Reviewer #1 (Public review):
In this paper, the authors use a doxycycline-inducible DLD1 cell line expressing a Clover-tagged RNA-binding-defective TDP-43 2KQ mutant that forms nuclear "anisosomes" (TDP-43 shell with HSP70 core) to carry out a small-molecule screen using the LOPAC 1280 library to identify compounds that reduce anisosome number or shift their morphology and dynamics. They also conducted a genome-wide siRNA screen to identify genetic modifiers of anisosome formation and dynamics. From these screens, the authors identify pathways in RNA splicing, translation, proteostasis (proteasome and HSP90), and nuclear transport, including XPO1. They then focus on XPO1 as their primary hit. Pharmacological inhibition of XPO1 using KPT-276, Verdinexor, and Leptomycin B reduces anisosome number while enlarging remaining condensates, which retain liquid-like behavior by FRAP and fusion assays. XPO1 overexpression causes fewer, enlarged TDP-43 puncta, including cytoplasmic puncta, with little or no FRAP recovery, interpreted as gel or solid-like aggregates. Anisosome induction reduces detectable nucleoplasmic XPO1 staining. Finally, the authors examine a homozygous TDP-43 K181E iPSC-derived forebrain organoid model, showing increased cytosolic pTDP-43 in K181E/K181E organoids compared to wild-type controls. Chronic low-dose KPT-276 reduces cytoplasmic pTDP-43 without changing total TDP-43 levels. Bulk RNA-seq shows only a modest fraction of dysregulated genes in K181E/K181E organoids are rescued by KPT-276. They conclude that nuclear export, via XPO1, is a key regulator of TDP-43 liquid-to-solid phase transitions and that cytoplasmic aggregation per se may contribute only modestly to TDP-43 proteinopathy, with RNA-processing defects being dominant.
The study presents well-executed chemical and genome-wide siRNA screens in a DLD1 TDP-43 2KQ anisosome model and follows up on nuclear transport, particularly XPO1, as a modulator of TDP-43 phase behavior and cytoplasmic aggregation. The screens are impressive in scale, and the microscopy and fluorescence recovery after photobleaching (FRAP) work is technically strong. However, the central mechanistic and disease-relevance claims are not yet sufficiently supported. There are major concerns about the heavy reliance on non-physiological, RNA-binding-defective, and acetylation-mimetic TDP-43 (2KQ) and a homozygous TDP-43 K181E organoid model. An underdeveloped and partly contradictory mechanistic link exists between XPO1 and TDP-43 phase transitions in the context of prior work showing TDP-43 is not a canonical XPO1 cargo. The paper also appears to overinterpret organoid data to conclude that cytoplasmic TDP-43 aggregation plays only a minor role in pathology, based largely on pTDP-43 antibody staining with limited sensitivity and relatively modest rescue readouts. A deeper mechanistic analysis and additional, more physiological validation are needed for this to reach the level of rigor and impact implied by the title and abstract. The work feels screen-rich but conceptually underdeveloped, with key claims outpacing the data. A major revision with substantial new data and tempering of conclusions is warranted. I outline several problematic areas below:
(1) The central mechanistic discoveries are derived almost entirely from a DLD1 colon cancer cell line overexpressing an RNA-binding-defective, acetylation-mimetic TDP-43 2KQ mutant and homozygous TDP-43 K181E iPSC-derived organoids. Both systems are far from physiological. The 2KQ mutation is a synthetic double lysine-to-glutamine mutant originally designed to mimic acetylation and disrupt RNA binding. In this study, essentially all cell-based mechanistic data on phase behavior, screens, and XPO1 effects rely on 2KQ. Yet there is no quantification of how much endogenous TDP-43 is acetylated in degenerating human neurons, nor whether a 2KQ-like acetylation state is ever achieved in vivo. It is not established that the phase behavior of 2KQ recapitulates the physiological or pathological phase behavior of wild-type TDP-43 or genuine disease-linked mutants, which may retain partial RNA binding and different post-translational modification patterns. As a result, it is difficult to know whether the modifiers identified here regulate a highly artificial 2KQ condensate or physiologically relevant TDP-43 condensates. To address this concern, the paper would benefit from quantifying endogenous TDP-43 acetylation at the relevant lysines in control and ALS/FTD patient tissue or more disease-proximal models such as heterozygous TARDBP mutant iPSC neurons, which would justify the focus on an acetyl-mimetic mutant. Key phenomena, including XPO1 dependence of phase behavior, effects of proteasome and HSP90 inhibition, and effects of splicing and translation inhibitors, should be tested for wild-type TDP-43 expressed at near-physiological levels and for one or more bona fide ALS/FTD-linked TARDBP mutants that are not acetyl mimetics. At a minimum, the authors should show that endogenous TDP-43 in neuronally differentiated cells exhibits qualitatively similar responses to XPO1 modulation, rather than exclusively relying on DLD1 2KQ overexpression.
(2) The organoid model is based on a homozygous K181E knock-in line. However, in patients, TARDBP mutations are overwhelmingly heterozygous. Homozygosity is thus a severe, arguably non-physiological sensitized background that may exaggerate nuclear RNA mis-splicing and phase defects and alter the relative contribution of cytoplasmic aggregation versus nuclear loss-of-function. In addition, it is not fully clear from this manuscript whether the structures in K181E organoids are bona fide anisosomes as defined in Yu et al. 2021, characterized by HSP70-enriched central liquid cores with TDP-43 shells and similar FRAP and fusion behavior to anisosomes in the DLD1 model. At present, the organoid section is framed as validation of "anisosome-bearing organoids," but the figures in this manuscript mainly show pTDP-43 puncta and total TDP-43 immunostaining, without detailed structural or biophysical characterization. The authors should explicitly compare heterozygous K181E/+ organoids or another heterozygous TARDBP mutant line with homozygous K181E/K181E organoids to assess whether XPO1 inhibition has similar effects in a genotype that more closely resembles patient genetics. They should provide direct evidence that the K181E condensates in organoids are anisosomes through HSP70 core immunostaining, three-dimensional reconstruction, and FRAP measurements, and clarify whether KPT-276 is acting on anisosome-like structures or more generic cytoplasmic aggregates or puncta. Without this, the leap from a DLD1 2KQ cancer cell model to human ALS/FTD-relevant neurons is not convincingly supported.
(3) The title and framing assert that "nuclear export governs TDP-43 phase transitions." However, prior studies such as Pinarbasi et al. 2018 and Duan et al. 2022 indicate that TDP-43 is not a canonical XPO1 cargo and that its export is largely passive, with active nuclear import being the dominant determinant of nuclear localization. The authors cite these studies but still position XPO1 as a central, quasi-direct regulator. The data presented are largely correlative or based on pharmacologic manipulation and overexpression in an overexpression mutant background, with no direct evidence that XPO1 engages TDP-43 in a specific, regulated manner. Even if XPO1 does not engage WT TDP-43, it could still engage the 2KQ variant, which needs to be tested.
(4) The XPO1 perturbations yield somewhat confusing phenotypes. XPO1 inhibition using Leptomycin B, KPT-276, and Verdinexor reduces anisosome number and enlarges remaining anisosomes, which remain liquid-like by FRAP recovery and fusion assays and stay nuclear. XPO1 overexpression causes fewer, enlarged puncta, but these are FRAP-impaired (gel-like) and redistribute to the cytoplasm. Thus, both decreased and increased XPO1 activity reduce anisosome number and enlarge puncta, but with opposite phase behaviors and subcellular localizations. The model presented in Figure 5L is relatively qualitative and does not resolve these issues. Moreover, XPO1 inhibition globally impairs nuclear export of many cargos and profoundly alters the nuclear environment, transcription, RNA processing, and chromatin. It is therefore difficult to conclude that the observed effects are specific to TDP-43 phase regulation as opposed to secondary consequences of broad nuclear export blockade.
(5) The authors show that anisosome induction depletes nucleoplasmic XPO1 signal and that mCherry-XPO1 can be seen in some TDP-43 puncta. However, antibody penetration into anisosomes is limited, so XPO1 depletion from nucleoplasm could reflect sequestration in the anisosome shell or core, but this is not demonstrated. There is no demonstration of physical interaction, even indirect interaction, between XPO1 and TDP-43 or a defined adaptor, nor identification of a specific mutant of XPO1 that selectively disrupts this putative interaction while preserving other functions. The known TDP-43 NES has been shown to be weak and not a functional XPO1-dependent NES in multiple studies. If XPO1 is acting through an adaptor that recognizes 2KQ or K181E specifically, that by itself would bring into question the generality of the mechanism for wild-type TDP-43.
(6) To support a mechanistic claim that nuclear export governs TDP-43 phase transitions, more targeted evidence is needed. The authors should test whether siRNA knockdown or CRISPR interference of XPO1 in the DLD1 2KQ model reproduces the effects seen with Leptomycin B and KPT-276, including FRAP and fusion phenotypes, and verify on-target effects by rescue with an siRNA-resistant XPO1 construct. They should demonstrate that canonical XPO1 cargos behave as expected under the inhibitor conditions used, as a positive control, and that the concentrations used are not grossly toxic. They should attempt to identify or at least constrain candidate adaptors that might enable XPO1-dependent export of TDP-43 through proteomic analysis of XPO1 co-purifying with 2KQ condensates or loss-of-function studies of candidate adaptors from the siRNA screen. Finally, they should test whether a TDP-43 mutant that cannot bind the proposed adaptor still responds to XPO1 manipulation.
(7) Even with these data, what is currently shown is that global modulation of nuclear export capacity can alter the phase behavior and localization of a highly overexpressed RNA-binding-defective TDP-43 mutant and of K181E in organoids. This is important, but it is weaker than asserting that XPO1 directly governs TDP-43 phase transitions in physiological contexts. The title, abstract, and Discussion should be tempered to reflect that nuclear export is one of several pathways, alongside RNA splicing, translation, and proteostasis, that influence TDP-43 phase states in this model, and that the specific mechanism and cargo relationship between XPO1 and TDP-43 remain unresolved and may be indirect.
(8) The authors conclude that cytoplasmic TDP-43 aggregation plays only a modest role in TDP-43 proteinopathies because in homozygous K181E organoids, chronic KPT-276 treatment almost abolishes cytoplasmic pTDP-43 puncta, yet bulk RNA-seq shows only a relatively small fraction of dysregulated genes are rescued. There are several issues with this inference. Relying primarily on pTDP-43 antibody staining to define cytoplasmic TDP-43 aggregation is limiting. pTDP-43 antibodies label only phosphorylated species and may miss non-phosphorylated, oligomeric, or amorphous TDP-43 species that could still be toxic. Different pTDP-43 antibodies vary in epitope accessibility depending on aggregate conformation and subcellular location. More sensitive approaches, such as high-affinity TDP-43 RNA aptamer probes developed by Gregory and colleagues, biochemical fractionation for SDS-insoluble and urea-soluble TDP-43, and filter-trap assays, would provide a more quantitative assessment of cytoplasmic aggregation and its reduction by KPT-276. Without these, it is not safe to assume that cytoplasmic aggregation has been eliminated, as opposed to one antigenic subclass.
(9) The treatment window, spanning from day 87 to 122 with 20 nanomolar KPT-276, may be too late or too mild to reverse entrenched nuclear RNA-processing defects, even if cytoplasmic inclusions are cleared. Once widespread cryptic exon inclusion and alternative polyadenylation misregulation are established, many downstream changes may become self-sustaining or only partially reversible. Moreover, XPO1 inhibition will massively rewire nucleocytoplasmic transport of many transcription factors, splicing factors, and RNA-binding proteins. Thus, the lack of full transcriptomic rescue cannot be cleanly interpreted as evidence that cytoplasmic aggregates are only modest contributors. It may instead reflect that nuclear dysfunction is primary and XPO1 inhibition does not correct, and may even exacerbate, certain nuclear defects.
(10) To support a causal statement about the modest contribution of cytoplasmic aggregates, one would want more direct measures of neuronal health and function, such as cell death, neurite complexity, synaptic markers, and electrophysiology before and after KPT-276, not only transcriptomics. A way to selectively reduce cytoplasmic aggregation without globally inhibiting nuclear export would allow comparison of outcomes.
(11) Given these caveats, the concluding statements that cytoplasmic TDP-43 aggregation is only a modest contributor should be substantially softened. A more defensible interpretation is that in this homozygous K181E organoid model, chronic global XPO1 inhibition reduces pTDP-43-positive cytoplasmic puncta but only partially normalizes the steady-state transcriptome, suggesting that persistent nuclear RNA-processing defects and other pathways continue to drive pathology.
(12) The screens are a major strength but need more rigorous validation for key hits, especially nuclear transport factors. For the siRNA screen, hits are filtered by anisosome number per nucleus, but there is no direct demonstration in the main text that XPO1 or CSE1L knockdown is efficient at the messenger RNA or protein level. For the highlighted genes, Western blot or quantitative polymerase chain reaction validation and phenotypic rescue would strengthen confidence. For small-molecule hits, it is not systematically shown that anisosome modulation is independent of changes in total TDP-43 2KQ expression or gross toxicity. Translation inhibitors are tested for this, but for many other hits, including proteasome, HSP90, and kinase inhibitors, expression and general nuclear structure should be monitored. Given the reliance on anisosome count as a readout, secondary screens that specifically distinguish changes in TDP-43 expression levels, changes in nuclear morphology or cell cycle, and specific changes in anisosome phase behavior, including FRAP and fusion for top hits, would greatly increase interpretability.
(13) The classification of condensates as liquid versus gel-like or solid is based almost entirely on FRAP recovery or lack thereof. While FRAP is appropriate, interpretations could be made more robust by including half-region-of-interest bleach controls and assessing mobile fractions and recovery kinetics more quantitatively across conditions. Complementing FRAP with other phase-behavior assays such as sensitivity to 1,6-hexanediol, shape relaxation after deformation, and coarsening behavior over longer timescales would strengthen the analysis. At present, some assignments, such as that XPO1 overexpression drives a gel-like transition, are reasonable but somewhat qualitative.
(14) For the Leptomycin B and KPT-276 experiments in cells and organoids, it would be important to confirm that canonical XPO1 cargo proteins accumulate in the nucleus and that the concentrations used are within a range that is not overtly toxic over the experimental timeframe. Assessing nuclear morphology, chromatin condensation, and general transcriptional activity through global RNA synthesis or key reporter genes would ensure that observed effects are not secondary to severe global nuclear export collapse.
(15) In the organoid section, it is not clear how many independent iPSC clones and organoid batches were used per condition, nor whether batch effects were assessed in the bulk RNA-seq analysis. This should be fully specified and ideally controlled with isogenic wild-type and K181E clones. For transcriptional rescue, it is important to know whether the changes in wild-type organoids treated with KPT-276 are negligible. A direct wild-type comparison with or without KPT-276 is important to disentangle general drug effects from K181E-specific rescue. More detailed quantification of total TDP-43 and pTDP-43 in both nuclear and cytoplasmic fractions, including biochemical fractionation if possible, would strengthen the assertion that KPT-276 specifically reduces cytosolic pTDP-43 aggregates while sparing nuclear TDP-43.
(16) Beyond the core issues above, several additions could greatly enhance the impact. The manuscript currently emphasizes XPO1, but the genetic and chemical data clearly implicate RNA splicing, translation, and proteostasis as equally strong or stronger regulators of TDP-43 phase states. A more integrated model that explains how these pathways intersect, for example, how splicing factor availability, ribosome loading, and proteasome capacity co-govern anisosome nucleation, growth, and hardening, would be valuable.
(17) A key unresolved question is whether XPO1 is acting directly on TDP-43, or instead primarily regulates anisosomes by exporting other factors that more proximally control TDP-43 phase behavior. Given that TDP-43 is not a canonical XPO1 cargo and prior work indicates that its nuclear export is largely passive, it seems at least as plausible that XPO1 inhibition alters the nuclear concentration or localization of splicing factors, RNA-binding proteins, chaperones, or other modifiers identified in the screens, and that changes in these proteins secondarily reshape anisosome dynamics. In other words, XPO1 may be exporting a more direct regulator of anisome formation and hardening, rather than exporting TDP-43 itself in a specific, regulated way. The current data do not distinguish between these possibilities. Systematic identification of XPO1-dependent cargos that colocalize with or biochemically associate with anisosomes, combined with targeted perturbation of their nuclear export, would be needed to determine whether the relevant XPO1 substrate in this system is actually TDP-43 or an upstream modulator of its phase behavior.
(18) Testing whether identified modifiers converge on nuclear TDP-43 concentration would be informative. Since phase separation is concentration-dependent, measuring nuclear versus cytoplasmic TDP-43 levels across key perturbations, including splicing inhibition, translation inhibition, proteasome inhibition, HSP90 inhibition, and XPO1 modulation, would help determine whether modifiers mainly work by changing nuclear TDP-43 concentration or by altering interaction networks and the material properties of condensates.
(19) Examining other ALS-relevant RNA-binding proteins would be valuable. Given the role of XPO1 and other hits, it would be informative to briefly test whether similar principles apply to FUS, hnRNPA1, or other ALS-relevant RNA-binding proteins in the same cellular context, to argue for generality versus TDP-43-specific idiosyncrasies of the 2KQ system.
(20) The Introduction sometimes implies that anisosomes are common and well-established intermediates en route to pathology. It would be helpful to more clearly state that, to date, anisosomes are primarily observed in overexpression and mutant systems and have not yet been unequivocally demonstrated in human patient tissue. The link between PDGFRβ, PAK4, GSK-3β, and YAP and TDP-43 phase dynamics is intriguing but only briefly mentioned. The authors should either expand on this or tone down the emphasis in the Results section.
(21) In the organoid methods, the authors should consider clarifying whether doxycycline is continuously used, which might alter TDP-43 expression and nuclear transport in a non-negligible way.
(22) For statistical methods, it would be beneficial to indicate whether multiple-comparison corrections were applied for the many FRAP, anisosome count, and size comparisons beyond DESeq2 internal corrections for RNA-seq.
(23) Some figure legends could more clearly indicate whether the images shown are single z-planes or maximum intensity projections and how the thresholding for anisosome detection was performed.
(24) In its current form, the manuscript contains an impressive set of screens and some nicely executed imaging of TDP-43 condensates, highlighting nuclear export among other pathways as a modulator of TDP-43 phase behavior. However, the physiological relevance is undercut by heavy reliance on an acetylation-mimetic, RNA-binding-defective TDP-43 mutant and a homozygous K181E organoid model. The mechanistic link between XPO1 and TDP-43 remains largely inferential and partly at odds with prior work. The conclusion that cytoplasmic TDP-43 aggregation is only a modest contributor to disease is not firmly supported by the available data.
(25) With substantial additional mechanistic work, particularly around XPO1, rigorous validation in more physiological TDP-43 contexts, more sensitive detection of cytoplasmic TDP-43 aggregates, and a tempering of the central claims, this study could make a meaningful contribution to understanding how nucleocytoplasmic transport and other cellular pathways influence TDP-43 phase transitions and aggregation. The work should be reframed as an important screening study that identifies nuclear export as one among several cellular processes that modulate TDP-43 phase behavior in a model system, rather than as a definitive demonstration that nuclear export governs pathological TDP-43 aggregation in disease.
