Nuclear export governs TDP-43 phase transitions and cytoplasmic aggregation

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.

Reviewer #2 (Public review):

Summary:

This manuscript addresses an important and timely question in TDP-43 biology by systematically identifying regulators of TDP-43 anisosome formation, with a particular focus on nuclear export via XPO1. Using a combination of unbiased chemical screening, genetic perturbation, and advanced imaging approaches, the authors propose that inhibition of nuclear export modulates the abundance and biophysical properties of TDP-43 anisosomes. The study is conceptually innovative and has potential relevance for neurodegenerative diseases characterized by TDP-43 pathology. However, significant concerns regarding experimental controls, reporting transparency, and model translatability currently limit the strength of the conclusions and the interpretability of several key findings.

Strengths:

(1) The study employs an unbiased, hypothesis-free compound screen to identify regulators of TDP-43 anisosome formation, which is a major strength and reduces confirmation bias.

(2) The authors combine chemical and genetic screening approaches, providing orthogonal validation of key pathways and increasing confidence in the biological relevance of top hits.

(3) The focus on biophysical properties of TDP-43 assemblies, assessed through imaging and FRAP, moves beyond simple presence/absence of aggregates and provides mechanistic insight into the biophysical states of TDP-43.

(4) The use of multiple experimental modalities, including live-cell imaging, FRAP, pharmacological perturbation, and transcriptomic analysis, reflects a technically sophisticated and ambitious study design.

(5) The authors attempt to extend findings beyond immortalized cancer cell lines by incorporating organoid models, demonstrating awareness of disease relevance and translational importance.

Overall, the manuscript is clearly written and logically structured, making complex experimental workflows accessible and the central hypotheses easy to follow.

Weaknesses:

Despite its strengths, the manuscript has several major limitations that affect data interpretation and confidence in the conclusions.

(1) Lack of appropriate controls for overexpression experiments:

A central concern is the absence of proper controls for TDP-43 and XPO1 overexpression. Prior studies (including those cited by the authors, Archbold et al.2018) show that overexpression of WT TDP-43 alone is toxic to neurons. Thus, the experimental system itself may induce anisosome formation independently of the mechanisms under study. Similarly, XPO1 overexpression lacks a suitable control (e.g., mCherry alone or mCherry fused to a protein known to be independent of TDP-43). The near-complete colocalization of XPO1 with TDP-43 anisosomes upon overexpression raises the possibility that these structures reflect non-physiological protein accumulation rather than regulated assemblies.

1. Insufficient experimental and analytical transparency:

The manuscript frequently lacks clear reporting of experimental details. In multiple figures, the stated number of independent experiments does not match the number of data points shown, making it difficult to assess statistical validity. Concentrations used in the compound screen are not clearly defined, nor is it stated whether multiple concentrations were tested. It is unclear how many wells, cells, or independent cultures were analyzed. The criteria used to reduce 1,533 screening hits to 211 candidates via STRING analysis are not explained. Knockdown and overexpression efficiencies are not reported.

(3) RNA-seq concerns:

The RNA-seq experiments are particularly problematic. The number of biological replicates per condition is not stated, and heatmaps suggest that only one sample per group may have been used, which would preclude statistical analysis. No baseline comparison between WT and mutant TDP-43 is shown. Given that TDP-43 is an RNA-binding protein, splicing analyses would be far more informative than gene expression alone, yet no splicing data are presented. Moreover, nuclear retention of TDP-43 does not preclude nuclear aggregation, which may still impair its splicing function.

(4) Limited translatability to neuronal biology:

All anisosome analyses are performed in a cancer cell line, raising concerns about relevance to post-mitotic neurons. While organoids are used as a secondary model, the assays performed do not overlap with those used in cancer cells, making it difficult to assess whether anisosome-related mechanisms are conserved. Neuronal toxicity, a critical outcome given known TDP-43 biology, is not assessed. Prior work has shown that WT TDP-43 overexpression alone is toxic to neurons, yet this is not addressed.

(5) Conceptual and interpretational gaps:

The authors quantify anisosome number but also report conditions in which anisosome number decreases while size increases. The biological interpretation of larger anisosomes is not discussed, and whether this reflects improvement or worsening of pathology is unclear. Compounds targeting the same mechanism (e.g., nuclear export inhibition) are inconsistently used across experiments (KPT compounds, verdinexor, leptomycin B), raising concerns about reproducibility. In organoids, the experimental paradigm shifts to long-term treatment (35 days vs. 16 hours), further complicating interpretation.

(6) Overinterpretation of rescue effects:

Although the authors state that they aim to test whether nuclear export inhibition rescues neuronal defects, no functional neuronal readouts are provided (e.g., viability, morphology, axon outgrowth, or electrophysiological measures). RNA-seq alone is insufficient to support claims of rescue.

(7) Finally, the model does not appear to exhibit cytosolic TDP-43 aggregation at baseline. It remains unclear whether longer induction would produce cytosolic gel-like assemblies and whether these would be prevented by nuclear export inhibition. Long-term data are shown only in organoids, yet anisosome formation is not assessed there.

Reviewer #3 (Public review):

Summary:

TDP-43 proteinopathy is broadly found in neurodegenerative diseases. This manuscript investigates how nuclear export influences the biophysical properties of TDP-43. The authors use a combination of chemical screening and genome-wide siRNA screening to identify pathways that modulate TDP-43 liquid-to-solid transitions. Overall, the study employs a broad array of approaches and addresses an important question in TDP-43 pathobiology. The identification of nuclear export as a central regulator is compelling and conceptually aligns with the emerging view that TDP-43 nucleocytoplasmic trafficking is a major defect in neurodegeneration.

Strengths:

This work integrates chemical and genetic screening to identify novel modifiers. The candidates were validated in both reporter cell lines and iPS-differentiated organoids. The findings support the nucleocytoplasmic transport is important for the biophysical properties of TDP-43.

Weaknesses:

The mechanisms underlying the connection between nuclear export and phase transition need further clarification. Broader consequences of XPO1 inhibition are not addressed.

Author response:

Public Reviews:

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.

We thank the reviewer for carefully summarizing our study.

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.

Acetylation of endogenous TDP-43 was reported by several studies. Although it occurs at low levels under normal conditions, TDP-43 acetylation is upregulated under stress conditions (e.g. oxidative stress and proteotoxic stress) (PMID: 25556531; PMID: 28724966). Importantly, Cohen et al. reported the identification of acetylated TDP-43 in ALS patient spinal cord (PMID: 25556531), while Yu et al. showed that endogenous wildtype TDP-43 undergoes demixing when neurons were treated with either a deacetylase inhibitor or proteasome inhibitors (PMID: 33335017). These studies also show that acetylated TDP-43 is defective in RNA binding and more prone to aggregation. Furthermore, ectopic expression of acetylated TDP-43 mimetics in cells and mice induces cellular defects similar to those observed in disease models (PMID: 28724966). Thus, our findings, based on previously established TDP-43 mimetics, should provide valuable information regarding the regulation of TDP-43 phase behavior. We agree with the reviewers that the model used in this study has its limitations, and we will be happy to revise the manuscript to tone down some conclusions, and include more background information to justify the use of TDP-43 acetylation mimetics.

(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.

The reviewer is correct that the use of homozygous K181E organoids generates a homogenous background that is more sensitive for detecting phosphor-TDP43. The goal of the experiment was to test whether XPO1 inhibition mitigates the aggregation of a TDP-43 disease mutant. For this purpose, we believe that our experimental setup is suitable. We agree that we should not extrapolate the result to overemphasize on its disease connections. We will revise the paper to tone down this part.

Regarding the immunostained signals in K181E organoids, we did not report them as anisosomes. As widely documented in the literature, p-TPD-43 is widely used as a marker of pathological TDP-43 aggregation. P-TDP-43 is enriched in pathological aggregates in human ALS and FTLD patients, colocalized with other aggregation signatures such as ubiquitin and other aggregation prone proteins (PMID: 36008843), and is being used as a diagnostic marker for neurodegeneration (PMID: 31661037). Figure 7A showed that inhibiting nuclear export mitigates the accumulation of p-TDP-43 in mutant tissues. We will revise the subheading and the corresponding text to avoid the confusion.

(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.

We did not conclude or imply the regulation of TDP-43 by XPO1 is direct. In fact, we explicatively mentioned on page 8 that the regulation is likely indirect and mediated by other factors. The sentence reads as “Since XPO1 does not bind TDP-43 directly (Pinarbasi et al., 2018), additional factors likely facilitate XPO1-mediated TDP-43 nuclear egression under this condition.” We can revise the part to make it clearer. We will also revise the title and change the framing accordingly.

(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.

The reviewer correctly summarizes our data and interpretation: XPO1 loss-of-function and gain-of-function generate opposite phenotypes regarding TDP-43 phase behavior. We agree that additional studies are needed to elucidate the underlying mechanism (e.g. direct or indirect), but we feel that belong to a separate study. We plan to re-test the effect of nuclear export inhibition on the subcellular distribution of WT TDP-43 and the acetylation mimetics. We will also add more discussions about the potential indirect effect of XPO-1 inhibition on TDP-43 phase behavior.

(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.

We agree that our observation does not demonstrate an interaction between XPO1 and TDP-43. As mentioned above, we did discuss that the regulation of TDP-43 by XPO1 is likely indirect. We will revise our paper further to separate any speculative statements from the data and narrow our mechanistic claim.

(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.

The anisosome enlargement phenotype upon XPO1 depletion was seen in our siRNA screend, which was identified by machine-based image analyses using 6 distinct siRNAs. This, together with the chemical inhibition experiments, convinced us that the phenotype is specifically caused by XPO1 inactivation.

When characterizing the effect of XPO1 inhibition on anisosome dynamics, we preferred chemical inhibitor because the effect is acute, and is therefore, less likely to be caused by secondary effects.

Regarding the inhibitor concentration, a literature survey suggested that 50-200nM of Leptomycin B was commonly used. We chose 200nm to ensure a quick and complete inhibition of XPO1-mediated nuclear export (see Figure 3 in PMID: 9628873). This dose is also well tolerated by our cells, at least during the chosen time window.

We did not propose any specific adaptor that mediates XPO1 interaction with TDP-43. The identification of such adaptor is out of the scope of this study. We will revise our paper to avoid this confusion.

(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.

We will revise the title, abstract, and discussion to temper the conclusion.

(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.

We agree with the reviewer that p-TDP-43 may not represent all aggregate species. However, p-TDP-43 antibodies detect the pathologically validated species most tightly associated with TDP-43 proteinopatheis. In human ALS and FTLD-TDP tissues, cytoplasmic inclusions are strongly immunoreactive for phosphorylated TDP-43 (typically S409/410, as used here). Additionally, p-TDP-43 immunohistochemistry is a routine diagnostic criterion in neuropathology. For these reasons, we believe that the observation that inhibition of XPO1 significantly reduces p-TDP-43 is a very significant finding, as it suggests that an improvement in TDP-43 proteinopathy can be achieved by the inhibition of nuclear transport. We plan to revise the text to better explain the significance of p-TDP-43 staining.

(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.

We agree with the reviewer that the lack of rescue may be caused by technical issues. We will remove the RNAseq data and related texts since it is not essential for our main conclusion.

(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.

We will remove the discussion regarding the role of cytoplasmic aggregates in disease.

(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.

We agree with the review and will revise this part accordingly.

(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.

For the siRNA screen, each positive hit was confirmed by two rounds of screen with 6 independent siRNAs in total. Although we did not validate the knockdown efficiency due to the large number of hits, we routinely include a positive siRNA control in our study (siRNAdeath), which targets an essential gene. Transfection efficiency was controlled by measuring cell viability after knocking down this essential gene. In addition, the identification of XPO1 as a positive regulator of TDP-43 phase behavior was independently validated by our chemical genetic screens. We feel confident that XPO1 is a key modulator of TDP-43 phase behavior. For chemical treatment experiments, the anisosome fusion phenotypes could be detected as early as 5 h post treatment. Given the short treatment, we do not expect a significant change in protein level or toxicity.

(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.

In this study, we described two types of condensates formed by TDP-43 2KQ, one characterized previously as nuclear anisosome and the other as cytosolic puncta in XPO1 over-expressing cells. The two can be clearly distinguished by several features including the subcellular localization, shape, and mobility. We feel that our FRAP data clearly segregate these puncta into two distinctive types of assemblies. The difference in fluorescence recovery rate is huge. The proposed half-region-of-interest bleach is technically challenging for small anisosomes under normal conditions. When they were enlarged by Leptomycin B treatment, we did perform both whole anisosome bleach and partial bleach (Figure 5D, I). Both assays demonstrate that TDP-43 in these enlarged anisosomes is highly mobile.

(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.

In Leptomycin B treatment experiments, we carefully chose a dose that was previously validated (see Figure 3 in PMID: 9628873). Based on our DAPI staining, the nuclear morphology appears normal (Figure 5A). Additionally, in cell line-based experiment, the effect of Leptomycin B on anisosomes was detected 6-8 hours post treatment. The change in global protein synthesis should be relatively minor at this time point. In the organoid experiment, the drug dose was determined by a pre-experiment in which the morphology of organoids was evaluated after prolonged treatment with different doses of the inhibitors.

(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.

The organoid experiment was performed with two batches per condition. This is to reduce the effect of batch variation. The wildtype cells and K181E mutant are derived from the same genetic background. We will revise the text to clarify these issues. Given the cost of this experiment, we did not include drug-treated wild-type as a control. Given the criticisms by review 1 and 2 on the RNAseq data, we will remove this non-essential data from our revision.

(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.

We agree with the reviewer that these are important directions for future studies. We will include some discussions on a possible model that integrate these factors.

(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.

The reviewer raises an important point. We did include some discussions along this line in our paper. We can add more to further clarify this issue. Again, as mentioned in the original draft, we did not conclude there is an interaction between TDP-43 and XPO1.

(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.

We will measure the nuclear TDP-43 concentration in our imaging experiments and add the data to a revised version.

(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.

We agree that this is an important issue but we feel the proposed experiments are beyond the scope of the study.

(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.

We will revise the introduction accordingly.

(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.

The organoid model does not involve protein overexpression or doxycycline treatment. We measured endogenous p-TDP-43. We will revise to paper to avoid the confusion.

(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.

We will add this information to the figure legends during revision.

(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.

We will revise the figure legends to include this information. As for anisosome detection, because they are so obvious, standard thresholding was sufficient to identify them.

(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.

We agree with the reviewer that the strength of the study is our unbiased approach that identify pathways capable of modulating TDP-43 phase separation behavior. We will revise our paper to carefully discuss the potential physiological relevance of our study and tone down some mechanistic conclusions, as suggested by the reviewer.

(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.

We will reframe the study as an important screening study that identifies nuclear export among several other pathways as modulators of TDP-43 phase behavior.

Reviewer #2 (Public review):

Summary:

This manuscript addresses an important and timely question in TDP-43 biology by systematically identifying regulators of TDP-43 anisosome formation, with a particular focus on nuclear export via XPO1. Using a combination of unbiased chemical screening, genetic perturbation, and advanced imaging approaches, the authors propose that inhibition of nuclear export modulates the abundance and biophysical properties of TDP-43 anisosomes. The study is conceptually innovative and has potential relevance for neurodegenerative diseases characterized by TDP-43 pathology. However, significant concerns regarding experimental controls, reporting transparency, and model translatability currently limit the strength of the conclusions and the interpretability of several key findings.

We thank the reviewer for acknowledging the significance and innovation of our study.

Strengths:

(1) The study employs an unbiased, hypothesis-free compound screen to identify regulators of TDP-43 anisosome formation, which is a major strength and reduces confirmation bias.

(2) The authors combine chemical and genetic screening approaches, providing orthogonal validation of key pathways and increasing confidence in the biological relevance of top hits.

(3) The focus on biophysical properties of TDP-43 assemblies, assessed through imaging and FRAP, moves beyond simple presence/absence of aggregates and provides mechanistic insight into the biophysical states of TDP-43.

(4) The use of multiple experimental modalities, including live-cell imaging, FRAP, pharmacological perturbation, and transcriptomic analysis, reflects a technically sophisticated and ambitious study design.

(5) The authors attempt to extend findings beyond immortalized cancer cell lines by incorporating organoid models, demonstrating awareness of disease relevance and translational importance.

Overall, the manuscript is clearly written and logically structured, making complex experimental workflows accessible and the central hypotheses easy to follow.

Weaknesses:

Despite its strengths, the manuscript has several major limitations that affect data interpretation and confidence in the conclusions.

(1) Lack of appropriate controls for overexpression experiments:

A central concern is the absence of proper controls for TDP-43 and XPO1 overexpression. Prior studies (including those cited by the authors, Archbold et al.2018) show that overexpression of WT TDP-43 alone is toxic to neurons. Thus, the experimental system itself may induce anisosome formation independently of the mechanisms under study. Similarly, XPO1 overexpression lacks a suitable control (e.g., mCherry alone or mCherry fused to a protein known to be independent of TDP-43). The near-complete colocalization of XPO1 with TDP-43 anisosomes upon overexpression raises the possibility that these structures reflect non-physiological protein accumulation rather than regulated assemblies.

As mentioned in our response to reviewer 1, point 1, we will add more discussion regarding the use of acetylation mimetics in our study. We agree with the reviewer that these large puncta (both anisosomes and gel-like structures) likely resulted from TDP-43 overexpression. Nevertheless, in a titration experiment done by Yu et al. 2020 (PMID: 33335017), they showed that ectopic TDP-43 undergo demixing even at concentrations lower than endogenous TDP-43, although the demixed puncta were very small. Their result suggested that overexpression per se does not change TDP-43 phase behavior, only enlarging the demixed TDP-43 structures. This is necessary for our screen and imaging-based characterization. We will revise the text to clarify this point.

For XPO1, we did include mCherry alone control in the study but due to space limit in Figure 5, we did not include it. We can put the data in a Supplementary Figure during revision.

(2) Insufficient experimental and analytical transparency:

The manuscript frequently lacks clear reporting of experimental details. In multiple figures, the stated number of independent experiments does not match the number of data points shown, making it difficult to assess statistical validity. Concentrations used in the compound screen are not clearly defined, nor is it stated whether multiple concentrations were tested. It is unclear how many wells, cells, or independent cultures were analyzed. The criteria used to reduce 1,533 screening hits to 211 candidates via STRING analysis are not explained. Knockdown and overexpression efficiencies are not reported.

We apologize for these omissions. We will add more experimental details to the figure legends and the method part. For the imaging experiments, data points reflect randomly selected individual cells imaged in 2-3 independent biological repeats. For chemical screens, we screened against NCATS libraries first at top concentration (10 mM) to ensure inhibitory efficacy for all compounds. In the follow-up study, we validated the top hits using a series of concentrations, as shown in Figure 1B.

We will explain the STRING analysis in more detail. We did not check XPO1 knockdown efficiency in high through-put screens (HTS) for several reasons. Firstly, the large number of positive hits makes it impossible to check knockdown efficiency for all these hits. Secondly, the effect of XPO1 knockdown on anisosomes was seen with 6 different siRNAs in two rounds of screens. Thirdly, in the HTS protocol, we routinely included a transfection control (siRNAdeath) to indicate high transfection efficiency. We would only process the data if siRNAdeath control killed > 90% of the cells.

(3) RNA-seq concerns:

The RNA-seq experiments are particularly problematic. The number of biological replicates per condition is not stated, and heatmaps suggest that only one sample per group may have been used, which would preclude statistical analysis. No baseline comparison between WT and mutant TDP-43 is shown. Given that TDP-43 is an RNA-binding protein, splicing analyses would be far more informative than gene expression alone, yet no splicing data are presented. Moreover, nuclear retention of TDP-43 does not preclude nuclear aggregation, which may still impair its splicing function.

We apologize for the lack of clarity regarding the RNA-seq design. For each condition, organoids of two independently differentiated batches were treated in triplicate. We pooled the organoids of the same treatment from the two batches to reduce the impact of batch variation.

Given the criticisms from both reviewer 1 and 2 on the limitation of the RNAseq study, we plan to remove this data from the revised manuscript.

(4) Limited translatability to neuronal biology:

All anisosome analyses are performed in a cancer cell line, raising concerns about relevance to post-mitotic neurons. While organoids are used as a secondary model, the assays performed do not overlap with those used in cancer cells, making it difficult to assess whether anisosome-related mechanisms are conserved. Neuronal toxicity, a critical outcome given known TDP-43 biology, is not assessed. Prior work has shown that WT TDP-43 overexpression alone is toxic to neurons, yet this is not addressed.

We agree with the reviewer that the model used in this study is not directly relevant to neurodegeneration. However, as pointed out by the reviewer, neurons are much more sensitive to TDP-43-associated toxicity. By contrast, the cell line used in this study can tolerate TDP-43 overexpression with no detectable cytotoxicity. This feature makes it feasible to evaluate how different cellular processes modulate TDP-43 phase behavior without the confounding effect from toxicity. The fact that TDP-43 expression was induced for a short period of time also help minimize the impact of toxicity. Notably, the processes identified by our screens are all house-keeping pathways that is present in neurons. Thus, we believe that the reported findings are likely applicable to neurons, though we will revise our paper to make sure that we don’t overstate the clinical relevance of our work.

(5) Conceptual and interpretational gaps:

The authors quantify anisosome number but also report conditions in which anisosome number decreases while size increases. The biological interpretation of larger anisosomes is not discussed, and whether this reflects improvement or worsening of pathology is unclear. Compounds targeting the same mechanism (e.g., nuclear export inhibition) are inconsistently used across experiments (KPT compounds, verdinexor, leptomycin B), raising concerns about reproducibility. In organoids, the experimental paradigm shifts to long-term treatment (35 days vs. 16 hours), further complicating interpretation.

As pointed out by the reviewer 1 in point 4 above, we do not have evidence to establish a convincing correlation between the size of anisosomes and clinical phenotypes. Regarding the use of different drugs for different experiments, the initial screen identified KPT and Verdinexor because Leptomycin B was not in our library. In the follow-up studies, we switched to Leptomycin B because 1) it is commercially available; 2) it is highly potent and specific; 3) it was more commonly used as inhibitors of XPO1 according to the literature. However, for the organoid study, we had to switch back to KPT because of the toxicity issue associated with long-term application of Leptomycin B.

(6) Overinterpretation of rescue effects:

Although the authors state that they aim to test whether nuclear export inhibition rescues neuronal defects, no functional neuronal readouts are provided (e.g., viability, morphology, axon outgrowth, or electrophysiological measures). RNA-seq alone is insufficient to support claims of rescue.

Our interpretation of the RNA-seq data was that the rescue effect by nuclear export inhibition was limited and likely insignificant. Given that this negative data is not conclusive, we will remove it from the revised manuscript.

(7) Finally, the model does not appear to exhibit cytosolic TDP-43 aggregation at baseline. It remains unclear whether longer induction would produce cytosolic gel-like assemblies and whether these would be prevented by nuclear export inhibition. Long-term data are shown only in organoids, yet anisosome formation is not assessed there.

The expression system used in the study reaches a steady state after 48 h of induction. At this point, we did not observe any gel-like structures. We can clarify this point during revision.

Reviewer #3 (Public review):

Summary:

TDP-43 proteinopathy is broadly found in neurodegenerative diseases. This manuscript investigates how nuclear export influences the biophysical properties of TDP-43. The authors use a combination of chemical screening and genome-wide siRNA screening to identify pathways that modulate TDP-43 liquid-to-solid transitions. Overall, the study employs a broad array of approaches and addresses an important question in TDP-43 pathobiology. The identification of nuclear export as a central regulator is compelling and conceptually aligns with the emerging view that TDP-43 nucleocytoplasmic trafficking is a major defect in neurodegeneration.

Strengths:

This work integrates chemical and genetic screening to identify novel modifiers. The candidates were validated in both reporter cell lines and iPS-differentiated organoids. The findings support the nucleocytoplasmic transport is important for the biophysical properties of TDP-43.

We thank the reviewer for acknowledging the significance and strength of our study.

Weaknesses:

The mechanisms underlying the connection between nuclear export and phase transition need further clarification. Broader consequences of XPO1 inhibition are not addressed.

We agree that our study does not address how nuclear export inhibition affect TDP-43 phase behavior. As discussed in the paper, we proposed that the effect of nuclear export inhibition on TDP-43 phase separation is likely indirect. The most likely scenario is that inhibition of nuclear export changes the nuclear environment over time, which affects TDP-43 phase separation. We have tried to isolate nuclear extracts from control and LMB-treated cells and used mass spec to identify proteins that are differentially present in the nucleus. However, knockdown of the identified top candidates did not abolish LMB-induced phase alteration. Considering our observation that RNA splicing is another modulator of TDP-43 phase behavior, it is possible that it is the combined change of RNA and protein composition in the nucleus that alters TDP-43 phase behavior. However, defining the mechanism would require substantial work that is beyond the scope of the current study.