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Title: "Towards deciphering the Nt17 code: How the sequence and conformation of the first 17 amino acids in Huntingtin regulate the aggregation, cellular properties, and neurotoxicity of mutant Httex1".
Tracking #: RC-2021-00675
Authors: Vieweg et al.
MAJOR COMMENTS from Referees #1, #2, and #3
Referee #1
General comments
« The manuscript by Vieweg, Mahul-Mellier, Ruggeri et al., describes the role of the sequence and conformation of the extreme N-terminus of the Huntingtin protein in terms of aggregation and toxicity together with its relation to the polyglutamine length. The authors use some outstanding methods to ensure that the conclusions are based on good quality data. Overall, this is an excellent study.
We thank the referee for the very positive feedback and for recognizing the quality of our work and his/her appreciation of our systematic approach to dissect the role of the Nt17 domain in regulating the aggregation, cellular properties, and neurotoxicity of mutant Httex1.
“The manuscript is generally well written although it might benefit from reducing the length of the discussion section ».
We thank the referee for his/her valuable comment. We have reduced by 10% the discussion as per requested.
Major comments
1) “For their in vitro data, the authors do not go beyond 42 polyglutamines. Is there any particular reason for that? The authors see a clear difference between 36Q and 42Q, but although not critical, it would have been useful to use longer repeats. In my view, the authors should at least discuss the rationale for this, particularly as in cellular models they do use 72Q constructs.”
We thank the referee for raising this point.
Most HD patients have a polyQ repeat stretch of 40-45 glutamines (1-4).
In vitro, the use of Httex1 constructs consisting of 42 polyglutamine residues is sufficient to induce mutant Httex1 aggregation and fibril formation. Mutant Httex1 proteins with polyQ repeats of 72Q or higher are highly aggregation-prone and difficult to purify, handle, or disaggregates. This is why all of the in vitro aggregation studies are based on mutant Htt proteins with polyQ ranging from 23Q-53Q (5-14). We have reviewed the literature carefully and were unable to identify any in vitro studies with recombinant Htt proteins containing polyQ repeats of 72 or greater.
In cells, induction of mature Htt inclusions requires much longer polyQ repeats. This is clearly reflected by the fact that most cellular studies use mutant Htt with polyQ repeats above 64Q and up to 160Q to induce the formation of cellular aggregates (10, 15-30).
We have recently conducted a systematic study on the effect of the polyQ repeat length on Htt inclusion formation in cells https://www.biorxiv.org/content/10.1101/2020.07.29.226977v1 (21). Characterization of the inclusions by EM revealed that the polyQ tract length dramatically influences the ultrastructure properties and the architecture of the Httex1 inclusions in cells. The dark shell structure that delimitated the core from the periphery of the Httex1 72Q inclusions was absent in the Httex1 39Q inclusions. Also, the Httex1 39Q inclusions appeared less dense compared to that of the Httex1 72Q. Finally, no significant cell death was observed in HEK cells overexpressing 39Q constructs while overexpression of Httex1 72Q was toxic. For these reasons, we and others select to use Httex1 with polyQ repeat of 75 or higher.
2) « The role of the N-terminus 17 aminoacids of huntingtin (Nt17) is addressed by comparing peptides with and without the Nt17 and their relation to the adjacent polyglutamine tract. Using this approach, the peptide without the Nt17 is composed of pure polyglutamines in its N-terminus, followed by the rest of exon 1 in its C-terminus. This is clearly the key comparison to address the role of the Nt17 in the context of an exon1 containing polyQ. »
Yes. In fact, we did perform this experiment and assessed if the addition of the Nt17 would be sufficient to inhibit mutant Httex1 aggregation or make ΔNt17-Httex1 aggregate similar to Httex1. This data is included in the original version of the manuscript as Figure S5 in supporting material. We observed that that __the presence of the Nt17 peptide during the aggregation of ΔNt17-exon1 fibrils did not interfere with the aggregation kinetic of mutant Httex1 or alter the fibril morphology of ΔNt17-exon1,__ indicating that intramolecular interactions between the Nt17 domain and the adjacent polyQ tract are key determinants of mutant HTtex1 fibrillization and fibril morphology.
Referee #2
General comments
This article describes the results from studies into mechanisms of the aggregation and toxicity of Htt Exon1 protein. The authors investigated the role of N17, polyQ length, M9C mutation, and phosphorylation. Multiple approaches were used that included biochemical protein design, biophysical measurements, and cell biological experiments with cultured mammalian cells. The authors demonstrate the effects of protein context on aggregation. Furthermore, the authors were able to visualize the aggregates in mammalian cells and in neurons using multiple methods. These are interesting data.
We greatly appreciate the positive feedbacks on our data and our systematic and integrative approaches.
There are several major weaknesses in the study. First problem is that most of the results related to aggregation mechanisms and toxicity are not original and incremental when compared to many previously published articles.
We respectfully disagree with this assessment and suggestion that our studies are not original and represent only incremental advances.
Originality
Our study provides novel mechanistic insights into the role of not only the sequence but also the conformational properties of the Nt17 domain in regulating the dynamics of Httex1 fibrillization, the kinetic fibril growth, the structure and morphology of Httex1 fibrils. Besides, we also addressed for the first time how the Nt17 domain and phosphorylation at different residues within this domain regulate the cellular uptake, subcellular localization (phosphorylated proteins) and toxicity of extracellular monomeric and fibrillar forms of mutant Httex1 in primary neurons. We are not aware of previous reports that have conducted similar studies addressing these questions and using multiple methods. Also, some of our findings using native Httex1 sequences are not in agreement with previous reports using Httex1 proteins fused to peptide/protein tags, thus underscoring the limitations of previous studies.
1) We demonstrated that the Nt17 domain plays an important role in shaping the surface properties of mutant Httex1 fibrils and regulate their lateral association and cellular uptake. Our findings are not in agreement with previous findings published in eLife by Shen et al. __(10)__, where they reported that removal of the Nt17 domain has the opposite effect of what we observed in our study, i.e., ∆Nt17 promotes the formation of fibrils that exhibit a low tendency to laterally associated and form a “bundled” architecture (10). Careful examination of their constructs revealed that all the proteins they used contained a highly charged 15-mer peptide tag (S-tag: Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-Asp-Ser) at the C-terminus of Httex1, which we believe would strongly influence the aggregation properties of the mainly uncharged ∆Nt17-Httex1 and Httex1 protein, thus possibly explaining the discrepancy between our findings and those of Shen et al (10). In fact, a previous study has shown that adding short peptide tags such as the HA or the LUM tag to mutant Htt171 changed the toxicity dramatically. In addition, we show that the subcellular localization of Htt171 expressed in cells (e.g: expression of Htt171 carrying the LUM tag was more toxic than untagged Htt171 and induced the formation of nuclear aggregates rather than the classical cytoplasmic aggregates) (31). The reference to this paper is now included and discussed in the main manuscript (page 11).
Our observations __highlight__ the critical importance of using tag-free proteins to investigate the sequence and structural determinants of Httex1 aggregation and structure.
2) Our study is the first to demonstrate that the Nt17 domain influences the relationship between fibril length and polyQ repeat length. This correlation disappears when the Nt17 is removed. This aspect of our work was not explored by Shen et al. (10), who limited their in vitro aggregation study to Httex1 wild-type and mutants (∆Nt17 or ∆PRD for Polyn Rich Domain).
3) This is also the first study to assess the effect of modulating the helicity of Nt17 on fibrils growth and morphology and Httex1 cellular properties.
- a) Using the helix and membrane-binding disrupting mutation (M8P), we showed that disrupting the Nt17 helix (M8P mutation) slows the aggregation propensity of Httex1 in vitro but does not alter the morphology of the fibrils. In contrast, removing the Nt17 domain leads to a strong lateral association of the fibrillar aggregates with ribbon-like morphology. This demonstrated that the __Nt17 sequence, but not its helical structure, is the key determinant of the quaternary packing of Httex1 fibrils. Shen et al. (10) did not investigate the effect of modulating the helicity of Nt17 on fibrils growth and morphology using M8P mutant__.
- b) Our cellular studies comparing the membrane association and uptake of extracellularly added Httex1 43Q and M8P Httex1 43Q fibrils in primary rat striatal neurons showed that disrupting the Nt17 helix promotes the internalization of M8P Httex1 while Httex1 stays bound to the plasma membrane. These findings suggest that the Nt17 helical conformation persists in the fibrillar state or that the Nt17 domain regains its helical structure upon interaction with the plasma membrane resulting in the sequestration of Httex1 fibrils at the membrane and impeding their uptake. This aspect of our study has never been explored in previous studies.
- c) Using the site-specific bona fide phosphorylation on T3, S13, SS16, and both S13/S16, this is the first study that shows that modulation of the overall helicity of Httex1 through site-specific phosphorylation of the Nt17 domain (pT3 stabilizes the alpha-helical conformation of Nt17 while pS13 and/or pS16 disrupts it (9)) enhance the rapid uptake of extracellular Httex1 monomeric species into neurons and their nuclear accumulation. Previous studies relied on phosphomimetic mutations (32), which we have shown do not reproduce the effect of phosphorylation at these residues on the structure of Nt17 (8, 9). Shen et al. __(10) did not investigate the effect of modulating the helicity of Nt17 on fibrils growth and morphology using site-specific __phosphorylation of the Nt17 domain.
4) Our overexpression model in HEK cells showed that removing the Nt17 domain or disrupting its helical structure (M8P mutation) was sufficient to prevent the cell death induced by Httex1 72Q overexpression and reduce the number of cells with inclusions drastically. Our data indicate that the cell death level correlates with the number of cells that contain inclusions or the number of inclusions formed in the cells or/and their subcellular localization. In contrast to our results, Shen et al.,__(10)__ demonstrated that the overexpression of ΔN17-Httex1 induced toxicity at a similar level as the full-length Httex1 in striatal-derived neurons or neurons from cortical rat brain slices culture, although ΔN17-Httex1 led to a significant reduction of punctate structures in these cells. The discrepancy between these studies and our Httex1 overexpression model in HEK cells may be due to the fact that in neurons, Httex1 lacking the Nt17 domain accumulates in the nucleus. In contrast, in HEK, it stayed mostly cytosolic. In line with this hypothesis, it has been recently shown that cytosolic inclusions (Httex1 200Q) and nuclear aggregates (Httex1 90Q) contribute – to various extents – to the onset and the progression of the disease in a transgenic HD mice model (33). Thus, the difference in cellular localization but also the cell type (HEK vs. neurons) could influence the toxic response of the cells to the overexpression of ∆Nt17-Httex1, with toxicity triggered only by the nuclear ∆Nt17-Httex1 species.
5) This is also the first study to investigate the role of the Nt17 domain and Nt17 PTMs in influencing the uptake, the subcellular localization, and the toxicity of extracellular Httex1 species (monomers and fibrils) in primary neurons. We showed that the helical propensity of Nt17 strongly influences the uptake of Httex1 fibrils into primary striatal neurons. At the same time, phosphorylation (at T3 or S13/S16) or removal of the Nt17 domain increased the uptake and accumulation of Httex1 fibrils into the nucleus and induced neuronal cell death. Our findings suggest that the Nt17 domain is exposed in the fibrillar state and is sufficiently dynamic to mediate fibril-membrane interactions and internalization.
Altogether our results, combined with previous findings from our groups and others demonstrating the role of Nt17 in regulating Htt degradation (34-36), suggest that this domain serves as one of the key master regulators of Htt aggregation subcellular localization of the pathological aggregates, and their toxicity. They further demonstrate that targeting Nt17 represents a viable strategy for developing disease-modifying therapies to treat HD.
Limitations of previous studies:
Although the effects of the Nt17 domain in regulating Httex1 aggregation and cellular properties have been studied and reported on by other groups, we would like to stress that most of the published studies had major limitations and used protein constructs that do not share the sequence of native Httex1 and exhibit biophysical and cellular properties that differ from those of native Httex1 sequences.
1) Many of the studies used Httex1-like model peptides (13, 37), which do not contain the complete sequence of Httex1 (e.g., Nt17 peptide (37)), contain additional solubilizing amino acids such as lysine residues(38-43) or are fused to large proteins (e.g., GST, YFP) (37).
2) Other studies relied on artificial fusion constructs whereby the polyQ domain (44-46) or Httex1 itself (12, 47-61) are fused to large solubilizing protein tags, such as glutathione-S-transferase (GST), maltose-binding protein (MBP) or thioredoxin (TRX) or C-terminal S-tag (10, 62, 63) or fluorescent proteins (e.g., GFP or YFP) (10, 15, 49, 64, 65) for the cellular studies.
One of the major limitations of using fusion constructs as precursors for the generation of Httex1 (12, 47-61) is the requirement to cleave the fusion protein in situ by adding a protease to release and initiate the aggregation of Httex1. Enzyme-mediated cleavage of Httex1-fusion proteins often results in the incorporation of additional amino acids at the N- or C-terminus of the protein. This could alter the biophysical and biochemical properties of Httex1 because of the important role of the Nt17 domain and the proline-rich domain in regulating the conformational and aggregation properties of the protein (38, 40, 43, 65, 66). Moreover, it has been shown that commonly used enzymes such as trypsin and thrombin may lead to cleavages within the Nt17 domain and result in the generation of undesired Httex1 fragments (7, 42, 60). The net effect of incomplete and/or unspecific enzymatic cleavage of Httex1 fusion proteins is the generation of heterogeneous protein mixtures, which precludes accurate interpretation and comparison of aggregation and structural data across different laboratories.
Moreover, several studies have shown that the fusion of small peptide tags or large fusion protein alters the aggregation of mutant Httex1 in vitro and in cells.
- We have previously shown that the presence of such tags (e.g., GST) alters the ultrastructural and biochemical properties of Httex1 as well as its aggregation properties in vitro (11).
We have also recently completed a comprehensive assessment of the GFP tag's impact on the aggregation, inclusion formation, and cellular properties of Httex1 (preprint paper available in BioRxiv https://www.biorxiv.org/content/10.1101/2020.07.29.226977v1 (21)). In this paper, we show that inclusions produced by mutant Httex1 72Q-GFP exhibit striking differences in terms of organization, ultrastructural properties, composition, and their impact on mitochondria functions as compared to the inclusions formed by the tag-free mutant Httex1 72Q. These findings highlight the critical importance of developing new tools that minimize the impact of large fluorescent proteins and/or label-free imaging methods and monitoring Htt aggregation in inclusion formation in cells.
From Riguet et al., __(21)__. Influence of GFP on the ultrastructural properties of Httex1 cellular inclusions by Correlative light electron microscopy (CLEM). CLEM of Httex1 72Q (+/-GFP) transfected in HEK 293 cells after 48h. Confocal images of A. Httex1 72Q and. B Httex1 72Q GFP, 48h after transfection. Httex1 expression (red) was detected using a specific primary antibody against the N-terminal part of Htt (amino acids 50-64) and the nucleus was stained with DAPI (blue). Electron micrographs of C. Httex1 72Q and D. Httex1 72Q GFP inclusions corresponding to confocal images panel A, and B (white square), respectively. Add-in binary images generated from electron micrographs by median filtering and Otsu intensity threshold. E. **Schematic depictions and original electron micrographs of cytoplasmic inclusions formed by native (tag-free) mutant Huntington exon1 proteins (Httex1 72Q, left) and the corresponding GFP fusion protein (Httex1 72Q-GFP).
A recent study by Chongtham et al. (31) also supports our findings and shows that adding short peptide tags such as the HA or the LUM tag to mutant Htt171 changed dramatically the toxic properties of Htt171 as well as its subcellular localization and the compactness of the aggregates formed in cells (e.g.: expression of Htt171 carrying the LUM tag was more toxic than untagged Htt171 and induce the formation of nuclear aggregates rather than the classical cytoplasmic aggregates, See Figure 4).
Figure 4 from Chongtham et al., __(31)__. The influence of peptide modifications on HTT171 fragment behavior. (A) When expressed ubiquitously with da>Gal4, the HTT171-120Q fragment exhibits little or no lethality, but appending either an HA ( ... YPYDVPDYA∗)oraLUMtag ( ... GCCPGCCGG∗) to the C-terminus dramatically increases the toxicity of the fragment. (B) Surviving adult flies expressing an HA-tagged HTT171 transgene exhibit about half the life span of those expressing untagged 171. Flies expressingLUM-tagged HTT171 do not survive to adulthood. (C) Flies expressing HA- or LUM-tagged 171 in tracheal cells show only modest increases in lethality that do not rise to the level of significance (P=0.12; 0.09), but the inclusion of tags changes the subcellular behavior significantly. (D) In contrast, in the prothoracic gland, expression of LUM-tagged 171 shows a significant increase in toxicity compared to 171 alone, while the HA-tagged 171 borders on significance (P=0.051). (E) In trachea, pure 171 forms cytoplasmic aggregates, while the inclusion of HA causes some HTT to become nuclear diffuse, and inclusion of the LUM tag causes the bulk of the HTT to appear as diffuse nuclear material with some cytoplasmic aggregates remaining when expressed with btl>Gal4 at 29◦C. (F) In the prothoracic gland, addition of the LUM tag causes aggregated- **cytoplasmic HTT to become weakly staining diffuse-cytoplasmic material while HTT171HA remains as extensive aggregates in the cytoplasm with a haze of diffuse staining as well. Scale bars are 10 μm
Therefore, in this study, we aimed to investigate for the first time the role of the sequence and the conformational properties of the Nt17 domain in regulating the dynamics of Httex1 fibrillization, the structure and morphology of Httex1 fibrils using a tag-free Httex1 constructs. In our studies, we used multiple methods to examine the structural and cellular properties of these proteins under the same conditions and in the same cellular systems, thus making it possible to correlate the sequence, structural and cellular properties of the different Httex1 proteins (monomers and fibrils). We are happy to see that this was nicely recognized and appreciated by the referee.
Referee #1 “The authors use some outstanding methods to ensure that the conclusions are based on good quality data. Overall, this is an excellent study.” Referee #3 “Their findings provided the precise information for the role of tag-free Nt17. The paper advanced our knowledge of Nt17, especially in the Huntington disease field.”
Major comments
Referee #2 raised the following concerns:
1) « The main hypothesis of this study solely depends on the ability of N17 domain to enhance aggregation (Fig 1 and Fig 2). According to the method for the protein solubilization 1mM TCEP was added to ∆Htt-Ex1, but not to Htt-Ex1 proteins. It is necessary to rule out the potential effects of TCEP on aggregation assay. »
We thank the referee for raising this important point. Indeed, we were also concerned about the potential effect of TCEP and conducted experiments to address this point. Our data show that TCEP does not affect our aggregation assay. This new panel is now included in supporting information as Figure S2A-B and mentioned in the corresponding section of the Material and method (page 29).
2) « The author needs to provide biophysical data of the mutation and phosphorylated proteins with/without Tag. »
All the proteins used in this study have been extensively characterized in recent publications from our lab __(9, 11, 21)__. All these papers are cited throughout our manuscript as well as in the material and method section.
The expression, purification and characterization of native tag-free Httex1 with polyQ repeats ranging from 7 to 49Q has been fully described in Vieweg et al., 2016 __(11)__. In this paper, the aggregation properties of tag-free Httex1 and Httex1 fused to GST or MBP tags were compared by sedimentation assay, while the morphology and length of the resulting fibrils were compared by EM.
The semisynthesis, purification, and characterization of Httex1 42Q phosphorylated at Ser-13 and/or Ser-16 or at T3 was described respectively in Deguire et al., 2018 __(9) and Chiki et al., 2017 (8)__. These studies include kinetics of aggregation and morphological assessment (i.e: heights and lengths) by EM and AFM of the fibrils formed by phosphorylated or unphosphorylated mutants Httex1.
The Httex1 mutants carrying the GFP tag were not used in the in vitro studies but were studied in our overexpression-based cellular model. The direct comparison characterization of inclusion formation by tag-free and GFP-tagged mutant Httex1 and their impact on cellular homeostasis are fully described in a preprint paper available in BioRxiv https://www.biorxiv.org/content/10.1101/2020.07.29.226977v1 (21). In this paper, we show that inclusions produced by mutant Httex1 72Q-GFP exhibit striking differences in terms of organization, ultrastructural properties, composition, and their impact on mitochondria functions as compared to the inclusions formed by the tag-free mutant Httex1 72Q. These findings highlight the critical importance of developing new tools that minimize the impact of large fluorescent proteins and/or label-free imaging methods and monitoring Htt aggregation in inclusion formation in cells.
Referee #3
General comments
“Their findings provided the precise information for the role of tag-free Nt17.__ The paper advanced our knowledge of Nt17, especially in the Huntington disease field.”__
We thank referee #3 for the very positive feedback and for recognizing the quality, depth and significance of our work and its potential impact in the field of Huntingtin disease.
“However, the conceptual advance is limited.”
We respectfully disagree with this assessment that the conceptual advance of our study is limited.
Please see our detailed response to Referee #2 regarding our work's originality and novelty (pages 3-8, in our referees' letter).
Major comments
Referee #3 raised the following concerns:
1) Finding of lateral association (bundling) of __Δ__Nt17-Httex1 fibrils is interesting.
We agree and thank the referee for further highlighting this point.
However, pathological significance is not clear
We agree that the significance for delta 17 is not clear as we do not know whether this cleavage occurs in vivo or not. This is why we decided to extend our studies beyond the removal of Nt17 and investigated the effect of natural PTMs that are known to alter the sequence of Nt17 and modulate its helicity. One additional distinguishing feature of our work is that we used proteins (monomers and fibrils) that bear site-specific bona fide phosphorylation on T3, S13, SS16, and both S13/S16.
a) Does even non-truncated form also increase this kind of bundling when polyQ is expanded?
We have addressed this specific question in a previous study (11) in which we have compared the morphology and length of fibrils formed from Httex1 with polyQ tract from 23Q to 43Q. The increased lateral association was not observed for the fibrils generated from Httex1 43Q or Httex1 23Q, 29Q, or 37Q (Figure 5F) (11). Besides, in this paper, we were the first to show an inverse correlation between the polyQ-length and fibril length, which suggests structural differences between Httex1 proteins with different polyQ repeat lengths. Others have investigated Httex1 with different polyQ repeat, but not using tag-free Httex1 proteins, and they did not observe this inverse relationship between polyQ lenth and fibril length, as we did here and in our previous studies (11).
b) When fibrils are added to striatal neurons like in Fig.5, is this structural feature preserved on the membrane or inside of the cells?
We agree with the referee that this is an important point to address. However, deciphering the structural properties of the membrane-bound and internalized fibrils is not trivial, especially given the limited amount of unlabelled fibrils that are taken up by the cells. This would require extensive optimization of the CLEM technique or the use of an alternative approach such as tomography. Due to the resources and time required to address this important question, this part of the project will be included as part of future projects aimed at investigating the mechanisms of Htt seeding and propagation. We are not aware of any reports by other groups that monitor the structural changes of exogenous fibril after internalization into cells.
c) When Httex1 fibrils species are expressed, is this bundling also observed?
In fact, we have recently completed a comprehensive analysis of the comparison of the inclusions formed by mutant Httex1-72Q and ΔNt17 Httex1.
In this study, we have shown that the expression of Httex1 72Q and the truncated form ΔNt17 Httex1 72Q form cytosolic inclusions of similar size and shape in HEK 293 (Figure 4). We have further characterized the architecture and organization of these inclusions at the ultrastructural level in the context of another project. Our findings are now available online (see Riguet et al., 2021, BioRxiv (21)).
Using correlative light electron microscopy, we showed that the inclusions formed by Httex1 full length or lacking the Nt17 domain exhibited similar architecture and a ring-like organization. Interestingly, we showed the inclusions are composed of highly organized fibrillar network at the core and periphery of the inclusions. In cells inclusion formation is a multiphasic process driven by different phases of polyQ dependent aggregation processes and complex interactions with lipids, proteins and organelles (ER).
Although CLEM approach in neurons provides very good contrast of cytosolic or nuclear inclusions, the resolution of this method is not sufficient to allow imaging at the level of individual fibrils and assessing their morphology. Differences between CLEM and EM resolution can be explained as the slices of the cellular objects are much thicker (~ 50 nm) than the fibrils prepared in vitro and directly deposited on the EM grids (the height of Httex1 pre-formed fibrils is between 5 and 7 nm). To improve the imaging and get a stronger contrast of de novo fibrils in our CLEM samples, we used a double-contrast method based on uranyl acetate and lead citrate stains. Nevertheless, the complex cellular environment and the presence of various cellular objects (e.g: organelles and proteins) surrounding the de novo fibrils might prevent the optimal stain penetration from allowing imaging at the level of individual fibrils. Finally, the preparation of the neuronal samples for CLEM imaging includes ethanol and detergents incubation and resin embedding. These steps can limit the ultrastructure detection of the de novo fibrils at the level of individual fibrils and therefore does not allow to determine their organization and their lateral association.
- d) What function (cell death, membrane integrity or others) is most correlated with this structural feature?
In our extracellular model, we have shown that the conformation and sequence properties of the Nt17 domain are key determinants of the internalization and the subcellular localization of Httex1 fibrils in primary striatal neurons. Httex1 43Q fibrils mostly accumulate at the outer side of the neuronal plasma membrane, Httex1-ΔNt17 43Q fibrils were detected primarily in the nucleus and the M8P-Httex1 43Q fibrils were equally distributed in the cytosol and nucleus.
Despite exhibiting completely different subcellular distribution and internalization levels, the 3 types of fibrils induced neuronal cell death with the highest toxicity observed for ∆Nt17-Httex1 (Figure 7).
Our data suggest that the neurotoxic response is primarily dependent on the subcellular localization of the Httex1 species: 1) accumulation of the Httex1 43Q fibrils on the plasma membrane is likely to induce loss of membrane integrity, based on previous observation with aSyn fibrils; 2) the nuclear accumulation of ∆Nt17-Httex1 aggregates has been previously shown to be highly toxic in several cellular and animal models (10, 67, 68).
Nevertheless, we could not rule out that the high toxicity of ΔNt17-Httex1 fibrils could also be due to their distinct biophysical and structural properties. ΔNt17-Httex1 forms broad fibrils characterized by lateral association, which could provide a surface for the sequestration of intracellular proteins.
2) The authors claimed, “we investigated for the first time, the role of the Nt17 sequence, PTMs and conformation in regulating the internalization and cell-to-cell propagation of monomeric and fibrillar forms of mutant Httex1”. However, so far this reviewer understands that the authors studied the internalization but not cell-to-cell propagation.
We agree and apologize for this mistake as we indeed only limited our study to the uptake, subcellular localization, and toxicity of extracellular Httex1 species in our primary neuronal model. The text has been amended, and cell-to-cell propagation has been removed from the abstract as well as in pages 2, 4, 12, and 17.
Minor points for Referees #1, #2 and #3
Referee #1
- « On page 6, the data on how the Nt17 domain affects Httex1 aggregation, the information on which figure it is referring to is missing.
Done. The information regarding the Figure related to this data has now been added page 6.
In Figure 1A, it is difficult to compare the data on Nt17 and DNt17, particularly for 36Q and 42Q, as the time axis are different. I understand that the kinetics are different, but particularly for the 42Q peptides (Nt17 and DNt17) as their kinetics are not that different, it may be useful to show them in the same panel. »
Done. The new panel that combined the data on Nt17 and DNt17 has now been added as Figure S1B.
Referee #2
1) “Fig 8 the color codes for PolyQ and PolyP need to be corrected. »
Done.
2) “It is a challenging technical problem to produce proteins which are rich in Pro and Gln content. But there is not enough experimental details provided in the methods. Please add detailed procedures for expression and purification of these proteins. »
We thank referee #2 for recognizing the technical challenges to express and produce Httex1 proteins and mutants. The expression, purification and characterization methods of all the proteins used in this manuscript have been extensively detailed in our previous studies (8, 9, 11, 69-71). We have now added the relevant references in the method section (page 29).
Referee #3
1) « Fig.3B arrowhead could not be seen. »
Done. Arrowheads are now added to Fig. 3B.
2) « Fig.4A: what do arrows mean? No scale bars? »
The arrows indicate the aggregates formed in HEK cells overexpressing Httex1 39Q and 72Q. This now added to the legend section of the Figure 4.
The scale bars are already present in both the main and the insets images.
3) « Fig.5A:no scale bars? »
Done. Scale bars were added in the 4 images where they were missing.
4) « Fig.S3. Height and length seem to be wrong. »
The measurement of height and length are performed as in literature (72), and are consistent with previous studies (8, 9, 11).
5) « Fig.S6C: hard to compare. D: What is Htt2-90? Also in Fig.S13. »
We thank the referee for bringing this to our attention and apologize for the lack of consistency in the names used for the proteins studied in Figures S6 and S13. We realized that in Figures S6 and S13 the names of the proteins have been either mislabelled due to the dash that was misplaced or the same proteins have been named in different ways. We agree that this makes it difficult to compare the data between the different panels. We have now corrected our mistakes and Figures S6B and S13 have been updated accordingly.
The name Htt2-90 corresponds to Httex1 expressed from amino acid 2 to amino acid 90, with the first N-terminal methionine removed.
6) « There are many abbreviations difficult to understand in supplement. » Fig.S1 Htt18-90(Q18C) etc.
His6-Intein Ssp stands for the Intein tagged with Histidine amino acid (6 units)
Htt18-90(Q18C) means Httex1 expressed from amino acid 18 to amino acid 90 with the Glutamine (Q) in position 18 mutated in a Cysteine (C).
Htt2-17 means Httex1 synthesized from amino acid 2 to amino acid 17, with the first N-terminal methionine removed.
Htt18-90(Q18A) corresponds to Httex1 expressed from amino acid 18 to amino acid 90 with the Q in position 18 mutated in an Alanine (A).
References
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