differential effects we anticipated (Fig. 6h), as was seen with THZ531 (Fig. 6i). SR4835 is a non-covalent inhibitor, while THZ531 is a covalent binder, so there are some functional differences between these compounds that might explain the lack of differential effects in the isogenic lines in a 4 day in vitro assay. We included the SR4835 in vitro data because it was used for the in vivo experiment. THZ531 is not suited for animal use.
Could the authors comment on SR4835 response in vivo as a function of tumor growth rate?
The in vivo SR4835 treated LuCaP189.4 did show signs of reduced proliferation with decreased Cell Cycle and DNA Replication in the RNA-seq signatures, but a more detailed investigation into cell cycle arrest vs apoptotic response has yet to be fully explored. We plan to conduct additional PDX experiments if we can obtain a selective CDK13 inhibitor.
Do the authors explore TDPs in their isogenic cell lines?
We performed low coverage WGS on the 22Rv1 KO clones and added that to the paper (Fig. S5c). We did not see any obvious signs of TDP. We suspect the phenotype takes longer to accumulate and is not apparent within the ~20 passages our clones underwent in culture. This would be consistent with the tumor analysis (Fig. 2b) showing increase in TDs from primary to metastatic tumors, suggesting TDs accumulate over time.
A future study may allow for screening synthetic lethals in the context of CDK12 loss in the presence or absence of SR4835 inhibition.
We are actively pursuing experiments to identify new synthetic lethal targets by CRISPR and drug screens in CDK12 loss models and hope to report those in a future study.
Reviewer #3 (Recommendations for the authors):
As discussed above, the authors may wish to adjust their terminology to "CDK12-altered" rather than "CDK12 lost" or "CDK12-inactivated" to leave open the possibility that some tumors may retain residual CDK12 function or adopt neomorphic functions.
Thank you for the additional comments and feedback. The possibility of neomorphic CDK12 allele function is important. As our models were all complete protein loss mutations, we decided to retain “biallelic loss” as our preferred nomenclature, but the note is well taken.
The plots in Figures 1f-h are interesting and suggest that certain cancer genes (especially oncogenes) are recurrently gained in CDK12-altered tumors. It may be interesting to look at this on the individual level rather than the cohort level to see whether any groups of oncogenes tend to be gained together in an individual patient - this could inform whether certain combinations of cancer drivers cooperate with CDK12 alteration to drive oncogenesis.
Thank you for the idea of looking at the patient-level for TDP-enriched oncogenes. A preliminary assessment did not identify recurrent co-gained oncogenes. We will continue these analyses as additional patient tumors with CDK12 alterations are identified.
The finding that AR gene or enhancer are recurrently gained with TDP is interesting and I am curious whether the authors have thoughts on whether these alterations can also be seen in the 1-2% of CDK12altered primary prostate cancers that are treatment naïve, and where AR pathway alterations are not as frequently seen.
We did not focus on CDK12 altered primary prostate cancers, but we did check if there is AR amplification enrichment in the 6 CDK12<sup>BAL</sup> cases of the TCGA-PRAD dataset and did not identify enrichment. However, with such small numbers we would hesitate to draw any hard conclusions.
It could be interesting to more comprehensively characterize some of the CDK12 KO-adapted lines in Figure 5 (e.g. by WES or WGS) to determine whether they exhibit the TDP and/or whether they have acquired any secondary mutations that allow them to adapt to CDK12 loss.
We are planning to do further genomics characterization of the CDK12-KO lines and will hopefully include that in a future study. Genomic analyses of the 22Rv1 clones (see copy number plots in Fig. S5c) did not identify a TDP. We plan to repeat the genomic assessments over additional cell passages and we have planned additional experiments designed to understand why some cells tolerate CDK12 loss and others do not.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Hurtado et al. show that Sox9 is essential for retinal integrity, and its null mutation causes the loss of the outer nuclear layer (ONL). The authors then show that this absence of the ONL is due to apoptosis of photoreceptors and a reduction in the numbers of other retinal cell types such as ganglion cells, amacrine cells, and horizontal cells. They also describe that Müller Glia undergoes reactive gliosis by upregulating the Glial Fibrillary Acidic Protein. The authors then show that Sox9+ progenitors proliferate and differentiate to generate the corneal cells through Sox9 lineage-tracing experiments. They validate Sox9 expression and characterize its dynamics in limbal stem cells using an existing single-cell RNA sequencing dataset. Finally, the authors argue that Sox9 deletion causes progenitor cells to lose their clonogenic capacity by comparing the sizes of control and Sox9-null clones. Overall, Hurtado et al. underline the importance of Sox9 function in retinal and corneal cells.
Strengths:
The authors have characterized a myriad of striking phenotypes due to Sox9 deletion in the retina and limbal stem cells which will serve as a basis for future studies.
Weaknesses:
Hurtado et al. investigate the importance of Sox9 in the retina and limbal stem cells. However, the overall experimental narrative appears dispersed.
(1) The authors begin by characterizing the phenotype of Sox9 deletion in the retina and show that the absence of the ON layer is due to photoreceptor apoptosis and a reduction in other retinal cell types. The authors also note that Müller glia undergoes gliosis in the Sox9 deletion condition. These striking observations are never investigated further, and instead, the authors switch to lineage-tracing experiments in the limbus that seem disconnected from the first three figures of the paper. Another example of this disconnect is the comparison of Sox9 high and Sox9 low populations using an existing scRNA-seq dataset and the subsequent GO term analysis, which does not directly tie in with the lineage-tracing data of the succeeding Sox9∆/∆ experiments.
We thank the reviewer for their thoughtful observations. We would like to clarify the rationale behind the structure of our study and how the different parts are conceptually connected.
Our central aim was to investigate the role of Sox9 in the adult eye. Given that Sox9 has been extensively studied during embryonic development, we specifically chose to use an inducible conditional knockout strategy (CAG-CreERTM) in order to assess its function postnatally, in the adult eye. This approach revealed a severe retinal phenotype, whereas the cornea showed no overt phenotype. A major strength of our experimental design is that it allowed us to examine the role of Sox9 specifically in the adult eye, avoiding confounding effects from embryonic development. Nevertheless, this approach entails an inherent limitation: the mosaic nature of the CAG-CreERTM system leads to substantial variability in both the extent and distribution of Sox9 inactivation among individual animals. We invested considerable effort over extended periods to obtain reliable and biologically meaningful data despite this variability. We did not proceed further because this mosaicism poses a significant limitation when attempting to dissect downstream mechanisms in a consistent and reproducible manner, making it extremely challenging to perform in-depth mechanistic studies.
Regarding the cornea, given the absence of a clear phenotype upon Sox9 deletion, we expanded our investigation by adding lineage-tracing and transcriptomic analyses to better understand Sox9’s potential role in adult limbal epithelial stem cells. These additional experiments provided valuable insight into Sox9 function in the adult cornea, even in the absence of gross morphological changes. Thus, while the retinal and corneal data stem from different experimental approaches, they are unified by a shared goal: understanding the celltype-specific and tissue-specific functions of Sox9 in the adult eye.
To ensure that other readers do not perceive this apparent disconnect, and overstate our conclusions, we have modified the manuscript. In the Introduction section, we have included the main findings from studies conducted to date on the role of Sox9 in the cornea and retina, and we have removed the corresponding section from the Discussion. We believe it is now clear that our study focuses on the role of Sox9 in the adult eye, in contrast to previous studies, which focused on the developing eye.
In the Discussion section, we have added a new paragraph at the beginning and end that explicitly addresses the relationship between the retinal and limbal findings, illustrating how a single transcription factor can play distinct roles in different tissues within the same organ.
Regarding the reviewer’s comment that the scRNA-seq analyses appear disconnected from the lineage-tracing data, we respectfully disagree. These analyses provide independent transcriptional confirmation that Sox9 is a marker of limbal stem cells, reinforcing the conclusions drawn from our in vivo experiments. These approaches are complementary and they converge on the same biological insight: Sox9 marks a population with stem-like properties in the adult limbus. Nevertheless, we acknowledge the reviewer’s concern and have moderated the tone of our statements in the revised version of the manuscript to better reflect the supporting nature of the scRNA-seq data, without overstating its functional implications.
(2) A major concern is that a single Sox9∆/∆ limbal clone has a sufficiently large size, comparable to wild-type clones, as seen in Figure 6D. This singular result is contrary to their conclusion, which states that Sox9-deficient stem cells minimally contribute to the maintenance of the cornea.
We thank the reviewer for this important observation.
Ligand-independent activity of Cre-ER fusion proteins has been repeatedly reported in various mouse models (Vooijs et al., 2001; Kemp et al., 2004; Haldar et al., 2009). This basal recombinase activity is thought to arise from inappropriate nuclear translocation or proteolysis of the Cre-ER fusion protein, leading to low-level recombination even in the absence of tamoxifen. Consistent with this, prior studies using the same CAGG-CreERTM; R26R-LacZ system for clonal analysis in the cornea have observed sparse reporter expression before tamoxifen administration (Dorà et al., 2015).
In line with these findings, we also detected minimal background LacZ staining in Sox9Δ/ΔLacZ corneas (mean surface area: 0.85%; n = 8 eyes). This low-level staining likely reflects recombination events in transient amplifying or more differentiated cells, which are not expected to generate long-lived clones. However, in the rare instance of a large clone, as shown in Figure 6D, we believe that a spontaneous recombination event may have occurred in a bona fide limbal stem cell, giving rise to a sustained contribution. To rigorously address this potential artefact and assess the true contribution of Sox9-deficient stem cells, we conducted a comparative analysis of 8 control (Sox9Δ/+-LacZ) and 5 mutant (Sox9Δ/ΔLacZ) corneas. This analysis revealed a highly significant 8-fold reduction in the LacZpositive surface area in mutant samples (Sox9Δ/+-LacZ: 6.65 ± 1.77%; Sox9Δ/Δ-LacZ: 0.85 ± 0.85%; paired t-test, p = 0.00017; Figs. 6E and F; Table S12).
We chose to include the image of the large clone in the main figure precisely because it does not align with our working hypothesis. We believe that showing such exceptions transparently is scientifically important and may be valuable for other researchers using similar inducible systems. Nonetheless, based on previous literature, the number of samples analyzed, and the statistically significant reduction in clonal contribution, we maintain that the observed phenotype reflects a true biological effect of Sox9 loss, supporting our conclusion that Sox9-deficient stem cells contribute minimally to corneal maintenance. To make that point clearer, we have introduced the following sentence in lines 462-464 of the revised version of the manuscript.
“A possible explanation for this clone may be that spontaneous ligand-independent activity of Cre-ER fusion may have occurred in a bona fide limbal stem cell, as previously reported (Vooijs et al., 2001; Kemp et al., 2004; Haldar et al., 2009, Dorà et al., 2015).”
Reviewer #2(Public revciew):
Sox9 is a transcription factor crucial for development and tissue homeostasis, and its expression continues in various adult eye cell types, including retinal pigmented epithelium cells, Müller glial cells, and limbal and corneal basal epithelia. To investigate its functional roles in the adult eye, this study employed inducible mouse mutagenesis. Adult-specific Sox9 depletion led to severe retinal degeneration, including the loss of Müller glial cells and photoreceptors. Further, lineage tracing revealed that Sox9 is expressed in a basal limbal stem cell population that supports stem cell maintenance and homeostasis. Mosaic analysis confirmed that Sox9 is essential for the differentiation of limbal stem cells. Overall, the study highlights that Sox9 is critical for both retinal integrity and the differentiation of limbal stem cells in the adult mouse eye.
Strengths:
In general, inducible genetic approaches in the adult mouse nervous system are rare and difficult to carry out. Here, the authors employ tamoxifen-inducible mouse mutagenesis to uncover the functional roles of Sox9 in the adult mouse eye.
Careful analysis suggests that two degeneration phenotypes (mild and severe) are detected in the adult mouse eye upon tamoxifen-dependent Sox9 depletion. Phenotype severity nicely correlates with the efficiency of Cre-mediated Sox9 depletion.
Molecular marker analysis provides strong evidence of Mueller cell loss and photoreceptor degeneration.
A clever genetic tracing strategy uncovers a critical role for Sox9 in limbal stem cell differentiation.
Weaknesses:
(1) The Introduction can be improved by explaining clearly what was previously known about Sox9 in the eye. A lot of this info is mentioned in a single, 3-page long paragraph in the Discussion. However, the current study's significance and novelty would become clearer if the authors articulated in more detail in the Introduction what was already known about Sox9 in retina cell types (in vitro and in vivo).
We appreciate this insightful comment. Following the reviewer`s suggestion, we have reorganized the manuscript to provide a clearer scientific context in the Introduction. Specifically, we have moved the relevant background information on Sox9 in different retinal cell types—previously included in a single, extended paragraph in the Discussion—into the Introduction. This helps to better frame our study within the context of existing knowledge.
Additionally, we have emphasized more explicitly that our work does not focus on embryonic development, as most previous studies on Sox9 have done, but instead investigates its role in the adult mouse retina and limbus/cornea. We believe this represents an important and novel aspect of our study, as the mechanisms of retinal maintenance and limbal stem cell differentiation in the adult have been less extensively studied.
(2) Because a ubiquitous tamoxifen-inducible CreER line is employed, non-cell autonomous mechanisms possibly contribute to the observed retina degeneration. There is precedence for this in the literature. For example, RPE-specific ablation of Otx2 results in photoreceptor degeneration (PMID: 23761884). Have the authors considered the possibility of non-cell autonomous effects upon ubiquitous Sox9 deletion?
Given the similar phenotypes between animals lacking Otx2 and Sox9 in specific cell types of the eye, the authors are encouraged to evaluate Otx2 expression in the tamoxifen-induced Sox9 adult retina.
We appreciate the insightful comment of the reviewer regarding the potential contribution of non-cell autonomous mechanisms to the retinal degeneration observed upon ubiquitous Sox9 deletion. We agree that this is an important consideration, particularly in the context of findings showing that RPE-specific deletion of Otx2 results in secondary photoreceptor degeneration.
However, we would like to emphasize that RPE-specific deletion of Sox9 does not lead to photoreceptor loss or retinal degeneration, as previously shown (Masuda et al., 2014; Goto et al., 2018; Cohen-Tayar et al., 2018) [PMID: 24634209; PMID: 29609731; PMID: 29986868]. In addition, it was shown that Sox9 deletion in the RPE caused downregulation of visual cycle genes but did not compromise photoreceptor integrity or survival. Interestingly, Otx2 expression was found to be upregulated in the absence of Sox9, further supporting the view that Sox9 is not a simple upstream regulator of Otx2 in the adult RPE (Matsuda, 2014). These findings suggest that RPE dysfunction alone cannot account for the severe retinal phenotype we observe in our model.
In our study, we observed that photoreceptor degeneration correlates strongly with the depletion of Sox9 Müller glial cells. Given the well-established supportive and neuroprotective roles of Müller glia, we interpret the retinal degeneration in our model to be primarily a consequence of Müller cell dysfunction (confirmed by the loss of Müller glia markers, such as SOX8 and S100). This interpretation is further supported by previous studies showing that selective ablation of Müller glia can lead to photoreceptor degeneration through cell-autonomous mechanisms (Shen et al., 2012) [PMID: 23136411].
Nevertheless, we agree that this possibility deserves further investigation, and we have acknowledged it in the following paragraph that has been added to the Discussion section (lines 511-523 of the revised ms):
“An important consideration in our model is the potential contribution of non-cell autonomous mechanisms to photoreceptor degeneration. Sox9 is expressed in both MG and RPE cells, and both cell types are known to support photoreceptor viability (Poché et al., 2008; Masuda et al., 2014). Notably, Sox9 and Otx2 cooperate to regulate visual cycle gene expression in the RPE (Masuda et al., 2014), and loss of Otx2 specifically in the adult RPE leads to secondary photoreceptor degeneration through non-cell autonomous mechanisms (Housset et al., 2013). However, RPE-specific deletion of Sox9 does not induce retinal degeneration and in fact results in Otx2 upregulation (Masuda et al., 2014; Goto et al., 2018; Cohen-Tayar et al., 2018), suggesting that Sox9 is not an upstream regulator of Otx2 in this context. Further investigation into the molecular and cellular interactions between MG, RPE, and photoreceptors may help to clarify the indirect pathways contributing to degeneration in the absence of Sox9.”
Consistent with the above, a new citation has been included:
Housset M, Samuel A, Ettaiche M, Bemelmans A, Béby F, Billon N, Lamonerie T. 2013. Loss of Otx2 in the adult retina disrupts retinal pigment epithelium function, causing photoreceptor degeneration. J Neurosci 33:9890–904. doi:10.1523/JNEUROSCI.1099-13.2013.
(3) The most parsimonious explanation for the dual role of Sox9 in retinal cell types and limbal stem cells is that the cell context is different. For example, Sox9 may cooperate with TF1 in photoreceptors, TF2, in Mueller cells, and TF3 in limbal stem cells, and such cell typespecific cooperation may result in different outcomes (retinal integrity, stem cell differentiation). The authors are encouraged to add a paragraph to the discussion and share their thoughts on the dual role of Sox9.
We thank the reviewer for this thoughtful and constructive suggestion. In , we have added a paragraph at the end of the Discussion addressing the potential dual role of Sox9 in the cornea and retina. In this new section, we discuss how Sox9 might exert distinct functions depending on the cellular context, possibly through interactions with different transcriptional partners in specific cell types. This may help explain the contrasting roles of Sox9 in maintaining retinal integrity versus regulating stem cell differentiation in the limbal epithelium.
(4) One more molecular marker for Mueller glial cells would strengthen the conclusion that these cells are lost upon Sox9 deletion.
We thank the reviewer for this constructive suggestion. To reinforce our conclusion that most Müller glial cells are lost following Sox9 deletion, we analysed the expression of S100, a well-established cytoplasmic marker of Müller glia. As S100 is primarily localized to the innermost Müller cell processes and not restricted to cell bodies, direct cell counting was not feasible. Instead, we quantified the S100+ signal intensity across defined retinal surface areas. This analysis revealed a statistically significant reduction in S100 signal in Sox9<sup>Δ/Δ</sup> retinas compared to controls. These new data, included in the revised Figure 1 (panels F and G), support and extend our previous observations using SOX8, further confirming the loss of Müller glial cells in Sox9-deficient retinas.
We have also modified the manuscript based on this new evidences as follows:
In the Results section, lines 168-177 of the revised ms, we have added the following paragraph: “To independently validate the loss of MG cells in Sox9-deficient retinas, we examined the expression of S100, a cytoplasmic marker that labels the processes of adult Müller cells. In control retinas, strong S100 immunoreactivity was observed across the inner retina, outlining the typical radial projections of Müller glia (Fig. 1F). In contrast, Sox9Δ/Δ retinas with an extreme phenotype exhibited a marked reduction in S100 signal (Fig. 1G). Given the diffuse cytoplasmic localization of S100, we quantified its expression by measuring the fluorescence signal within a defined surface area of the retina. This analysis revealed a statistically significant reduction in S100 signal intensity in mutant samples (including both mild and extreme phenotypes) compared to controls (Fig. 1G; Table S4), further supporting the loss of MG cells upon Sox9 deletion.”
In Methods, line 684 of the revised ms, the anti-S100 antibody reference and its working dilution have been added.
(5) Using opsins as markers, the authors conclude that the photoreceptors are lost upon Sox9 deletion. However, an alternate possibility is that the photoreceptors are still present and that Sox9 is required for the transcription of opsin genes. In that case, Sox9 (like Otx2) may act as a terminal selector in photoreceptor cells. This point is particularly important because vertebrate terminal selectors (e.g., Nurr1, Otx2, Brn3a) initially affect neuron type identity and eventually lead to cell loss.
We perfectly understand the reviewer’s point. However, we believe that the possibility that Sox9 regulates opsin gene expression without affecting photoreceptor survival is very unlikely in our model. The primary evidence comes from the histological analysis shown in Figure 1B, where hematoxylin and eosin staining clearly demonstrates the complete loss of the ONL in Sox9<sup>Δ/Δ</sup> retinas exhibiting the extreme phenotype. Similarly, DAPI counterstain also evidences the lack of the ONL in many of our immunofluorescence images of these samples. This morphological disappearance of the ONL strongly supports the conclusion that photoreceptor cells are not merely transcriptionally silent but are physically absent.
Furthermore, TUNEL assays in two retinas with a mild phenotype revealed extensive apoptosis within the ONL, suggesting a progressive degeneration process rather than a selective transcriptional effect. While we acknowledge that transcriptional regulation of opsin genes by Sox9 cannot be entirely ruled out, the observed phenotype is more consistent with a structural loss of photoreceptors rather than a change in their molecular identity alone. Therefore, our data support the interpretation that Sox9 is required for photoreceptor survival, likely through non-cell autonomous mechanisms related to Müller glia dysfunction, rather than acting as a terminal selector within photoreceptor cells themselves.
(6) Quantification is needed for the TUNEL and GFAP analysis in Figure 3.
We have quantified the GFAP immunofluorescence signal across defined surface areas of the retina and found a statistically significant increase in GFAP expression in Sox9<sup>Δ/Δ</sup> mutants compared to controls (Mann-Whitney U test, P = 0.0240; n = 4 controls, 10 mutants). These quantification data are now included in the revised Figure 3.
Regarding the TUNEL assay, although extensive apoptosis was clearly observed in two Sox9<<sup>Δ/Δ</sup> retinas with a mild phenotype (as shown in Figure 3A), this pattern was not consistent across the full study mouse cohort. Out of 15 mutant samples analyzed (5 of them previously analyzed and 10 additional ones that have been newly analyzed), only two exhibited this pronounced apoptotic pattern. However, in the remaining 13 mutants, we did observe a small but statistically significant increase in the number of TUNEL+ cells compared to controls (zero-inflated Poisson test, P = 0.028, n = 5 controls, 13 mutants). These results are now included in Figure 3 and in Tables S7 and S8.
This pattern likely reflects the transient nature of apoptosis in the degenerative process, which may occur rapidly and thus be difficult to capture consistently at a single time point. Nevertheless, the quantification supports our conclusion that Sox9 loss is associated with increased photoreceptor cell death.
Based on the above, we have included the following paragraphs in the Results section of the manuscript:
In lines 224-252 of the revised ms, the final version of the paragraph is as follows: “Since photoreceptors are absent in severely affected Sox9-mutant retinas, we conducted TUNEL assays to study the role of cell death in the process of retinal degeneration. In control samples (n=5), almost no TUNEL signal was observed in the retina. In contrast, Sox9<sup>Δ/Δ</sup> mice (n=15) showed numerous TUNEL+ cells, mainly located in the persisting ONL, indicating that photoreceptor cells were dying (Fig. 3A). Although extensive TUNEL staining in the ONL was clearly observed in two Sox9<sup>Δ/Δ</sup> retinas with mild phenotypes, this pattern was not consistently present across the full cohort. In the remaining 13 mutant retinas, we observed a modest but noticeable increase in the number of apoptotic cells compared to controls (Fig. 3B; Table S7). Despite a high frequency of zero counts (particularly among controls), the difference between groups reached statistical significance when analyzed using a zeroinflated Poisson model (P = 0.028; n = 5 controls, 13 mutants). These findings suggest that photoreceptor apoptosis following Sox9 deletion may occur acutely and within a narrow temporal window, making it challenging to capture the full degenerative process at a single time point”.
Lines 263-269 of the revised ms: “To support these observations quantitatively, we measured GFAP fluorescence intensity across defined retinal surface areas in control and Sox9<sup>Δ/Δ</sup> mice (Fig. 3D; Table S8). This analysis revealed a statistically significant increase in GFAP signal in mutant retinas compared to controls (Mann-Whitney U test, P = 0.0240; n = 4 controls, 10 mutants). These results are consistent with a progressive gliotic following Sox9 deletion and provide further evidence that MG cells become reactive in the absence of Sox9”.
Similarly, the section “Estimation of the percentage of tamoxifen-induced, Cre-mediated recombination” has been expanded as follows:
Lines 660-665 of the revised ms: “In parallel, to quantify GFAP expression as a measure of MG reactivity, we analyzed GFAP immunofluorescence intensity across defined retinal surface areas. Given the cytoplasmic distribution of GFAP within glial processes, direct cell counting was not feasible. Instead, fluorescence intensity was measured using ImageJ, within full-thickness retinal regions in 20x microphotographs of a retinal sections stained for GAFP. The total GFAP signal was normalized to the measured area for each section”.
(7) Line 269-320: The authors examined available scRNA-Seq data on adult retina. This data provides evidence for Sox9 expression in distinct cell types. However, the dataset does not inform about the functional role of Sox9 because Sox9 mutant cells were not analyzed with RNA-Seq. Hence, all the data that claim that this experiment provides insights into possible Sox9 functional roles must be removed. This includes panels F, G, and H in Figure 5. In general, this section of the paper (Lines 269-320) needs a major revision. Similarly, lines 442-454 in the Discussion should be removed.
We thank the reviewer for this important observation. We agree that the scRNA-Seq dataset used in this section does not include Sox9 mutant cells and therefore does not allow us to assess the consequences of Sox9 loss-of-function. However, we believe that this analysis still provides valuable complementary information. Specifically, it confirms that Sox9 is expressed in a distinct population of limbal stem cells, and that its expression dynamically changes along differentiation trajectories. Although we do not infer causality or phenotypic consequences, the ability to observe how gene expression programs shift as Sox9 is downregulated offers insights into potential transcriptional programs associated with Sox9 activity.
We have carefully revised Lines 269–320 to remove any overinterpretations, and eliminated the corresponding lines in the Discussion (Lines 442–454). However, we have retained Panels G, and H in Figure 5 with updated text that reflect the descriptive nature of these findings, specifically to illustrate that the Sox9-positive cell signature is consistent with a stem cell genetic program, and that when Sox9 is downregulated some gene pathways involved in stem cell differentiation are upregulated.
Reviewer #1 (Recommendations for the authors):
Major points
(1) Figure 1C shows the proportions of Sox9+cells that express Sox8 in control, mild and extreme phenotypes. However, as no quantitative classification of mild and extreme phenotypes is reported along with Figure 1A, the large standard deviation for Sox9∆/∆ mild retina might be due to a misclassification of the sample. Therefore, the authors must ascribe each sample to "mild" or "extreme" based on a quantitative metric.
We appreciate the reviewer’s suggestion to clarify the classification criteria used to distinguish “mild” and “extreme” phenotypes in Sox9<sup>Δ/Δ</sup> retinas. As noted, our classification was based on a qualitative, phenotypic assessment of retinal morphology in hematoxylin/eosin-stained sections. Specifically, retinas were classified as “extreme” when the outer nuclear layer (ONL) was completely absent, and as “mild” when the ONL was present, although often reduced in thickness. This classification reflects the observable structural depletion of the ONL and aligns well with the extent of Sox9 loss in Müller glial cells, as shown in Figure 1. We acknowledge that some variability exists within the “mild” group, likely due to differences in recombination efficiency and the mosaic nature of tamoxifen-induced deletion.
The phenotypic classification of each individual sample is explicitly provided in Supplementary Table S1. We have also added a statement in the Results section clarifying that this classification was based on qualitative histological criteria rather than a numerical threshold.
Lines 104-113 of the revised ms: “We categorized Sox9<sup>Δ/Δ</sup> retinas into “mild” and “extreme” phenotypes in order to facilitate interpretation of our data. Clasification was based on a qualitative assessment of ONL integrity in histological sections. Specifically, samples were classified as “extreme” when the ONL was completely depleted, and as “mild” when the ONL persisted, albeit variably reduced in thickness. This phenotypic classification reflects observable structural differences rather than a fixed quantitative threshold. Some variability exists within the “mild” group, likely due to differences in recombination efficiency and the mosaic nature of tamoxifen-induced Cre-mediated Sox9 deletion”
(2) The authors infer Sox9 high and Sox9 low groups of limbal stem cells using an existing scRNA-seq dataset. However, an immunohistology-based validation of this difference is missing. Given that limbal stem cells express Sox9, the authors must examine the heterogeneity in Sox9 levels within the Sox8+ population to demonstrate their claim: "...Sox9 expression decreases as transiently amplifying progenitors undergo progressive differentiation from limbal to peripheral corneal cells." in Line 292. Ideally, this must be further validated using differentiation markers corresponding to CB and ILB populations that show lower Sox9 expression according to the pseudotime graph.
To validate the Sox9 expression results obtained with scRNA-seq, we performed double immunofluorescence for Sox9 and P63, the latter expressed by the basal cells of the limbal epithelium, but not by transient amplifying cells covering the corneal surface (Pellegrini et al., 2001, https://www.pnas.org/doi/abs/10.1073/ pnas.061032098). These results can be observed in the new panel 5F. Accordingly we have included a new paragraph in lines 369-396 of the revised version of the ms:
“To validate these results, we decided to closely examine Sox9 expression in the limbus using immunofluorescence. Previous analyses revealed that the outer limbus is approximately 100 μm wide, while the inner limbus is wider, around 240 μm (Altshuler 2021). We observed that in the region corresponding to the OLB, most cells showed strong Sox9 expression. In the area corresponding to the ILB, this immunoreactivity appeared weaker in the basal layer (corresponding to the ILB proper), and no expression was detected in the suprabasal layers (flattened cells; Fig 5F left). Double immunofluorescence for SOX9 and P63, which is expressed in basal cells of the limbal epithelium, but not by transient amplifying cells covering the corneal surface (Pellegrini et al., 2001) revealed that Sox9 expression was restricted to P63-positive cells (Fig 5F right). These observations confirm that Sox9 is expressed in a basal cell population within both the OLB and ILB, and that its expression decreases in differentiated transient amplifying cells. ”
We also have deleted “This expression pattern is consistent with our immunofluorescence observations" from line 356 of the revised ms.
(3) The authors' claim of "...Sox9-null cells cannot survive or proliferate as well as their wildtype neighbors, and are hence outcompeted over time, leading to an essentially wild-type cornea" does not seem very convincing in the light of Fig.6D and S3B where Sox9 deletion can still allow for a large LacZ+ clone. Their claim of wild-type cornea due to out-competing neighbors must be validated by increasing the number of Sox9-null progenitors, which can be tested by administering tamoxifen for a significantly longer duration, leading to a majority Sox9 deficient progenitor population, and then examining limbal and corneal defects.
As previously discussed, we observed only one instance of a large LacZ+ clone across 8 Sox9<sup>Δ/Δ</sup>-LacZ eyes. Based on prior reports of ligand-independent Cre activity (Vooijs et al., 2001; Kemp et al., 2004; Haldar et al., 2009; Dorà et al., 2015), we believe this rare event likely resulted from spontaneous recombination in a bona fide limbal stem cell, independent of tamoxifen administration. For this reason, we do not expect that increasing the dose or duration of tamoxifen would eliminate such rare events. Furthermore, due to the mosaic and highly variable recombination efficiency of the CAGG-CreERTM system in the adult eye (see McMahon et al., 2008), attempting to increase the TX dosage would likely lead to systemic toxicity or lethality, without guaranteeing full inactivation of the gene in the limbus. Thus, this system is not well-suited for generating a fully Sox9-deficient limbal epithelium. To overcome this limitation, we crossed our mice with the R26R-LacZ reporter line to track the clonal behavior of Sox9-deficient cells. In control animals (Sox9Δ/+-LacZ), LacZ+ stripes originating from limbal stem cells are readily observed. In contrast, in Sox9Δ/Δ-LacZ mutants, these clones are either absent or drastically reduced. This suggests that Sox9-null cells have a severely impaired ability to form and sustain clones. To rigorously quantify this effect, we compared 8 control and 5 mutant corneas, revealing a highly significant 8-fold reduction in LacZ-positive area in the mutants (6.65 ± 1.77% vs. 0.85 ± 0.85%; p = 0.00017; Fig. 6F; Table S12; Supp. Fig. X???), supporting our claim that Sox9null cells cannot survive or proliferate as well as their wild-type neighbors, and are hence outcompeted over time, leading to an essentially wild-type cornea.
Minor points
(1) Quantification for Figure 2C and 2D is missing.
We have now included quantification of BRN3A+ retinal ganglion cells (Figure 2E) across control and Sox9<sup>Δ/Δ</sup> retinas. Cell counts were performed on matched retinal sections, and the difference between groups was found to be statistically significant through Mann–Whitney U test (Table S5).
Regarding PAX6/AP2a, we quantified inner retinal neurons by analyzing AP2α+ amacrine cells and PAX6+/AP2α- horizontal cells as distinct subpopulations, rather than simply comparing total PAX6 or AP2α immunoreactivity. This approach allowed us to better resolve specific neuronal subtype changes. Both populations showed a statistically significant reduction in Sox9-deficient retinas relative to controls. The quantification for these analyses has now been incorporated into the revised Figure 2F and G (Table S6).
Consequently with the above, the following paragraph of the Results section (line 210 of the revised ms:
“We also studied the status of other retinal cell types. The transcription factor BRN3A was used to identify ganglion cells (Nadal-Nicolás et al., 2009), which were shown to decrease in number in the mutant retinas, compared to control ones (Fig. 2C). Similarly, double immunodetection of the transcription factors PAX6 and AP2A was used to identify both amacrine and horizontal cells, as previously described (Marquardt et al., 2001; Barnstable et al., 1985; Edqvist and Hallböök, 2004), showing a similar reduction in both cell types in degenerated retinas (Fig. 2D).”
Has been modified as follows:
“We also studied the status of other retinal cell types. The transcription factor BRN3A was used to identify ganglion cells (Nadal-Nicolás et al., 2009), which were shown to decrease in number in the mutant retinas, compared to control ones (Figs. 2C and 2D and Table S5; n = 5 controls, n = 12 mutants; Mann-Whitney U test, P = 3 × 10<sup>-4</sup>). Similarly, double immunodetection of the transcription factors PAX6 and AP2A was used to identify both amacrine and horizontal cells (Fig. 2E), as previously described (Marquardt et al., 2001; Barnstable et al., 1985; Edqvist and Hallböök, 2004), showing a similar reduction in both cell types in degenerated retinas (Figs. 2F and 2G and Table S6; AP2α+ amacrine cells: n = 3 controls, n = 8 mutants; 2-sample T-tests P = 0.029; PAX6+/AP2α− horizontal cells: n = 3 controls, n = 8 mutants; Mann-Whitney U test P = 0.021). These findings indicate that the loss of Sox9 in the adult retina ultimately leads to the degeneration of multiple inner retinal neuronal populations, beyond the previously described effects on photoreceptors and Müller glia.
(2) Figure 4G & H: The authors must mention that the dashed lines enclose the limbal area.
Done
(3) The authors infer from an existing scRNA-seq dataset that OLB cells have high Sox9 expression as compared to ILB and corneal populations. However, Figures 4A and B do not indicate the anatomical positions of these cell types. The authors must label these for the reader's reference as they state that "[Sox9] expression pattern is consistent with our immunofluorescence observations" in Line 280.
As previously indicated, we have generated a new panel 5F and a corresponding paragraph to illustrate Sox9 expression pattern in the limbus. Accordingly, we have removed the sentence from line 280.
(4) Quantification for Figures 6A and 6B is missing.
We have quantified the number of Sox9 and P63 positive cells in the limbus between mutant and control corneas and found no difference in the number of positive cells. We have included these data in new panel 6C and Table S11.
Reviewer #2 (Recommendations for the authors):
Line 24: "synapsis" should be "synapses".
Done
(1) Consider starting a new paragraph after line 30.
Done
(2) Lines 42-48: make clear that this paragraph provides information only for HUMAN SOX9.
We now distinguish which studies were conducted in humans and which in mice.
(3) Line 55: explain to the non-expert reader what the "visual cycle" is.
Done (lines 64-65 of the revised ms)
(4) Line 66: consider "inactivate" instead of "suppress".
We substituted “suppress” with “inactivate”
(5) Line 90-92: ONLY PCR for the cGMP will provide formal evidence that this is not present in the mouse line.
We agree with the reviewer that PCR genotyping is the most straightforward method to exclude the presence of the Pde6<sup>brd</sup>1 allele. Although retinal degeneration was never observed in untreated or control animals in our study, we have now removed the term “formal possibility” from the text to better reflect this limitation.
We have modified the following paragraph (page 116 in the revised version of the manuscript): “Retinal degeneration was never observed in mice that had not been tamoxifen-treated, nor any other controls, eliminating the formal possibility that the retinal degeneration allele of photoreceptor cGMP phosphodiesterase 6b (Pde6brd1) was present in our mice (Bowes et al., 1990).”
As follows: “Retinal degeneration was never observed in mice that had not been tamoxifentreated, nor any other control groups, making the presence of the retinal degeneration allele of photoreceptor cGMP phosphodiesterase 6b (Pde6<sup>brd1</sup>) unlikely in our mice (Bowes et al., 1990). However, we acknowledge that definitive exclusion of this possibility would require PCR-based genotyping.”
(6) Line 160-166: This paragraph needs a conclusion.
We agree with the reviewer and have added the following sentence at the end of the paragraph:
“These findings indicate that the loss of Sox9 in the adult retina ultimately leads to the degeneration of multiple inner retinal neuronal populations, beyond the previously described effects on photoreceptors and Müller glia”
(7) Line: 240-265: This paragraph ends without a conclusion.
We have include the following conclusion:
“Thus, Sox9 is expressed in a basal limbal stem cell population with the ability to form two types of long-lived cell clones involved in stem cell maintenance and homeostasis.”
(8) In Results, it needs to be specified when exactly in adulthood the tamoxifen treatment started. This information is only provided in the Methods.
We have specified the age of the mice at the onset of tamoxifen treatment (two months) and included it in the schemes of Figs 1A, 4C, 4H, 6E.
(9) Line 250: Because live imaging is not conducted, the word "dynamics" is not suitable.
We substituted “dynamics” with “contribution”
(10) Panel C in Figure 6 is nice and helpful. Consider adding a similar panel in Figure 1.
Done.
(11) Line 420: is this the human Sox9 enhancer?
Yes. It is a human enhancer. We have indicated it in the text.
(12) Line 459: typo "detected tissue".
Corrected
(13) Line 448 and 468: citations are needed.
Line 448 is deleted in the revised version of the ms.
(14) 479: typo "clones clones'.
Corrected.
Reviewer #3 (Public review):
Summary:
The manuscript presents computational modelling of the behaviour of mice during encounters with novel and familiar objects, originally reported in Akiti et al. (Neuron 110, 2022). Mice typically perform short bouts of approach followed by retreat to a safe distance, presumably to balance exploration to discover possible reward with the potential risk of predation. However, there is considerable heterogeneity in this exploratory behaviour, both across time as an individual subject becomes more confident in approaching the object, and across subjects; with some mice rapidly becoming confident to closely explore the object, while other timid mice never become fully confident that the object is safe. The current work aims to explain both the dynamics of adaptation of individual animals over time, and the quantitative and qualitative differences in behaviour between subjects, by modelling their behaviour as arising from model-based planning in a Bayes adaptive Markov Decision Process (BAMDP) framework, in which the subjects maintain and update probabilistic estimates of the uncertain hazard presented by the object, and rationally balance the potential reward from exploring the object with the potential risk of predation it presents.
In order to fit these complex models to the behaviour the authors necessarily make substantial simplifying assumptions, including coarse-graining the exploratory behaviour into phases quantified by a set of summary statistics related to the approach bouts of the animal. Inter-individual variation between subjects is modelled both by differences in their prior beliefs about the possible hazard presented by the object, and by differences in their risk preference, modelled using a conditional value at risk (CVaR) objective, which focuses the subject's evaluation on different quantiles of the expected distribution of outcomes. Interestingly, these two conceptually different possible sources of inter-subject variation in brave vs timid exploratory behaviour turn out not to be dissociable in the current dataset as they can largely compensate for each other in their effects on the measured behaviour. Nonetheless, the modelling captures a wide range of quantitative and qualitative differences between subjects in the dynamics of how they explore the object, essentially through differences in how subject's beliefs about the potential risk and reward presented by the object evolve over the course of exploration, and are combined to drive behaviour.
Exploration in the face of risk is a ubiquitous feature of the decision-making problem faced by organisms, with strong clinical relevance, yet remains poorly understood and under-studied, making this work a timely and welcome addition to the literature.
Strengths:
- Individual differences in exploratory behaviour are an interesting, important, and under-studied topic.
- Application of cutting-edge modelling methods to a rich behavioural dataset, successfully accounting for diverse qualitative and qualitative features of the data in a normative framework.
- Thoughtful discussion of the results in the context of prior literature.
Limitations:
- The model-fitting approach used of coarse-graining the behaviour into phases and fitting to their summary statistics may not be applicable to exploratory behaviours in more complex environments where coarse-graining is less straightforward.
Comments on revisions:
All recommendations to authors from the first review were addressed in the revised manuscript.
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
This work computationally characterized the threat-reward learning behavior of mice in a recent study (Akiti et al.), which had prominent individual differences. The authors constructed a Bayes-adaptive Markov decision process model and fitted the behavioral data by the model. The model assumed (i) hazard function starting from a prior (with free mean and SD parameters) and updated in a Bayesian manner through experience (actually no real threat or reward was given in the experiment), (ii) risk-sensitive evaluation of future outcomes (calculating lower 𝛼 quantile of outcomes with free 𝛼 parameter), and (iii) heuristic exploration bonus. The authors found that (i) brave animals had more widespread hazard priors than timid animals and thereby quickly learned that there was in fact little real threat, (ii) brave animals may also be less risk-aversive than timid animals in future outcome evaluation, and (iii) the exploration bonus could explain the observed behavioral features, including the transition of behavior from the peak to steady-state frequency of bout. Overall, this work is a novel interesting analysis of threat-reward learning, and provides useful insights for future experimental and theoretical work. However, there are several issues that I think need to be addressed.
Strengths:
(1) This work provides a normative Bayesian account for individual differences in braveness/timidity in reward-threat learning behavior, which complements the analysis by Akiti et al. based on model-free threat reinforcement learning.
(2) Specifically, the individual differences were characterized by (i) the difference in the variance of hazard prior and potentially also (ii) the difference in the risk-sensitivity in the evaluation of future returns.
Weakness:
(1) Theoretically the effect of prior is diluted over experience whereas the effect of biased (risk-aversive) evaluation persists, but these two effects could not be teased apart in the fitting analysis of the current data.
(2) It is currently unclear how (whether) the proposed model corresponds to neurobiological ( rather than behavioral) findings, different from the analysis by Akiti et al.
We thank reviewer #1 for their useful feedback which we’ve used to improve the discussion, formatting and clarity of the paper, and for highlighting important questions for future extensions of our work.
Major points:
(1) Line 219
It was assumed that the exploration bonus was replenished at a steady rate when the animal was at the nest. An alternative way would be assuming that the exploration bonus slowly degraded over time or experience, and if doing so, there appears to be a possibility that the transition of the bout rate from peak to steady-state could be at least partially explained by such a decrease in the exploration bonus.
Section 2.2.3 explains the mechanism of the exploration bonus which motivates approach. We think that the mechanism suggested by the reviewer is, in essence, what is happening in the model. The exploration pool is indeed depleted over time or bouts of experience at the object. In the peak confident phase for brave animals and the peak cautious phase for timid animals, the rate of depletion exceeds the rate of regeneration, since the agent spends only a single turn at the nest between bouts. In the steady-state phase, the exploration pool has depleted so much previously that the agent must wait multiple turns at the nest for the pool to regenerate to a sufficiently high value to justify approaching the object again.
We have updated section 2.2.3 to explain that agents spend one turn at the nest during peak phase but multiple turns during steady-state phase. Hopefully, this makes our mechanism clear:
“In simulations, when 𝐺(𝑡) is high, the agent has a high motivation to explore the object, spending only a single turn in the nest state between bouts. In other words, the depletion from 𝐺0 substantially influences the time point at which approach makes a transition from peak to steady-state; the steady-state time then depends on the dynamics of depletion (when at the object) and replenishment (when at the nest). In particular, in the steady-state phases, the agent must wait multiple turns at the nest for 𝐺(𝑡) to regenerate so that informational reward once again exceeds the potential cost of hazard.“
(2) Line 237- (Section 2.2.6, 2.2.7, Figures 7, 9)
I was confused by the descriptions about nCVaR. I looked at the cited original literature Gagne & Dayan 2022, and understood that nCVaR is a risk-sensitive version of expected future returns (equation 4) with parameter α (α-bar) (ranging from 0 to 1) representing risk preference. Line 269-271 and Section 4.2 of the present manuscript described (in my understanding) that α was a parameter of the model. Then, isn't it more natural to report estimated values of α, rather than nCVaR, for individual animals in Section 2.2.6, 2.2.7, Figures 7, 9 (even though nCVaR monotonically depends on α)? In Figures 7 and 9, nCVaR appears to be upper-bounded to 1. The upper limit of α is 1 by definition, but I have no idea why nCVaR was also bounded by 1. So I would like to ask the authors to add more detailed explanations on nCVaR. Currently, CVaR is explained in Lines 237-243, but actually, there is no explanation about nCVaR rather than its formal name 'nested conditional value at risk' in Line 237.
Thank you for pointing out this error. We have corrected the paper to use nCVaR to refer to the objective and nCVaR's α, or sometimes just α, to refer to the risk sensitivity parameter and thus the degree of risk sensitivity.
(3) Line 333 (and Abstract)
Given that animals' behaviors could be equally well fitted by the model having both nCVaR ( free α) and hazard prior and the alternative model having only hazard prior (with α = 1), may it be difficult to confidently claim that brave (/timid) animals had risk-neutral (/risk-aversive) preference in addition to widespread (/low-variance) hazard prior? Then, it might be good to somewhat weaken the corresponding expression in the Abstract (e.g., add 'potentially also' to the result for risk sensitivity) or mention the inseparability of risk sensitivity and prior belief pessimism (e.g., "... although risk sensitivity and prior belief pessimism could not be teased apart").
Thank you for this suggestion, we have duly weakened the wording in the Abstract to say “potentially more risk neutral”:
“Some animals begin with cautious exploration, and quickly transition to confident approach to maximize exploration for reward; we classify them as potentially more risk neutral, and enjoying a flexible hazard prior. By contrast, other animals only ever approach in a cautious manner and display a form of self-censoring; they are characterized by potential risk aversion and high and inflexible hazard priors.”
Reviewer #2 (Public Review):
Shen and Dayan build a Bayes adaptive Markov decision process model with three key components: an adaptive hazard function capturing potential predation, an intrinsic reward function providing the urge to explore, and a conditional value at risk (CvaR, closely related to probability distortion explanations of risk traits). The model itself is very interesting and has many strengths including considering different sources of risk preference in generating behavior under uncertainty. I think this model will be useful to consider for those studying approach/avoid behaviors in dynamic contexts.
The authors argue that the model explains behavior in a very simple and unconstrained behavioral task in which animals are shown novel objects and retreat from them in various manners (different body postures and patterns of motor chunks/syllables). The model itself does capture lots of the key mouse behavioral variability (at least on average on a mouse-by-mouse basis) which is interesting and potentially useful. However, the variables in the model - and the internal states it implies the mice have during the behavior - are relatively unconstrained given the wide range of explanations one can offer for the mouse behavior in the original study (Akiti et al). This reviewer commends the authors on an original and innovative expansion of existing models of animal behaviour, but recommends that the authors revise their study to reflect the obvious challenges . I would also recommend a reduction in claiming that this exercise gives a normative-like or at least quantitative account of mental disorders.
We thank reviewer #2 for highlighting some of the strengths of our paper as well as pointing out important limitations of Akiti et al’s original study which we’ve inherited as well as some limitations of our own method. We address their concerns below.
We have added a paragraph to the discussion discussing the limitations of the state representation we adopted from Akiti’s study.
(Reviewer #1 had the same concern, see above) “Motivated by tail-behind versus tail-exposed in Akiti et al. (2022), we model approach using a dichotomy between cautious and confident approach states [...]”
We have reduced the suggestion that our model provides an account of mental disorders in the abstract.
Before:
“On the other hand, “timid” animals, characterized by risk aversion and high and inflexible hazard priors, display self-censoring that leads to the sort of asymptotic maladaptive behavior that is often associated with psychiatric illnesses such as anxiety and depression.”
After:
“By contrast, other animals only ever approach in a cautious manner and display a form of self-censoring; they are characterized by potential risk aversion and high and inflexible hazard priors. “
My main comment is that this paper is a very nice model creation that can characterize the heterogeneity rodent behavior in a very simple approach/avoid context (Akiti et al; when a novel object is placed in an arena) that itself can be interpreted in a multitude of ways. The use of terms like "exploration", "brave", etc in this context is tricky because the task does not allow the original authors (Akiti et al) to quantify these "internal states" or "traits" with the appropriate level of quantitative detail to say whether this model is correct or not in capturing the internal states that result in the rodent behavior. That said, the original behavioral setup is so simple that one could imagine capturing the behavioral variability in multiple ways ( potentially without evoking complex computations that the original authors never showed the mouse brain performs). I would recommend reframing the paper as a new model that proposes a set of internal states that could give rise to the behavioral heterogeneity observed in Akiti et al, but nonetheless is at this time only a hypothesis. Furthermore, an explanation of what would be really required to test this would be appreciated to make the point clearer.
We thought very hard about using terms that might be considered to be anthropomorphic such as ‘timid’ and ‘brave’. We are, of course, aware, of the concerns articulated by investigators such as LeDoux about this. However, we think that, provided that we are clear on the first appearance (using ‘scare’ quotes) that we are using them as indeed labels for latent characteristics that capture correlations in various aspects of behaviour, they are more helpful than harmful in making our descriptions understandable.
Reviewer #3 (Public Review):
Summary:
The manuscript presents computational modelling of the behaviour of mice during encounters with novel and familiar objects, originally reported by Akiti et al. (Neuron 110, 2022) . Mice typically perform short bouts of approach followed by a retreat to a safe distance, presumably to balance exploration to discover possible rewards with the potential risk of predation. However, there is considerable heterogeneity in this exploratory behaviour, both across time as an individual subject becomes more confident in approaching the object, and across subjects; with some mice rapidly becoming confident to closely explore the object, while other timid mice never become fully confident that the object is safe. The current work aims to explain both the dynamics of adaptation of individual animals over time, and the quantitative and qualitative differences in behaviour between subjects, by modelling their behaviour as arising from model-based planning in a Bayes adaptive Markov Decision Process (BAMDP) framework, in which the subjects maintain and update probabilistic estimates of the uncertain hazard presented by the object, and rationally balance the potential reward from exploring the object with the potential risk of predation it presents.
In order to fit these complex models to the behaviour the authors necessarily make substantial simplifying assumptions, including coarse-graining the exploratory behaviour into phases quantified by a set of summary statistics related to the approach bouts of the animal. Inter-individual variation between subjects is modelled both by differences in their prior beliefs about the possible hazard presented by the object and by differences in their risk preference, modelled using a conditional value at risk (CVaR) objective, which focuses the subject's evaluation on different quantiles of the expected distribution of outcomes. Interestingly these two conceptually different possible sources of inter-subject variation in brave vs timid exploratory behaviour turn out not to be dissociable in the current dataset as they can largely compensate for each other in their effects on the measured behaviour. Nonetheless, the modelling captures a wide range of quantitative and qualitative differences between subjects in the dynamics of how they explore the object, essentially through differences in how subject's beliefs about the potential risk and reward presented by the object evolve over the course of exploration, and are combined to drive behaviour.
Exploration in the face of risk is a ubiquitous feature of the decision-making problem faced by organisms, with strong clinical relevance, yet remains poorly understood and under-studied, making this work a timely and welcome addition to the literature.
Strengths:
(1) Individual differences in exploratory behaviour are an interesting, important, and under-studied topic.
(2) Application of cutting-edge modelling methods to a rich behavioural dataset, successfully accounting for diverse qualitative and qualitative features of the data in a normative framework.
(3) Thoughtful discussion of the results in the context of prior literature.
Limitations:
(1) The model-fitting approach used of coarse-graining the behaviour into phases and fitting to their summary statistics may not be applicable to exploratory behaviours in more complex environments where coarse-graining is less straightforward.
(2) Some aspects of the work could be more usefully clarified within the manuscript.
We thank reviewer #3 for their positive feedback and helping us to improve the clarity of our paper. We have added discussion they thought was missing.
Reviewer #1 (Recommendations for the authors):
(1) Line 25-28
This part of the Abstract might give an impression that timidity (but not braveness) is potentially associated with psychiatric illness and even that timidity is thus inferior to braveness. However, even though extreme timidity might indeed be associated with anxiety or depression, extreme braveness could also be associated with other psychiatric or behavioral problems. Moreover, as a population, the existence of both timid and brave individuals could be advantageous, and it could be a reason why both types of individuals evolutionarily survived in the case of wild animals (although Akiti et al. used mice, which may have no or very limited genetic varieties, and so things may be different). So I would like to encourage the authors to elaborate on the expression of this part of the Abstract and/or enrich the related discussion in the Discussion.
This is an important point. We note on line 38 that excessive novelty seeking (potentially caused by excessive braveness) could also be maladaptive.
Additionally, we have added a paragraph to the discussion discussing heterogeneity in risk sensitivity within a population.
“Our data show that there is substantial variation in the degrees of risk sensitivity across the mice. Previous works have reported substantial interpopulation and intrapopulation differences in risk-sensitivity in humans which depend on gender, age, socioeconomic status, personality characteristics, wealth and culture (Rieger et al., 2015; Frey et al., 2017). Despite the normative appeal of 𝛼 = 1, it is possible that a population may benefit from including individuals with $\alpha$ different from 1.0 or highly negative priors. For example, more cautious individuals could learn from merely observing the risky behavior of less cautious individuals. Furthermore, we have only considered risk-sensitivity under epistemic uncertainty in our work. Risk averse individuals, for instance with 𝛼 < 1 may be more successful than risk-neutral agents in environments where there are unexpected dangers ( unknown unknowns). Risk-aversion is thus a temperament of ecological and evolutionary significance (Réale et al., 2007).”
(2) Line 149
Section 2.2 consists of eight subsections. I think this organization may not be very appealing, because there are a bit too many subsections, and their relations are not immediately clear to readers. So I would like to encourage the authors to make an elaboration. For example, since 2.2.1 - 2.2.5 describes a summary of model construction and model fitting whereas 2.2.6-2.2.8 shows the results, it could be good to divide these into separate sections (2.2.1 - 2.2.5 and 2.3.1 - 2.3.3).
Thank you for pointing this out. We’ve renumbered the sections as you’ve suggested.
(3) Line 347-8
Theoretically, the effect of prior is diluted over experience whereas the effect of biased (risk-aversive) evaluation persists, as the authors mentioned in Lines 393-394. Then isn't it possible to consider environments/conditions in which the two effects can be separated?
We appreciate this suggestion. Indeed, our original thought in modeling this experiment was that this would be exactly the case here - with epistemic uncertainty reducing as the object became more familiar. However, proving to an animal that a single environment is completely stationary/fixed is hard - reflected in our conclusion here that the exploration bonus pool replenishes. Thus, we argued in the discussion that a series of environments would be necessary to separate risk sensitivity from priors.
(4) Line 407
It would be nice to add a brief phrase explaining how (in what sense) this model's assumption was consistent with the reported behavior. Also, should the assumption of having two discrete approach states (cautious and confident) itself be regarded as a limitation of the model? If the tail-behind and tail-exposure approaches were not merely operationally categorized but were indicated to be two qualitatively distinct behaviors in the experiment by Akiti et al., it is reasonable to model them as two discrete states, but otherwise, the assumption of two discrete states would need to be mentioned as a simplification/limitation.
We have now removed line 407, and now have an additional paragraph in the discussion discussing the limitations of the tail-behind and tail-exposure state representation: “Motivated by tail-behind versus tail-exposed in Akiti et al. (2022), we model approach using a dichotomy between cautious and confident approach states. This is likely a crude approximation to the continuous and multifaceted nature of animal approach behavior. For example, during approach animals likely adjust their levels of vigilance continuously (or discretely; Lloyd and Dayan (2018)) to monitor threat, and choose different velocities for movement, and different attentional strategies for inspecting the novel object. We hope future works will model these additional behavioral complexities, perhaps with additional internal states, and corroborate these states with neurobiological data.”
(5) Line 418
The authors contrasted their model-based analyses with the model-free analyses of Akiti et al. Another aspect of differences between the authors' model and the model of Akiti et al. is whether it is normative or mechanistic: while how the model of Akiti et al. can be biologically implemented appears to be clear (TS dopamine represents threat TD error, and TS dopamine-dependent cortico-striatal plasticity implements TD error-based update of model-free threat prediction), biological implementation of the authors' model seems more elusive. Given this, it might be a fruitful direction to explore how these two models can be integrated in the future.
We enthusiastically agree that it would be most interesting in the future to explore the integration of the two models - and, in the discussion ( Lines 537-548, 454-461) , point to some first steps that might be fruitful along these lines. There are two separate considerations here: one is that our account is mostly computational and algorithmic, whereas Akiti’s model is mostly algorithmic and implementational; the second is, as noted by the reviewer, that our account is model-based, whereas Akiti’s model is model-free (in the sense of reinforcement learning; RL). These are related - thanks in no small part to the work from the group including Akiti, we know a lot more about the implementation of model-free than model-based RL. However, our model-based account does reach additional features of behavior not captured in Akiti et al.’s model such as bout duration, frequency, and approach type. Thus, the temptation of unification.
(6) Line 426
Related to the previous point, it would be nice to more specifically describe what variable TS dopamine can represent in the authors' model if possible.
In the discussion (Lines 454-461) , we speculate that TS dopamine could still respond to the physical salience of the novel object and affect choices by determining the potential cost of the encountered threat or the prior on the hazard function. For example, perhaps ablating TS dopamine reduces the hazard priors which leads to faster transition from cautious to confident approach and longer bout durations, consistent with the optogenetics behavioral data reported in Akiti et al.
Reviewer #2 (Recommendations for the authors):
My guess is simpler versions of the model would not fit the data well. But this does not mean for example that the mice have probability distortions (CvaR) or that even probabilistic reasoning and the internal models necessary to support them are acting in the behavioral context studied by Akiti. So related to the above, I would ask what other models would fit and would not fit the data? And what does this mean?
These are good points. Our model provides an approximately normative account of the animals’ behavior in terms of what it achieves relative to a utility function. In practice, the animals could deploy a precompiled model-free policy (which does not rely on probabilistic computations) that is exactly equivalent to our model-based policy. With the current experiment, we cannot conclude whether or not the animals are performing the prospective calculations in an online manner. Of course, the extent to which animals or humans are performing probabilistic computations online and have internal models are on-going questions of study.
Model comparison is difficult because currently we do not know of any other risk-sensitive exploration models. We cannot directly compare to the model in Akiti et al. since our model explains additional features of behavior: bout duration, frequency, and approach type. Indeed, our model is as simple as it can be in the sense with the exception of nCVaR, removing any of the other parameters makes it difficult to fit some animals in our dataset. In the future, our model could be used to fit other datasets of risk-sensitive exploration and, ideally, be compared to other models.
Explaining why animals avoid the novel object in what the offers call benign environment is a very tricky issue. In Akiti et al, the readers are not yet convinced that the mice know that this environment is benign. Being placed in an arena with a novel object presents mice with a great uncertainty and we do not know whether they treat this as benign. Therefore, the alternative explanations in this study need to be carefully discussed in lieu of the limitations of the initial study.
It is certainly true that it is unclear if the arena is completely benign to the animals. However, the amount of time the animal spends in the center of the arena decreases significantly from habituation to novelty days. This suggests that the animals avoid the novel object largely because of the object itself, rather than the potential danger associated with the arena. Furthermore, the animals are not reported as exhibiting more extreme behaviours such as freezing. In any case, our account is relative in the sense that we are comparing the time the animal spends at the object versus elsewhere in the environment, driven by the relative novelty and relative risk of the environment versus the object. Trying to get more absolute measures of these quantities would require a richer experimental set-up, for instance with different degree of habituation or experience of the occurrence of (other) novel objects, in general.
We added a short note to the discussion to explain this:
“Fourth, we modeled the relative amount of time the animal spends at the object versus elsewhere in the environment which depends on the differential risk in the two states. However, it is likely the animals avoid the novel object largely because of the object itself, rather than the potential danger associated with the arena since they spend much less time at the center of the arena during novelty than habituation days.”
Figure 2 - how confident are the authors that each mouse differs from y=1? Related to this, the behavior in Akiti is very noisy and changes across time. I am not sure if the authors fully describe at what levels their model captures the behavior vs not in a detailed enough fashion.
We have performed a random permutation test on the minute-to-minute data. We have updated Figure 2 so that brave animals that pass the Benjamini–Hochberg procedure y>1 at level q=0.05 are represented with solid green dots and animals that don’t pass are represented with hollow dots. 8 out of 11 brave animals passed Benjamini–Hochberg.
Reviewer #3 (Recommendations for the authors):
(1) I could not find information in the preprint about code availability. Please consider making the code public to help others apply these modelling methods.
We have released code and included the url in the paper in the Methods section.
(2) Though the manuscript was generally clearly written, there were a number of places where some additional information or clarification would be useful:
a) Please define and explain the terms 'tail-behind' and 'tail-exposed' (used to describe approach bout types) when first used.
We have added definitions when we first mention these terms:
“[...] 'tail-behind' (bouts where the animal's nose was closer to the object than the tail for the entire bout) and 'tail-exposed' (bouts where the animal's tail is closer to the object than the nose at some point during the bout), associated respectively with cautious risk-assessment and engagement”
b) At lines 57-58 when contrasting the 'model-free' account of Akiti et al with the 'model-based' account of the current work, it would be worth clarifying that these terms are being used in the RL sense rather than e.g. a model-based analysis of the data.
We have updated the relevant lines to say “model-free/based reinforcement learning”.
c) Line 61, the phrase 'the significant long-run approach of timid animals despite having reached the "avoid" state' is unclear as the 'avoid' state has not been defined.
We updated the terminology to “avoidance behavior” to be consistent with Akiti et al. Avoidance refers to the animal routinely avoiding the object and therefore being unable to learn whether it is safe.
d) It was not completely clear to me how the coarse-graining of the behaviour was implemented. Specifically, how were animals assigned to the brave, intermediate, or timid group, and how were the parameters of the resulting behavioural phases fit?
Sorry that this was not clear. Section 2.1 explains how the minute-to-minute behavioral data was coarse-grained and how animal groups were assigned. We have added further explanation of Figure 2 to the main text:
“Fig 2 summarizes our categorization of the animals into the three groups: brave, intermediate, and timid based on the phases identified in the animal's exploratory trajectories. Timid animals spend no time in confident approach and are plotted in orange at the origin of Fig 2. Brave animals differ from intermediate animals in that their approach time during the first ten minutes of the confident phase is greater than the last ten minutes ( steady-state phase). Brave animals are plotted in green above and intermediate animals are plotted in black below the y=1 line in Fig 2.”
We also added extra information to outline the goal, and methodology of coarse-graining and animal grouping:
“We sought to capture these qualitative differences (cautious versus confident) as well as aspects of the quantitative changes in bout durations and frequencies as the animal learns about their environment. To make this readily possible, we abstracted the data in two ways:
averaging bout statistics over time, and clustering the animals into three groups with operationally distinct behaviors.”
e) What purpose does the 'retreat' state serve in the BAMDP model (as opposed to transitioning directly from 'object' to 'nest' states), and why do subjects not pass through it following 'detect' states?
Thank you for pointing this out. We have updated Figure 3 to note that the two “detected states” also point to the “retreat” state. The reviewer is correct that there could be alternative versions of the state diagram, and the ‘retreat’ state could indeed have been eliminated. However, we thought that it was helpful to structure the animal’s progress through state space.
f) Why was the hazard function parameterised via the mean and SD at each time step rather than with a parametric form of the mean and SD as a function of time?
Since the agent can only spend 2, 3, or 4 turns at the object states, we didn’t see a need to parameterize the mean and SD as a function of time. Doing so is a good solution to scaling up the hazard function to more time-steps.
(3) There were also a couple of points that could potentially be usefully touched on in the discussion:
a) What, if any, is the relationship between the CVaR objective and distributional RL? They seem potentially related due to both focussing on quantiles of the outcome distribution.
We have added a paragraph to the discussion discussing the connection between distributional RL and CVaR:
“CVaR is known to come in different flavors in the case of temporally-extended behavior. Gagne and Dayan (2021) introduces two alternative time-consistent formulations of CVaR: nested CVaR (nCVaR) and precommitted CVaR (pCVaR). nCVaR and pCVaR both enjoy Bellman equations which make it possible to compute approximately optimal policies without directly computing whole distributions of the outcomes. We use nCVaR in this study for its computational efficiency. There is, of course, great current interest in distributional reinforcement learning (Bellemare et al., 2023b) which does acquire such whole distributions, not the least because of prominent observations linking non-linearities in the response functions of dopamine neurons to methods for learning distributions of outcomes ( Dabney et al., 2020; Masset et al., 2023; Sousa et al., 2023). One functional motivation for considering entire outcome distributions is the possibility of using them to determine risk-sensitive policies (Gagne and Dayan, 2021).
While it is possible to compute CVaR directly from return distributions, Gagne and Dayan (2021) showed that this can lead to temporally inconsistent policies where the agent deviates from its original plans (the authors called this the fixed CVaR or fCVaR measure).
Rather further removed from our model-based methods is work from Antonov and Dayan (2023), who consider a model-free exploration strategy which exploits full return distributions to compute the value of perfect information which is used as a heuristic for trying actions with uncertain consequences. Future works can examine risk-sensitive versions of Antonov and Dayan (2023)'s computationally efficient model-free algorithm as one solution to the burdensome computations in our model-based method.”
b) Why normatively might subjects have non-neutral risk preference as captured by the CvaR?
We also added a paragraph to the discussion discussing the advantage of heterogeneity in risk sensitivity within a population:
(Reviewer #1 had the same question, see above) “Our data show that there is substantial variation in the degrees of risk sensitivity across the mice. Previous works have reported substantial interpopulation and intrapopulation differences in risk-sensitivity in humans which depend on gender, age, socioeconomic status, personality characteristics, wealth and culture [...]”
c) Relevance of the current modelling work to clinical conditions characterised by dysregulation of risk assesment (e.g. anxiety or PTSD).
We’ve added a paragraph to the discussion:
“Inter-individual differences in risk sensitivity are also of critical importance in psychiatry, reflected in a panoply of anxiety disorders (Butler and Mathews, 1983; Giorgetta et al., 2012; Maner et al., 2007; Charpentier et al., 2017), along with worry and rumination (Gagne and Dayan, 2022). Understanding the spectrum of extreme priors and extreme values of 𝛼 could have therapeutic implications, adding significance to the search for tasks that can more cleanly separate them.”
d) Is it surprising to see differences in risk preference (nCVaR) between the familiar object and novel object condition, given that risk preference might be conceptualised as a trait rather than a state variable?
Thank you for raising this point. You are right that we expected risk sensitivity (nCVaR alpha) to be the same between FONC and UONC animals on average. It is difficult to know if alpha is higher for FONC than UONC animals due to the non-identifiability between alpha and hazard priors. We have added this discussion to the paper:
“This is surprising if we interpret 𝛼 as a trait that is stable through time. Unfortunately, due to the non-identifiability between 𝛼 and hazard priors, we cannot verify whether 𝛼 is actually higher for FONC animals than UONC animals.”
Briefing : Les Violences Éducatives Ordinaires (VEO)
Résumé
Ce document de synthèse analyse les Violences Éducatives Ordinaires (VEO) en s'appuyant sur l'expertise de professionnels de l'enfance.
Il met en lumière le contexte historique, la définition, les impacts neuroscientifiques et les défis sociétaux liés à ces pratiques.
Les VEO, héritage d'une histoire millénaire de domination patriarcale, englobent non seulement les violences physiques (gifles, fessées) mais aussi des formes psychologiques et verbales (humiliations, chantage, cris) qui sont banalisées et profondément ancrées dans les schémas éducatifs.
La législation française n'a que très récemment, en 2019, interdit explicitement ces pratiques, marquant une rupture avec un passé où le "droit de correction" était légitimé.
L'impact des VEO sur l'enfant est désormais documenté par les neurosciences : loin de favoriser l'obéissance, le stress généré active les circuits cérébraux de la peur, inhibant les capacités de raisonnement et de coopération.
Cela compromet le "méta-besoin" fondamental de sécurité de l'enfant, essentiel à son développement.
Les parents actuels se trouvent dans une "période de transition éducative" complexe, cherchant à abandonner des modèles transmis sur des générations.
Il est crucial de distinguer l'éducation sans violence du laxisme : l'enjeu est de poser un cadre clair, prévisible et contenant, tout en instaurant un dialogue basé sur la confiance et le respect.
Ce document détaille ces concepts et recense les ressources disponibles pour accompagner les familles dans cette transition.
I. Contexte Historique et Sociétal : De la Domination aux Droits de l'Enfant
La notion de violences éducatives ordinaires est intrinsèquement liée à une longue histoire de domination et à l'évolution du statut de l'enfant dans la société.
A. L'Héritage Patriarcal
• Antiquité Romaine : Le concept du pater familias donnait au chef de famille un pouvoir absolu, y compris un droit de vie et de mort sur ses enfants et ses esclaves, afin de maintenir un ordre social fondé sur la domination.
• Code Civil Napoléonien (1804) : Cet héritage a été formalisé dans la loi française, qui a réaffirmé la "puissance paternelle" et le "droit de correction" du père sur ses enfants.
L'article 375 permettait même au père de faire enfermer sa progéniture au titre de la correction. Bien que datant de plus de deux siècles, ce code constitue encore la base du droit civil actuel.
• XIXe et début du XXe siècle : Le père conservait un pouvoir coercitif majeur, pouvant décider de l'enfermement d'un enfant jugé "rebelle" ou désobéissant dans des "maisons de correction" ou des "colonies pénitentiaires agricoles" (comme celle de Mettray en Indre-et-Loire), qui s'apparentaient davantage à des bagnes qu'à des lieux d'éducation.
B. L'Émergence Lente des Droits de l'Enfant
Le XXe siècle a vu une évolution progressive de la perception de l'enfant, qui passe d'un objet de correction à un sujet de droits.
• 1935 : Abolition de la "correction paternelle", mettant fin au droit d'enfermement parental.
• 1945 : L'ordonnance de 1945, dans le contexte de l'après-guerre, crée les juges pour enfants et pose les fondements d'une justice moderne pour les mineurs, axée sur la protection et l'éducation plutôt que sur la seule coercition.
• 1970 : La "puissance paternelle" est définitivement abolie et remplacée par l'autorité parentale, qui instaure des droits et devoirs égaux entre la mère et le père. C'est une étape majeure mais le droit de correction reste toléré dans la pratique.
En 1982, un juge en cour d'appel pouvait encore statuer que fessées et coups de règle ne constituaient pas une "brutalité excessive" s'ils ne laissaient pas de traces.
• 1989 : La Convention Internationale des Droits de l'Enfant (CIDE), ratifiée par la France en 1990, reconnaît enfin l'enfant comme un sujet de droit à part entière, devant être protégé de toute forme de violence.
C. La Loi de 2019 : Une Reconnaissance Tardive
Malgré la CIDE, la France a mis près de 30 ans à légiférer spécifiquement sur les VEO.
• 2015 : La France est condamnée par le Conseil de l'Europe pour l'absence d'une loi "suffisamment claire" interdisant les châtiments corporels.
• Juillet 2019 : Adoption de la loi, souvent surnommée péjorativement "loi anti-fessée". Cette loi, proposée par la députée Maud Petit, a fait l'objet de nombreuses moqueries et d'une forte résistance, illustrée par l'argument "j'ai pris des claques et je n'en suis pas mort".
• Contenu de la loi : Elle stipule de manière concise que "l'autorité parentale s'exerce sans violence physique ou psychologique".
L'introduction de la notion de violence psychologique est une avancée fondamentale, car elle reconnaît les impacts invisibles mais profonds de certaines pratiques éducatives.
II. Définition et Formes des Violences Éducatives Ordinaires (VEO)
Les VEO sont définies comme des pratiques punitives et coercitives, banalisées et courantes ("ordinaires"), utilisées au nom de l'éducation mais qui n'ont aucune valeur éducative et portent atteinte à la dignité et à l'intégrité de l'enfant.
Elles se classifient en trois grandes catégories.
Type de Violence Exemples Concrets Citées Violence Physique Gifles, fessées, tapes sur les mains, tirage de cheveux, pincements, secousses, jet d'objets, destruction de jouets, privation de nourriture, isolement forcé dans une pièce. Violence Psychologique Menaces ("tu vas voir..."), culpabilisation, chantage affectif, éducation par la peur, indifférence (ignorer l'enfant, notamment quand il pleure), créer un climat d'insécurité. Violence Verbale Humiliations, insultes, cris, dévalorisation ("tu es nul", "tu n'y arriveras jamais"), comparaisons (entre frères et sœurs ou avec d'autres enfants), moqueries.
Ces pratiques sont souvent des réactions automatiques de l'adulte face à un sentiment de débordement ou d'impuissance, et peuvent être la reproduction de schémas éducatifs subis durant sa propre enfance.
III. L'État des Lieux Actuel et la Perception Sociétale
Une enquête IFOP réalisée pour la Fondation pour l'enfance en 2024 révèle une évolution contrastée des mentalités depuis la loi de 2019. • Baisse des violences physiques : La loi "anti-fessée" semble avoir eu un impact positif, avec une diminution déclarée du recours aux châtiments corporels.
• Stagnation des violences psychologiques : Les violences psychologiques et verbales peinent à diminuer, voire augmentent pour certaines.
Cela traduit une difficulté à prendre conscience de la portée de ces actes et à modifier des modèles de communication profondément ancrés.
• Résistance parentale : Une part significative des parents interrogés exprime encore une réticence face à la loi, la percevant comme une ingérence de l'État dans la sphère privée ("de quoi se mêle l'État").
Cet argument de "l'intimité familiale" a historiquement freiné l'avancée de la législation.
IV. L'Impact des VEO sur le Développement de l'Enfant
Les neurosciences permettent de comprendre pourquoi les VEO sont non seulement néfastes, mais aussi contre-productives.
A. La Réponse Cérébrale à la Peur
Face à un adulte perçu comme menaçant (cris, gestes brusques), le cerveau de l'enfant active un mécanisme de survie.
1. Perception d'un danger : L'adulte devient une source de frayeur.
2. Court-circuit du raisonnement : Le signal de peur est traité directement par les zones archaïques du cerveau (système limbique), qui gèrent les émotions et le danger, en contournant le cortex préfrontal, siège de la réflexion et de l'apprentissage.
3. Réactions instinctives : Le cerveau déclenche l'une des trois réponses primaires au danger :
◦ L'attaque (Fight) : Rare envers un parent.
◦ La fuite (Flight) : Évitement.
◦ La sidération (Freeze) : L'enfant est "tétanisé", incapable d'agir ou de réagir.
C'est souvent interprété à tort par le parent comme de la provocation.
Ce processus s'accompagne d'une surproduction d'hormones de stress comme le cortisol, qui, en excès, est néfaste pour le développement cérébral.
B. L'Atteinte au Besoin Fondamental de Sécurité
Le "méta-besoin" de sécurité est le pilier du développement de l'enfant, comme l'a reconnu la loi de protection de l'enfance de mars 2016.
Ce besoin inclut :
• Les besoins physiologiques (sommeil, alimentation).
• La nécessité de relations affectives stables et prévisibles.
Les VEO créent une insécurité fondamentale : l'enfant perd confiance en son environnement et en les figures qui sont censées le protéger.
C. Les Risques à Long Terme
Bien qu'il n'y ait pas de causalité automatique, un enfant exposé de manière répétée aux VEO présente une vulnérabilité accrue à :
• Des difficultés scolaires et d'apprentissage : Un enfant en état d'alerte permanent n'est pas disponible pour apprendre.
• Une faible estime de soi : Les messages dévalorisants sont intériorisés.
• La reproduction des schémas de violence : Il peut devenir lui-même auteur de violences ou se retrouver en situation de victime à l'âge adulte (auto-maltraitance ou maltraitance subie).
V. Le Défi de la "Transition Éducative" pour les Parents
Les parents d'aujourd'hui sont à la charnière de deux modèles, ce qui crée une période de "transition éducative".
• Sortir de la reproduction : La plupart des adultes ont été éduqués avec des VEO. En situation de stress, la tendance est de reproduire inconsciemment ces schémas. Prendre conscience de cela est la première étape du changement.
• Réparer la relation : Il n'est jamais trop tard pour revenir sur un incident. Reconnaître son erreur devant l'enfant ("ma réaction était disproportionnée"), lui expliquer le contexte sans se défausser, permet de restaurer le lien de confiance et de lui montrer un modèle de gestion de conflit non-violent.
• Éduquer sans violence n'est pas du laxisme : C'est une confusion fréquente. L'enfant a un besoin essentiel de cadre.
Ce cadre doit être :
◦ **Clair et prévisible** : Les règles sont connues et cohérentes.
◦ Contenant : Il offre une sécurité affective qui permet à l'enfant d'explorer le monde.
◦ Adapté et évolutif : Il change avec l'âge et les compétences de l'enfant, et peut être discuté, notamment avec un adolescent.
• Sanction vs. Punition : La sanction, si elle est proportionnée à l'acte et à l'âge, peut être éducative si elle n'a pas pour but d'humilier ou de dominer, mais de poser une limite et de permettre une réparation.
VI. Cas Pratiques et Lignes Directrices
• Obliger à embrasser un proche : Cette pratique est une VEO car elle ne respecte pas le corps et le consentement de l'enfant.
C'est une occasion manquée d'enseigner le droit de dire "non", une compétence cruciale pour la prévention des abus. Il est préférable de proposer des alternatives ("tu peux dire bonjour d'une autre façon").
• Forcer à manger ou à goûter : Le forçage alimentaire peut générer des troubles du comportement alimentaire.
L'utilisation de la nourriture comme chantage (notamment le sucre comme récompense) crée une relation malsaine à l'alimentation.
Le rôle du parent est de proposer une alimentation variée, mais l'enfant doit rester maître de ce qu'il ingère.
• Refus de soins (médicaments, brossage de dents) :
La santé de l'enfant n'est pas négociable, et l'adulte doit poser un cadre ferme. Cependant, l'approche est essentielle : expliquer l'importance du soin, rester calme et convaincu, et utiliser des stratégies ludiques pour obtenir l'adhésion plutôt que de recourir à la force.
VII. Ressources et Soutien Disponibles
Il est essentiel pour les parents de ne pas rester isolés face à leurs difficultés. De nombreuses structures, gratuites et confidentielles, existent pour offrir écoute et accompagnement.
• Protection Maternelle Infantile (PMI) : Pour les parents d'enfants de 0 à 6 ans, dans les Maisons Départementales de la Solidarité.
• Lieux d'Accueil Enfants-Parents (LAEP) : Espaces de rencontre et de jeu pour les parents et enfants de 0 à 3 ans.
• Maison des Adolescents : Lieu dédié aux jeunes et à leurs parents.
• Espace Santé Jeune : Ligne d'écoute et accueil pour les 7-25 ans.
• Espaces Parents : Nouveaux lieux d'accueil, d'écoute et d'activités pour tous les parents.
• Associations spécialisées : Comme Les Établis, qui proposent prévention, écoute et orientation.
• Ressources en ligne : Le site stopveo.fr (Violences Éducatives Ordinaires) offre des articles, vidéos et témoignages.
L'Orientation Scolaire : Parcours, Psychologie et Stratégies
Synthèse Exécutive
L'orientation scolaire est présentée non pas comme une série de décisions ponctuelles et anxiogènes, mais comme un cheminement progressif et continu, un parcours singulier qui se construit tout au long de la vie.
Les psychologues de l'Éducation Nationale (PsyEN) jouent un rôle central dans cet accompagnement, en s'appuyant sur leur expertise en psychologie du développement de l'enfant et de l'adolescent pour aider les jeunes et leurs familles à naviguer dans ce processus.
Les points critiques qui émergent sont :
• La primauté de l'épanouissement : La réussite scolaire et professionnelle est directement conditionnée par l'épanouissement du jeune dans la voie choisie.
Le choix doit être en phase avec sa manière d'apprendre (théorique, pratique, par projet) et ses centres d'intérêt.
• La déconstruction des mythes : Il n'existe pas de "métier idéal" unique ni de trajectoire linéaire.
La voie professionnelle n'est pas une impasse et peut mener à des études supérieures.
L'apprentissage est une modalité d'excellence accessible à tous les niveaux, du CAP au diplôme d'ingénieur.
• La notion de "maturité vocationnelle" : La capacité à se projeter dans un avenir professionnel est une compétence cognitive qui mûrit progressivement, atteignant un pic vers 18 ans.
Il est donc normal pour un adolescent de ne pas avoir de projet défini. Le rôle des adultes est de rassurer, d'encourager et d'ouvrir des horizons.
• L'importance des expériences multiples : La construction du projet d'orientation se nourrit autant des temps formels (stages, séances en classe) que des expériences informelles (discussions en famille, loisirs, voyages).
"Se tromper" n'est pas un échec, mais une expérience qui affine la connaissance de soi.
• Parcoursup comme outil : Loin d'être un juge, Parcoursup est une plateforme de mise en relation entre les candidats et les établissements d'enseignement supérieur, qui conservent leurs propres critères de recrutement.
C'est également une ressource d'information précieuse, utilisable bien avant la classe de Terminale pour explorer les possibilités via sa "carte des formations".
En somme, l'approche préconisée est de dédramatiser l'orientation en la considérant comme un processus d'exploration, où la connaissance de soi, la flexibilité et l'ouverture aux opportunités sont les clés d'un parcours réussi et épanouissant.
Le Rôle Central du Psychologue de l'Éducation Nationale (PsyEN)
De Conseiller à Psychologue : Une Approche Globale
Le métier a évolué de "conseiller d'orientation" à "psychologue de l'Éducation Nationale" pour refléter un changement fondamental d'approche.
L'accompagnement proposé dépasse le simple conseil pour s'ancrer dans une compréhension profonde de la psychologie de l'enfant et de l'adolescent.
• Fondements psychologiques : Les entretiens s'appuient sur la connaissance du développement psychosocial, cognitif et affectif du jeune.
• Objectif : Aider le jeune à construire son projet d'orientation et à faire des choix éclairés lors des moments clés de sa scolarité.
• Citation clé : Géraldine Mignier précise : "Ce sont toutes ces notions sur lesquelles nous on appuie nos entretiens pour travailler avec le jeune son projet d'orientation, l'aider à faire des choix au moment où il doit prendre des décisions."
• Formation : Les PsyEN sont des psychologues de formation ayant suivi une année de spécialisation supplémentaire pour exercer au sein de l'Éducation Nationale.
Deux Spécialités pour un Accompagnement Adapté
Les PsyEN se divisent en deux spécialités pour couvrir l'ensemble du parcours scolaire :
1. EDA (Éducation, Développement, Apprentissage) :
Interviennent dans le premier degré, de l'école maternelle au CM2.
2. EDO (Éducation, Développement et Conseil en Orientation Scolaire et Professionnelle) :
Interviennent dans l'enseignement secondaire (collège, lycée) et supérieur. Ce sont ces professionnels qui sont au cœur du sujet de l'orientation.
Mission : Rassurer, Accompagner et Servir de Médiateur
Le PsyEN agit souvent comme une tierce personne neutre entre le jeune et ses parents, surtout lorsque les désirs de l'un ne correspondent pas aux attentes des autres.
Leur rôle est d'apaiser les angoisses, d'expliquer le fonctionnement du système et de permettre des prises de décision plus sereines.
Ils reçoivent les élèves, mais aussi les parents, que ce soit au sein des établissements scolaires ou dans les Centres d'Information et d'Orientation (CIO).
Les Parcours d'Orientation : Un Paysage aux Multiples Voies
L'orientation n'est pas limitée aux classes de 3ème et de Terminale. Elle est jalonnée de plusieurs moments de choix tout au long de la scolarité.
Au-delà de la 3ème et de la Terminale : Les Premiers Paliers
La question de l'orientation peut se poser bien avant les grandes échéances connues :
• Dès la 6ème : Des orientations adaptées peuvent être envisagées (SEGPA, dispositif ULIS).
• En 4ème ou 3ème : Des choix vers l'enseignement agricole ou une 3ème "prépa-métiers" sont possibles pour découvrir le monde professionnel plus tôt.
Après la 3ème : Choisir sa Modalité d'Apprentissage
Le choix post-3ème doit avant tout se fonder sur la manière dont l'élève apprend et s'épanouit.
L'épanouissement conditionne la réussite.
Voie Description Profil d'Apprenant Débouchés
Générale et Technologique (Seconde GT) Classe commune menant soit à un Bac Général (enseignements théoriques et généraux), soit à un Bac Technologique (apprentissage par projets).
Bac Général : À l'aise avec les apports théoriques. <br> Bac Technologique : Apprécie l'apprentissage par le biais de projets.
Nécessite une poursuite d'études supérieures.
Professionnelle
Prépare à deux diplômes : le CAP (en 2 ans) ou le Bac Pro (en 3 ans). Peut se faire sous statut scolaire (lycée professionnel) ou en apprentissage (entreprise).
Apprend de manière concrète, avec plus de pratique et d'expérience.
Permet une insertion professionnelle directe mais aussi une poursuite d'études supérieures. La voie pro n'est pas synonyme d'études courtes.
Focus sur l'Apprentissage : L'apprentissage n'est pas réservé aux filières courtes.
Il est possible de suivre un parcours complet en apprentissage, du CAP jusqu'au Bac+5 (Master, titre d'ingénieur), en alternant formation en école et en entreprise.
L'Enseignement Supérieur après le Baccalauréat
Le schéma des études supérieures n'est pas linéaire ni figé.
• Flexibilité des parcours : Il existe de nombreuses passerelles entre les filières (ex: de BTS/BUT vers une grande école ou un master universitaire).
• Trajectoires singulières : Les parcours sont propres à chacun et peuvent inclure des pauses (année de césure, service civique), des réorientations ou des reprises d'études.
• Diversité des diplômes : L'offre va du Bac+2 (BTS) au Bac+5 (Master) et au-delà (Doctorat), avec des durées et des modalités variées (alternance, etc.).
La Construction du Projet : Un Cheminement Personnel et Expérientiel
Le projet d'orientation s'élabore progressivement grâce à une multitude d'expériences qui nourrissent la réflexion.
• Expériences formelles : Visites d'entreprises, forums, stages (obligatoire en 3ème, puis en 2nde GT), mini-stages en lycée professionnel.
• Expériences informelles : Discussions sur le travail à la maison, voyages, loisirs, rencontres.
Il est crucial de permettre aux jeunes de se forger des représentations variées du monde du travail.
• Le stage : Il s'agit d'une étape de découverte du monde de l'entreprise (codes, fonctionnement, assiduité) et de soi-même, plus que d'une validation d'un choix de métier.
• La psychologie de l'adolescent et la "Maturité Vocationnelle" :
La capacité à définir un projet professionnel, appelée "maturité vocationnelle", se développe avec la maturation cognitive du cerveau, qui se poursuit bien après 18 ans.
Il est donc contre-productif de mettre la pression sur un jeune adolescent pour un choix de métier définitif.
Démystifier l'Orientation et Gérer l'Anxiété Une part importante de l'accompagnement consiste à rassurer les familles et à déconstruire certaines idées reçues.
• Le droit à l'erreur : Une réorientation n'est pas un échec mais une expérience enrichissante qui permet d'affiner son projet et d'acquérir de nouvelles compétences.
Comme le souligne une intervenante : "Ce ne sont que des expériences qui permettent d'alimenter la connaissance de soi."
• Le mythe du "métier idéal" : L'idée d'un métier unique et parfait pour une personne n'est pas réaliste. Les parcours professionnels sont aujourd'hui marqués par la reconversion et l'évolution.
• Un monde du travail en évolution : Avec l'émergence de l'IA et de l'écologie, de nombreux métiers de demain n'existent pas encore.
Il est donc plus pertinent de faire des choix basés sur ses intérêts actuels que de tenter de prédire l'avenir du marché du travail.
• Analyse Statistique : Environ 50% des jeunes n'exercent pas un emploi directement lié à leur formation initiale.
Cela ne signifie pas une erreur d'orientation, mais illustre la transférabilité des compétences (méthodologie, savoir-faire) acquises et l'évolution naturelle des trajectoires professionnelles.
Outils et Plateformes Clés : Le Cas de Parcoursup
Parcoursup : Une Plateforme de Candidature, pas de Décision
Parcoursup est souvent une source de stress, mais son rôle doit être bien compris :
• Mise en relation : C'est une plateforme qui met en relation les candidats avec les formations post-bac.
• Le recrutement par les écoles : Ce sont les établissements d'enseignement supérieur qui examinent les dossiers et effectuent le recrutement selon leurs propres critères (dossier scolaire, concours, entretiens, etc.).
Calendrier et Fonctionnement
• Période clé : La saisie des vœux se déroule généralement du 15 janvier au 13 mars.
• Importance de l'information : Il est essentiel que le jeune se renseigne en amont sur les "attendus" et les modalités de recrutement de chaque filière qui l'intéresse pour formuler des vœux ajustés à son profil.
Un Outil de Recherche Précieux dès le Collège
La plateforme n'est pas réservée aux élèves de Terminale. Sa "carte des formations" est un moteur de recherche accessible à tous, qui permet de :
• Explorer les formations par mots-clés ou diplômes au niveau national.
• Visualiser géographiquement les établissements.
• Consulter les spécificités de chaque formation, les critères de recrutement et les statistiques des années précédentes.
Lieux et Ressources pour S'orienter
Les Centres d'Information et d'Orientation (CIO)
Les CIO offrent des entretiens gratuits sur rendez-vous (environ 1 heure) avec des PsyEN, pour les jeunes seuls ou accompagnés de leur famille. Ils sont ouverts pendant les vacances scolaires.
• Sites en Indre-et-Loire : Tours (sur le site de Joué-lès-Tours), Joué-lès-Tours, Loches, Amboise, Chinon. Ressources Numériques de Référence Ces sites sont recommandés pour la fiabilité et l'actualisation de leurs informations :
• ONISEP : Informations sur les diplômes et filières. Propose un guide pour les parents sur l'onglet "Avenir(s)".
• Parcoursup.fr : Pour la carte des formations et les informations sur l'enseignement supérieur.
• Eduscol : Ressources pédagogiques de l'Éducation Nationale.
• Cléor : Informations sur les métiers et le monde économique régional.
• orientation.centre-valdeloire.fr : Calendrier des journées portes ouvertes et recherche de formations.
• CIDJ (Centre d'Information et de Documentation Jeunesse) : Fiches métiers détaillées avec les parcours d'études associés.
Acteurs Locaux en Indre-et-Loire
• Bureau Information Jeunesse (BIJ) : Situé à Tours, il propose accueil, écoute et ateliers. Des Points Information Jeunesse (PIJ) existent aussi en milieu rural.
• Maison de l'Orientation et de l'Insertion Professionnelle (MOIP) : Rattachée à l'Université, elle s'adresse principalement aux étudiants mais reçoit aussi des lycéens.
Editorial note: To ensure a thorough evaluation of the revised manuscript, we invited a third reviewer to assess whether the authors had sufficiently addressed the concerns raised in the initial round of peer review. This additional reviewer confirmed that the authors responded partially to the original reviewers requests. While he/she also provided a set of new comments, these do not alter the original assessment or editorial decision regarding the manuscript. For transparency and completeness, the additional comments are included below.
Reviewer #3 (Public Review):
Summary:
In this manuscript, Li and coworkers present experiments generated with human induced pluripotent stem cells (iPSCs) differentiated to astrocytes through a three-step protocol consisting of neural induction/midbrain patterning, switch to expansion of astrocytic progenitors, and terminal differentiation to astroglial cells. They used lineage tracing with a LMX1A-Cre/AAVS1-BFP iPSCs line, where the initial expression of LMX1A and Cre allows the long-lasting expression of BFP, yielding BFP+ and BFP- populations, that were sorted when in the astrocytic progenitor expansion. BFP+ showed significantly higher number of cells positive to NFIA and SOX9 than BFP- cells, at 45 and 98 DIV. However, no significant differences in other markers such as AQP4, EAAT2, GFAP (which show a proportion of less than 10% in all cases) and S100B were found between BFP-positive or -negative, at these differentiation times. Intriguingly, non-patterned astrocytes produced higher proportions of GFAP positive cells than the midbrain-induced and then sorted populations. BFP+ cells have enhanced calcium responses after ATP addition, compared to BFP- cells. Single-cell RNA-seq of early and late cells from BFP- and BFP+ populations were compared to non-patterned astrocytes and neurons differentiated from iPSCs. Bioinformatic analyses of the transcriptomes resulted in 9 astrocyte clusters, 2 precursor clusters and one neuronal cluster. DEG analysis between BFP+ and BFP- populations showed some genes enriched in each population, which were subject to GO analysis, resulting in biological processes that are different for BFP+ or BFP- cells.
Strengths:
The manuscript tries to tackle an important aspect in Neuroscience, namely the importance of patterning in astrocytes. Regionalization is crucial for neuronal differentiation and the presented experiments constitute a trackable system to analyze both transcriptional identities and functionality on astrocytes.
Weaknesses:
The presented results have several fundamental issues, to be resolved, as listed in the following major points:
(1) It is very intriguing that GFAP is not expressed in late BFP- nor in BFP+ cultures, when authors designated them as mature astrocytes.<br /> (2) In Fig. 2D, authors need to change the designation "% of positive nuclei".<br /> (3) In Fig. 2E, the text describes a decrease caused by 2APB on the rise elicited by ATP, but the graph shows an increase with ATP+2APB. However, in Fig. 2F, the peak amplitude for BFP+ cells is higher in ATP than in ATP+2APD, which is mentioned in the text, but this is inconsistent with the graph in 2E.<br /> (4) The description of Results in the single-cell section is confusing, particularly in the sorted CD49 and unsorted cultures. Where do these cells come from? Are they BFP-, BFP+, unsorted for BFP, or non-patterned? Which are the "all three astrocyte populations"? A more complete description of the "iPSC-derived neurons" is required in this section to allow the reader to understand the type and maturation stage of neurons, and if they are patterned or not.<br /> (5) A puzzling fact is that both BFP- and BFP- cells have similar levels of LMX1A, as shown in Fig. S6F. How do authors explain this observation?<br /> (6) In Fig. 3B, the non-patterned cells cluster away from the BFP+ and BFP-; on the other hand, early and late BFP- are close and the same is true for early and late BFP+. A possible interpretation of these results is that patterned astrocytes have different paths for differentiation, compared to non-patterned cells. If that can be implied from these data, authors should discuss the alternative ways for astrocytes to differentiate.<br /> (7) Fig. 3D shows that cluster 9 is the only one with detectable and coincident expression of both S100B and GFAP expression. Please discuss why these widely-accepted astrocyte transcripts are not found in the other astrocytes clusters. Also, Sox9 is expressed in neurons, astrocyte precursors and astrocytes. Why is that?<br /> (8) Line 337, Why authors selected a log2 change of 0.25? Typically, 1 or a higher number is used to ensure at least a 2-fold increase, or a 50% decrease. A volcano plot generated by the comparison of BFP+ with BFP- cells would be appropriate. The validation of differences by immunocytochemistry, between BFP+ and BFP-, is inconclusive. The staining is blur in the images presented in Fig. S8C. Quantification of the positive cells, without significant background signal, in both populations is required.<br /> (9) Lines 349-351: BFP+ cells did not show higher levels of transcripts for LMX1A nor FOXA2. This fact jeopardizes the claim that these cells are still patterned. In the same line, there are not significant differences with cortical astrocytes, indicating a wider repertoire of the initially patterned cells, that seems to lose the midbrain phenotype. Furthermore, common DGE shared by BFP- and BFP+ cells when compared to non-patterned cells indicate that after culture, the pre-pattern in BFP+ cells is somehow lost, and coincides with the progression of BFP- cells.<br /> (10) For the GO analyses, How did authors select 1153 genes? The previous section mentioned 287 genes unique for BFP+ cells. The Results section should include a rationale for performing a wider search for the enriched processes.<br /> (11) For Fig. 4C and 4D, both p values and the number of genes should be indicated in the graph. I would advise to select the 10 or 15 most significant categories, these panels are very difficult to read. Whereas the listed processes for BFP+ have a relation to Parkinson disease, the ones detected for BFP- cells are related to extracellular matrix and tissue development. Does it mean that BFP+ cells have impaired formation of this matrix, or defective tissue development? This is in contradiction of enhanced calcium responses of BFP+ cells compared to BFP- cells.<br /> (12) Both the comparison between midbrain and cortical astrocytes in Fig. S8A, and the volcano plot in S8B do not show consistent changes. For example, RCAN2 in Fig. S8A has the same intensity for cortical and midbrain cells, but is marked as an enriched gene in midbrain in the p vs log2FC graph in Fig. S8B.
Author response:
The following is the authors’ response to the current reviews.
Response to Reviewer #3:
We thank reviewer 3 for spending their valuable time on commenting on our revised paper.
We would like to reiterate the central conclusion of this work, which appears to have been missed by Reviewer 3. Using a BFP-expressing lineage tracer hPSC line for tracking LMX1A+ midbrain-patterned neural progenitors and their differentiated progeny, we discovered a loss of the LMX1A lineage during pluripotent stem cell differentiation into astrocytes, despite BFP+ neural progenitors were the dominant population at the onset of astrocyte induction.
Hence, the take-home message of this study is, as summarized in the abstract, ‘ the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population’ and that one should not take for granted that in vitro/stem cell-derived astrocytes are the descendants of the dominant starting neural progenitors (which is a general assumption in PSC publications as described in the paper and our response to reviewers).
Please find below our point-by-point response to reviewer comments. We have re-ordered the points according to their relative importance to our main conclusions.
‘ the lineage composition of iPSC-derived astrocytes may not accurately recapitulate the founder progenitor population’ and that one should not take for granted that in vitro/stem cell derived astrocytes are the descendants of the dominant starting neural progenitors (which is a general assumption in PSC publications as described in the paper and our response to reviewers).
Please find below our point-by-point response to their comments. We have re-ordered the points according to their relative importance to our main conclusions.
…. They used lineage tracing with a LMX1A-Cre/AAVS1-BFP iPSCs line, where the initial expression of LMX1A and Cre allows the long-lasting expression of BFP, yielding BFP+ and BFP- populations, that were sorted when in the astrocytic progenitor expansion. BFP+ showed significantly higher number of cells positive to NFIA and SOX9 than BFP- cells …
This is a misunderstanding by reviewer 3. As indicated in the first sentence of the second section, BFP- populations used for functional and transcriptomic analysis was not sorted BFP<sup>-</sup> cells, but those derived from unsorted, BFP<sup>+</sup> enriched populations. Our scRNAseq analysis indicated that they were transcriptomically aligned to human midbrain astrocytes. This finding is consistent with the fact that they are derived from midbrain-patterned neural progenitors, presumably minority LMX1A- progenitors.
Reviewer 3’s comments indicate that they misunderstood the primary aims of our study as a mere functional and transcriptomic comparison of the two astrocyte populations.
(9) BFP+ cells did not show higher levels of transcripts for LMX1A nor FOXA2. This fact jeopardizes the claim that these cells are still patterned. In the same line, there are not significant differences with cortical astrocytes, indicating a wider repertoire of the initially patterned cells, that seems to lose the midbrain phenotype. Furthermore, common DGE shared by BFP- and BFP+ cells when compared to non-patterned cells indicate that after culture, the pre-pattern in BFP+ cells is somehow lost, and coincides with the progression of BFP- cells.
The reviewer seems to assume that astrocytes derived from LMX1A+ ventral midbrain progenitors must retain LMX1A expression. We do not take this view and do not claim this in this study. Moreover, we have discussed in the paper that due to a lack of transcriptomic studies of in vivo track regional progenitors (such as LMX1A), it remains unknown whether and to what extent patterning gene expression is maintained in astrocytes of different brain regions.
Our findings on the lack of LMX1A and FOXA2 in BFP+ astrocytes are supported by several published single-cell transcriptomic studies of human midbrain astrocytes (La Manno et al. 2016; Agarwal et al. 2020; Kamath et al. 2022). We have a paragraph of discussion on this topic in both the original and updated versions of the paper with the relevant publications cited.
Other points raised by reviewer 3
(1) It is very intriguing that GFAP is not expressed in late BFP- nor in BFP+ cultures, when authors designated them as mature astrocytes.
We did not designate our cells as ‘mature’ astrocytes but ‘astrocytes’ based on their global gene expression with the human fetal and adult brain astrocytes as references.
Moreover, ‘mature’ only appeared once in the paper indicating that our cells lie in between the fetal and adult astrocytes in maturity.
(2) In Fig. 2D, authors need to change the designation "% of positive nuclei".
To be corrected in the version of record.
(3) In Fig. 2E, the text describes a decrease caused by 2APB on the rise elicited by ATP, but the graph shows an increase with ATP+2APB. However, in Fig. 2F, the peak amplitude for BFP+ cells is higher in ATP than in ATP+2APD, which is mentioned in the text, but this is inconsistent with the graph in 2E.
To be corrected in the version of record.
(4) The description of Results in the single-cell section is confusing, particularly in the sorted CD49 and unsorted cultures. Where do these cells come from? Are they BFP-, BFP+, unsorted for BFP, or non-patterned? Which are the "all three astrocyte populations"? A more complete description of the "iPSC-derived neurons" is required in this section to allow the reader to understand the type and maturation stage of neurons, and if they are patterned or not.
As previously reported in the reference cited, CD49 is a novel human astrocyte marker. This is independent of BFP expression. For all three astrocyte populations studied here (BFP+, BFP-, and non-patterned astrocytes), we included both CD49f+ sorted and unsorted samples to account for selection bias caused by FACS. iPSC-derived neurons were included in the sequencing study to provide a reference for cell-type annotation. They were generated following a GABAergic neuron differentiation protocol. However, their maturation stages and/or regional characteristics are not relevant to astrocytes.
(5) A puzzling fact is that both BFP- and BFP- cells have similar levels of LMX1A, as shown in Fig. S6F. How do authors explain this observation?
This figure panel shows that LMX1A, LMX1B and FOXA2 are essentially NOT expressed in these astrocytes.
(6) In Fig. 3B, the non-patterned cells cluster away from the BFP+ and BFP-; on the other hand, early and late BFP- are close and the same is true for early and late BFP+. A possible interpretation of these results is that patterned astrocytes have different paths for differentiation, compared to non-patterned cells. If that can be implied from these data, authors should discuss the alternative ways for astrocytes to differentiate.
Both BFP+ and BFP- astrocyte are from ventral midbrain patterned neural progenitors, while non-patterned neural progenitors are more akin to that of forebrain. Figure 3B is expected and confirms the patterning effect.
(7) Fig. 3D shows that cluster 9 is the only one with detectable and coincident expression of both S100B and GFAP expression. Please discuss why these widely-accepted astrocyte transcripts are not found in the other astrocytes clusters. Also, Sox9 is expressed in neurons, astrocyte precursors and astrocytes. Why is that?
S100B and GFAP are classic astrocyte markers in certain states. We are not relying only on two markers but the genome-wide expression profile as the criteria for astrocytes. As shown in the unbiased reference mapping to multiple human brain astrocyte scRNA-seq datasets, all our astrocyte clusters were mapped with high confidence to human astrocytes.
SOX9 is an important regulator for astrogenesis, so its expression is expected in precursors (doi.org/10.1016/j.neuron.2012.01.024). In addition, recent studies have uncovered that SOX9 expression is also reported in foetal striatal projection neurons and early postnatal cortical neurons, where SOX9 regulates neuronal synaptogenesis and morphogenesis (dois:10.1016/j.fmre.2024.02.019; 10.1016/j.neuron.2018.10.008). Therefore, the expression of SOX9 in multiple cell types was expected. Instead of using a few selected markers for cell-type annotation, we employed a genomic approach relying on an unbiased reference mapping approach and a combination of various markers to ascertain our annotation results.
(8) Line 337, Why authors selected a log2 change of 0.25? Typically, 1 or a higher number is used to ensure at least a 2-fold increase, or a 50% decrease. A volcano plot generated by the comparison of BFP+ with BFP- cells would be appropriate. The validation of differences by immunocytochemistry, between BFP+ and BFP-, is inconclusive. The staining is blur in the images presented in Fig. S8C. Quantification of the positive cells, without significant background signal, in both populations is required.
We used a lenient threshold owing to the following considerations: 1) High FC does not necessarily mean biological relevance, as gene expression does not necessarily translate to protein expression. Therefore, a smaller FC value could also be biologically meaningful. 2) Balance between noise and biological differences. Any threshold was chosen arbitrarily. 3) We are identifying a trend rather than pinpointing a specific set of
The quality was unfortunately reduced due to restrictions on file size upon submission. A high resolution Fig. S8C is available.
(10) For the GO analyses, How did authors select 1153 genes? The previous section mentioned 287 genes unique for BFP+ cells. The Results section should include a rationale for performing a wider search for the enriched processes.
GO enrichment using unique DEGS may not capture the wider landscape of the transcriptomic characteristics of BFP<sup>+</sup> astrocytes. The 287 unique genes were only differentially expressed in BFP<sup>+</sup> astrocytes. However, apart from these 287 genes, other genes among the 1187 DEGs were differentially expressed in BFP<sup>+</sup> astrocytes and in one other population.
(11) For Fig. 4C and 4D, both p values and the number of genes should be indicated in the graph. I would advise to select the 10 or 15 most significant categories, these panels are very difficult to read. Whereas the listed processes for BFP+ have a relation to Parkinson disease, the ones detected for BFP- cells are related to extracellular matrix and tissue development. Does it mean that BFP+ cells have impaired formation of this matrix, or defective tissue development? This is in contradiction of enhanced calcium responses of BFP+ cells compared to BFP- cells.
Information on all DEGs, including p values and numbers, is provided in Supplementary data 1-5.
BFP+ astrocytes do have enrichment for GO terms related to extracellular matrix and tissue development, although not as obvious as BFP- astrocytes. Previous work have shown that both in vitro and in vivo derived astrocytes are functionally heterogeneous, containing functionally distinct subtypes exhibiting different GO enrichment profiles (doi: 10.1016/j.ygeno.2021.01.008; 10.1038/s41598-024-74732-7).
(12) Both the comparison between midbrain and cortical astrocytes in Fig. S8A, and the volcano plot in S8B do not show consistent changes. For example, RCAN2 in Fig. S8A has the same intensity for cortical and midbrain cells, but is marked as an enriched gene in midbrain in the p vs log2FC graph in Fig. S8B.
These are integrated analyses of published human datasets. S8A and S8B show the same data in different formats. The differences are better shown in the volcano plot/easier detected by the human eye.
These are integrated analysis of published human datasets. S8A and S8B are the same data shown in different format. Differences are better shown in volcano plot /easier detected by the human eye. RCAN2 had a higher average expression in the midbrain than in the telencephalon, albeit small, and the difference was statistically significant (as shown in the volcano plot).
The following is the authors’ response to the original reviews
Reviewer 1:
In vitro nature of this work being the fundamental weakness of this paper
We disagree with this statement. As explained in the provisional response, the aim of this study was to test the validity of a general concept applied in pluripotent stem cell research that pluripotent stem cell-derived astrocytes faithfully represent the lineage heterogeneity of their ancestral neural progenitors and hence preserve the regionality of such progenitors. Our genetic lineage study is justified for addressing this in vitro-driven question. However, we have highlighted the rationale where appropriate in the revised paper.
If regional identity is not maintained, so what? Don't we already know that this can happen? The authors acknowledge that this is known in the discussion.
Importance of regional identity: Growing evidence demonstrates the functional heterogeneity of brain astrocytes in health and disease. Therefore, for in vitro disease modeling, it is believed that one should use astrocytes represent the anatomy of disease pathology; for example, midbrain astrocytes for studying dopamine neurodegeneration and Parkinson’s disease. Understanding the dynamics of stem cell-derived astrocytes and identifying astrocyte subtypes is important for their biomedical applications.
Regional identity change/Discussion: It seems that the reviewer misunderstood the context in which the ‘identity change’ was discussed. The literature referred to (in the Discussion) concerns shifts in regional gene expression in bulk-cultured cells. In the days of pre-single-cell analysis/lineage tracking, one cannot distinguish whether this was due to a change in the transcriptomic landscape in progenies of the same lineage or alterations in lineage heterogeneity, but to interpret at face value as regional identity was not maintained. In the revised paper, we have made an effort to indicate that ‘regional identity’ is used broadly to refer to lineage relationships and/or traits rather than static gene expressioin.
validation of the markers/additional work
The scNAseq analysis performed in this study compared the profiles of astrocytes derived from LMX1A+ and LMX1A- ventral midbrain-patterned neural progenitors. Since it is not possible to perform genetic lineage tracking in humans and an analogous mouse lineage tracer line is not available, in vivo validation of these markers with respect to their lineage relationship is not currently feasible. However, we took advantage of abundant single-cell human astrocyte transcriptomic datasets and validated our genes in silico. We also validated the differential expression of selected markers in late BFP+ and BFP- astrocytes using immunocytochemistry, where reliable antibodies are available. The results of the additional analyses are presented in Figure S8 and Supplemental Data 5.
Knowledge gaps concerning astrocyte development
Reviewer 1 pointed out a number of knowledge gaps concerning astrocyte development, such as the transcriptomic landscape trajectories of midbrain floor plate cells as they progress towards astrocytes. Indeed, the limited knowledge on regional astrocyte molecule heterogeneity restricts the objective validation of in vitro-derived astrocyte subtypes and the development of novel approaches for their generation in vitro. We agree with the need for in-depth in vivo studies using model organisms, although these are beyond the scope of the current work.
Reviewer 2:
(1) The authors argue that the depletion of BFP seen in the unsorted population immediately after the onset of astrogenic induction is due to the growth advantage of the derivatives of the residual LMX1A- population. However, no objective data supporting this idea is provided, and one could also hypothesize that the residual LMX1A- cells could affect the overall LMX1A expression in the culture through negative paracrine regulation.
We acknowledge the lack of evidence-based explanation for the depletion of BFP+ cells in mixed cultures. We were unable to perform additional experiments because of resource limitations. The design of the LMX1A-Cre/AAVS1-BFP lineage tracer line determines that BFP is expressed irreversibly in LMX1A-expressing cells or their derivatives regardless of their LMX1A expression status. Therefore, the potential negative paracrine regulation of LMX1A by residual LMX1A- cells should not affect cells that have already turned on BFP. We have highlighted the working principles of the LMX1A tracer line in the revised manuscript.
(2) Furthermore, on line 124 it is stated that: "Interestingly, the sorted BFP+ cells exhibited similar population growth rate to that of unsorted cultures...". In the face of the suggested growth disadvantage of those cells, this statement needs clarification.
To avoid confusion, we have removed the statement.
(3) Regarding the fidelity of the model system, it is not clear to me how the TagBFP expression was detected in the BFP+ population supposedly in d87 and d136 pooled astrocytes (Fig S6C) while no LMX1A expression was observed in the same cells (Fig S6F).
The TagBFP tracer is expressed in the progenies of LMX1A+ cells, regardless of their LMX1A expression status. We have gone through the MS text to ensure that this information has been provided.
(4) The generated single-cell RNASeq dataset is extremely valuable. However, given the number of conditions included in this study (i.e. early vs late astrocytes, BFP+ vs BFP-, sorted vs unsorted, plus non-patterned and neuronal samples) the resulting analysis lacks detail. For instance, from a developmental perspective and to better grasp the functional significance of astrocytic heterogeneity, it would be interesting to map the identified clusters to early vs late populations and to the BFP status.
We performed additional bioinformatics analysis, which provided independent support for the relative developmental maturity suggested by functional assays. The additional data are now provided in the revised Figure 3B, C, E.
Moreover, although comprehensive, Figure S7 is complex to understand given that citations rather than the reference populations are depicted.
The information provided in the revised Figure S7.
(5) Do the authors have any consideration regarding the morphology of the astrocytes obtained in this study? None of the late astrocyte images depict a prototypical stellate morphology, which is reported in many other studies involving the generation of iPSC-derived astrocytes and which is associated with the maturity status of the cell.
The morphology of our astrocytes was not unique to the present study. Many factors may influence the morphology of astrocytes, such as the culture media and supplements used, and maturity status. Based on the functional assays and limited GFAP expression, our astrocytes were relatively immature.
Reviewer #2 (Public review):
Summary:
DNA double-strand breaks (DSB) in repeated DNA pose a challenge for repair by homologous recombination (HR) due to the potential of generating chromosomal aberrations, especially involving repeats on different chromosomes. This conceptual caveat led to a long-held notion that HR is not active in repeated DNA, which was disproven in groundbreaking work by Chiolo showing in Drosophila that DSBs in pericentromeric repeats are mobilized to the nuclear periphery for repair by HR. A similar mechanism operates in mouse cells, as shown by the Gautier laboratory, but the mobilization goes to the nucleolar periphery, called nucleolar caps. In this manuscript, the authors reexamine the role of MDC1 in the mobilization of DSBs in rDNA in human cells. Previous work has shown that MDC1 is replaced by Treacle, the gene associated with Treacher Collins syndrome 1, in its role as the main adaptor of the DNA damage response, and these results are confirmed here. The novelty of this contribution lies in the discovery that MDC1 is required downstream in the recruitment of BRCA1 and RAD51 to nucleolar DSBs that were mobilized to the nucleolar cap. Using multiple MCD knockout models and DSBs induced by the nuclease PpoI, which cleaves at nuclear sites as well as in the 28S rDNA, convincingly documents this role of MDC1 and shows that it acts upstream of the RNF8-RNF168 ubiquitylation axis. Using a proxy assay of co-localization of EdU incorporation at DSBs (gammaH2AX), evidence is provided that MDC1 is required for HR in rDNA. MDC1 was not required for RAD51 recruitment to IR-induced foci, but it is unclear whether this is related to the different DSB chemistry (enzymatic versus IR) or to the localization of the DSB (rDNA versus unique sequence genome).
Strengths:
(1) The manuscript is well-written, and the experimental evidence is nicely presented.
(2) Multiple MDC1 knockout models are used to validate the results.
(3) Convincing back-complementation data clarify the relationship between MDC1 and RNF8.
Weaknesses:
(1) The recruitment of BRCA2 was not directly demonstrated. This caveat could be recognized, as IF for BRCA2 is challenging.
(2) PpoI also induces DSBs in the non-rDNA genome. These DSBs would be an ideal control to establish nucleolar specificity of the events described and clarify whether the difference between IR and PpoI is the chemical structure of the DSB or the location of the DSB.
Reviewer #1 (Public review):
The authors present an approach that uses the transformer architecture to model epistasis in deep mutational scanning datasets. This is an original and very interesting idea. Applying the approach to 10 datasets, they quantify the contribution of higher-order epistasis, showing that it varies quite extensively.
Suggestions:
(1) The approach taken is very interesting, but it is not particularly well placed in the context of recent related work. MAVE-NN, LANTERN, and MoCHI are all approaches that different labs have developed for inferring and fitting global epistasis functions to DMS datasets. MoCHI can also be used to infer multi-dimensional global epistasis (for example, folding and binding energies) and also pairwise (and higher order) specific interaction terms (see 10.1186/s13059-024-03444-y and 10.1371/journal.pcbi.1012132). It doesn't distract from the current work to better introduce these recent approaches in the introduction. A comparison of the different capabilities of the methods may also be helpful. It may also be interesting to compare the contributions to variance of 1st, 2nd, and higher-order interaction terms estimated by the Epistatic transformer and MoCHI.
(2) https://doi.org/10.1371/journal.pcbi.1004771 is another useful reference that relates different metrics of epistasis, including the useful distinction between biochemical/background-relative and background-averaged epistasis.
(3) Which higher-order interactions are more important? Are there any mechanistic/structural insights?
Reviewer #2 (Public review):
Summary:
This paper presents a novel transformer-based neural network model, termed the epistatic transformer, designed to isolate and quantify higher-order epistasis in protein sequence-function relationships. By modifying the multi-head attention architecture, the authors claim they can precisely control the order of specific epistatic interactions captured by the model. The approach is applied to both simulated data and ten diverse experimental deep mutational scanning (DMS) datasets, including full-length proteins. The authors argue that higher-order epistasis, although often modest in global contribution, plays critical roles in extrapolation and capturing distant genotypic effects, especially in multi-peak fitness landscapes.
Strengths:
(1) The study tackles a long-standing question in molecular evolution and protein engineering: "how significant are epistatic interactions beyond pairwise effects?" The question is relevant given the growing availability of large-scale DMS datasets and increasing reliance on machine learning in protein design.
(2) The manuscript includes both simulation and real-data experiments, as well as extrapolation tasks (e.g., predicting distant genotypes, cross-ortholog transfer). These well-rounded evaluations demonstrate robustness and applicability.
(3) The code is made available for reproducibility.
Weaknesses:
(1) The paper mainly compares its transformer models to additive models and occasionally to linear pairwise interaction models. However, other strong baselines exist. For example, the authors should compare baseline methods such as "DANGO: Predicting higher-order genetic interactions". There are many works related to pairwise interaction detection, such as: "Detecting statistical interactions from neural network weights", "shapiq: Shapley interactions for machine learning", and "Error-controlled non-additive interaction discovery in machine learning models".
(2) While the transformer architecture is cleverly adapted, the claim that it allows for "explicit control" and "interpretability" over interaction order may be overstated. Although the 2^M scaling with MHA layers is shown empirically, the actual biological interactions captured by the attention mechanism remain opaque. A deeper analysis of learned attention maps or embedding similarities (e.g., visualizations, site-specific interaction clusters) could substantiate claims about interpretability.
(3) The distinction between nonspecific (global) and specific epistasis is central to the modeling framework, yet it remains conceptually underdeveloped. While a sigmoid function is used to model global effects, it's unclear to what extent this functional form suffices. The authors should justify this choice more rigorously or at least acknowledge its limitations and potential implications.
(4) The manuscript refers to "pairwise", "3-4-way", and ">4-way" interactions without always clearly defining the boundaries of these groupings or how exactly the order is inferred from transformer layer depth. This can be confusing to readers unfamiliar with the architecture or with statistical definitions of interaction order. The authors should clarify terminology consistently. Including a visual mapping or table linking a number of layers to the maximum modeled interaction order could be helpful.
Reviewer #3 (Public review):
Summary:
Sethi and Zou present a new neural network to study the importance of epistatic interactions in pairs and groups of amino acids to the function of proteins. Their new model is validated on a small simulated data set and then applied to 10 empirical data sets. Results show that epistatic interactions in groups of amino acids can be important to predict the function of a protein, especially for sequences that are not very similar to the training data.
Strengths:
The manuscript relies on a novel neural network architecture that makes it easy to study specifically the contribution of interactions between 2, 3, 4, or more amino acids. The study of 10 different protein families shows that there is variation among protein families.
Weaknesses:
The manuscript is good overall, but could have gone a bit deeper by comparing the new architecture to standard transformers, and by investigating whether differences between protein families explain some of the differences in the importance of interactions between amino acids. Finally, the GitHub repository needs some more information to be usable.
eLife Assessment
This manuscript uses simulations to describe the dynamics of the Pseudomonas-derived cephalosporinase PDC-3 β-lactamase and its mutants to better understand antibiotic resistance. The finding that clinically observed mutations alter the flexibility of the Ω- and R2-loops, reshaping the cavity of the active site, is useful to the field. However, the evidence is considered incomplete; there is a lack of description of methods, and there is a need for additional analysis to demonstrate statistical significance, visualisation of the Markov states, analysis to explain changes due to the different mutations, and possible simulations in the presence of substrates to shed direct light on modulation mechanisms.
Reviewer #1 (Public review):
Summary:
This manuscript uses adaptive sampling simulations to understand the impact of mutations on the specificity of the enzyme PDC-3 β-lactamase. The authors argue that mutations in the Ω-loop can expand the active site to accommodate larger substrates.
Strengths:
The authors simulate an array of variants and perform numerous analyses to support their conclusions.
The use of constant pH simulations to connect structural differences with likely functional outcomes is a strength.
Weaknesses:
I would like to have seen more error bars on quantities reported (e.g., % populations reported in the text and Table 1).
Reviewer #1 (Public review):
Summary:
This manuscript uses adaptive sampling simulations to understand the impact of mutations on the specificity of the enzyme PDC-3 β-lactamase. The authors argue that mutations in the Ω-loop can expand the active site to accommodate larger substrates.
Strengths:
The authors simulate an array of variants and perform numerous analyses to support their conclusions.
The use of constant pH simulations to connect structural differences with likely functional outcomes is a strength.
Weaknesses:
I would like to have seen more error bars on quantities reported (e.g., % populations reported in the text and Table 1).
Reviewer #1 (Public review):
Summary:
The study is methodologically solid and introduces a compelling regulatory model. However, several mechanistic aspects and interpretations require clarification or additional experimental support to strengthen the conclusions.
Strengths:
(1) The manuscript presents a compelling structural and biochemical analysis of human glutamine synthetase, offering novel insights into product-induced filamentation.
(2) The combination of cryo-EM, mutational analysis, and molecular dynamics provides a multifaceted view of filament assembly and enzyme regulation.
(3) The contrast between human and E. coli GS filamentation mechanisms highlights a potentially unique mode of metabolic feedback in higher organisms.
Weaknesses:
(1) The mechanism underlying spontaneous di-decamer formation in the absence of glutamine is insufficiently explored and lacks quantitative biophysical validation.
(2) Claims of decamer-only behavior in mutants rely solely on negative-stain EM and are not supported by orthogonal solution-based methods.