Author response:
The following is the authors’ response to the original reviews.
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 inhibitor (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 phase regulation of a disease-relevant TDP-43 mutant. We have included more background information to justify the use of TDP-43 acetylation mimetics in the introduction.
(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 background that is more sensitive for detecting phospho-TDP-43. The goal was to test whether XPO1 inhibition mitigates the phosphorylation 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 over emphasize on its disease connection. We have revised the paper to tone down this section. We also remove the RNAseq data as it is not essential for our conclusions.
It is also noteworthy that TDP-43 disease mutations are usually loss-of-function alleles. Although heterozygous background is sufficient to induce disease phenotype in aged humans, heterozygous background in experimental settings is usually unable to generate severe defects. Thus, it is quite common to study TDP-43 disease-related defects in homozygous knockout or RNAi-mediated depletion conditions (e.g. PMID: 35197626; 41120751; 38277467).
Regarding the immunostaining signals in K181E organoids, we did not report them as anisosomes. As documented in the literature, p-TPD-43 is widely used as a marker to indicate pathological TDP-43 aggregation. P-TDP-43 is enriched in pathological aggregates in human ALS and FTD patients, colocalized with other aggregation signatures such as ubiquitin and other aggregation-prone proteins in the cytoplasm (PMID: 36008843), and is being used as a diagnostic marker for neurodegeneration (PMID: 31661037). The characterization of K181E organoid is reported in a pre-print by Zhang Q. et al., 2026 (PMID: 41292965), which is currently under revision for Science Advances. In Fig. 1I of this manuscript, we confirmed the cytosolic localization of p-TDP-43 in cells that were isolated from K181E organoids. In the current manuscript, Figure 7 is to show that nuclear export inhibition mitigates the accumulation of p-TDP-43 in a brain-like tissues. We 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 mean to conclude or imply that the regulation of TDP-43 by XPO1 is direct. In fact, we explicatively mentioned on page 8 of the original manuscript 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 might link XPO1-mediated nuclear export to TDP-43 nuclear egression.”
We now add new data in Figure 6, showing that in an in vitro reconstitution assay using semi-permeabilized cells, LMB treatment significantly stabilizes anisosomes in an RNA dependent manner. This new data suggests that XPO1 inhibition leads to increased nuclear RNA availability, which indirectly favors anisosome assembly and maturation (see discussion). We believe that this new finding has provided significant new insight into how nuclear transport modulates TDP-43 phase behavior. We have revised the title, the abstract and changed the framing according to the reviewer’s suggestion.
(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 regulation.
Regarding the mechanism underlying XPO1-dependent TDP-43 phase regulation, as mentioned above, we developed a semi-permeabilized cell-based assay in which we used the pore-forming toxin streptolysin O to damage the plasma membrane after anisosome induction. We noticed that upon cell permeabilization and cytosol loss, anisosomes were mostly lost (Figure 6B, C). This is probably due to a reversible partition of TDP-43 into a less fluorescent soluble fraction. Supporting this idea, when permeabilized cells were incubated with cytosol plus an energy regenerating system, small puncta containing TDP-43 2KQ could be reformed in an energy dependent manner (Figure 6D, E). Interestingly, in LMB-treated cells, anisosomes remained stable despite cell permeabilization（Figure 3F). Since LMB treatment did not increase TDP-43 nuclear concentration (Supplemental Figure 1), this data suggest that nuclear export inhibition likely alter the nuclear environment to stabilize anisosomes. Indeed, when cells were permeabilized in the presence of a small RNAase, LMB-stabilized anisosomes also collapsed (Figure 6G).
We now add more discussions on the potential effect of RNA on TDP-43 phase behavior in XPO-1 inhibited cells considering these new findings.
(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 data does not demonstrate an interaction between XPO1 and TDP-43. Considering our new data (mentioned above), it is possible that the effect of anisosome induction on endogenous XPO1 localization is also mediated by RNA. We now mention more explicitly that the regulation of TDP-43 by XPO1 is likely indirect (Page 8). We have revised our paper to separate any speculative statements from the data, and also discussed the possibility of alternative interpretations.
(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 screens, which was identified by machine-based image analyses using 6 different siRNAs. This, together with the chemical inhibition experiments, demonstrate 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 therefore less likely to be secondary.
Regarding the inhibitor concentration, according to the literature, Leptomycin B was commonly used at 50-200 nM. We chose 200 nM 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.
We did not suggest any specific adaptor that mediates XPO1 interaction with TDP-43. Whether there is an adaptor, and if so, the identity of such adaptor is out of the scope of this study. We revise our paper on page 8-9 to clarify these points.
(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 have revised the title, abstract, and main text to temper our conclusions.
(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 tightly associated with TDP-43 proteinopatheis. In human ALS and FTD-TDP tissues, cytoplasmic inclusions are strongly immunoreactive for phosphorylated TDP-43 (typically S409/410, as detected 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 significant finding, as it suggests that inhibition of nuclear transport may rescue TDP-43 proteinopathy. We revised the text on page 9 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 some technical issues. We have removed the RNAseq data and the related texts since it is not essential.
(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 have removed 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 have removed the RNAseq part.
(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 (Cell death siRNA), which targets several essential gene. Transfection efficiency was controlled by measuring cell viability after knocking down of these genes. In addition, the identification of XPO1 as a positive regulator of TDP-43 phase behavior was independently validated by our chemical genetic screens with three XPO-1 inhibitors. 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 relatively short treatment, we do not expect a significant change in protein level or toxicity. To alleviate this reviewer’s concern, we performed an immunoblotting experiment to measure the total TDP-43 protein levels in drug-treated cells. Except for VLX, we did not detect any significant changes in the level of TDP-43 after drug treatment (Supplemental Figure 1).
(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 used two types of FRAP assays. We either bleached TDP-43 within anisosomes or bleached the surrounding TDP-43 molecules（Figure 2）. The two complementary methods yield consistent results that allow unambiguously distinguish between TDP-43 LLPS state and gel-like condensation.
In XPO1-related experiments, the two types of condensates formed by TDP-43 2KQ can be distinguished by several features including their subcellular localization, shape, and the fluorescence recovery kinetics. We feel that these combined data clearly segregate these puncta into two distinct types of assemblies. The proposed half-region-of-interest bleach is technically challenging for small anisosomes under normal conditions. However, whenever possible, (e.g. anisosomes enlarged by Leptomycin B), 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 with no abnormal chromosome condensation (Figure 5A). Additionally, in cell line-based experiments, the effect of Leptomycin B on anisosomes was detected 6-8 hours post treatment. The change in global protein synthesis because of RNA changes should be relatively minor at this stage. Indeed, our new immunoblotting experiment showed that LMB treatment did not affect TDP-43 protein level (Supplemental Figure 1). Most importantly, the in vitro semi-permeabilized assay demonstrates a direct role for RNA in stabilizing anisosomes.
(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 to reduce the effect of batch variation. The wildtype cells and K181E mutant are derived from the same genetic background. This information is now included in the method section on page 14. Given the criticisms by review 1 and 2 on the RNAseq data, we have removed this non-essential data.
(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 now discuss a new model in discussion based on our new Figure 6, which integrates the role of RNA splicing and nuclear transport in TDP-43 phase regulation on page 10. We agree with the reviewer that other questions are also important for future studies.
(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.
As discussed above, our new data regarding the role of RNA in TDP-43 phase regulation should alleviate this concern, although we cannot exclude the possible involvement of splicing factors in this process. We also clearly state that there is no evidence to support a direct interaction between TDP-43 and XPO1 on page 8.
(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.
In the newly performed immunoblotting experiment, we measured the TDP-43 levels in drug-treated cells but found no effect by most drugs (Supplemental Figure 1).
(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 have revised the introduction and added the following sentence on page 4. “The 2KQ-containing anisosomes, observed mostly in the nucleus under overexpression conditions, have not been validated in human patient samples.”
(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, which is why we feel this experiment is very significant. Unlike many other p-TDP-43 detection studies that rely on TDP-43 overexpression or exposing cells to excess stressors, we could detect substantial p-TDP-43 in 3D organoids grown under normal conditions, whereas the same cells grown and differentiated in 2D culture do not show p-TDP-43 (Zhang Q. et al., BioRxiv 2025).
(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 have added more statistical information to the figure legends.
(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 revised the figure legends to include this information. As for anisosome detection, because they are so obvious, standard thresholding combined with automated counting 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 identifies pathways capable of modulating TDP-43 phase behavior. In the revised paper, we included several experiments using an in vitro semi-permeabilized cell system to further dissect the role of nuclear export in TDP-43 phase separation. We believe that these new results should provide significant mechanistic insight that links nuclear export and RNA transcription and splicing to TDP-43 phase regulation. Additionally, we have revised our paper carefully to discuss the physiological relevance and the limitation of our study.
(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 now reframe the study as an important screening study that identifies nuclear export among several other pathways as modulators of TDP-43 phase behavior. We also propose a model that links RNA splicing to nuclear export in TDP-43 phase regulation.
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 have added more discussions to justify 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 enlarge the demixed TDP-43 structures, which is necessary for our screen and imaging-based characterization.
For XPO1 overexpression, we have done the mCherry alone control but due to space limit in Figure 5, we did not include it. We now include the data in Supplemental Figure 4. This figure shows that overexpression of mCherry did not change TDP-43 localization or anisosome structures.
(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 have added more experimental details to the figure legends and the method. For the imaging experiments, data points reflect randomly selected individual cells imaged in 2-3 independent biological repeats. This is now stated in the figure legends. For chemical screens, we screened against NCATS libraries was first done at top concentration (10 mM) to ensure inhibitory efficacy for all potential hits. In the follow-up validation study, we validated the top hits using a series of concentrations, as shown in Figure 1B. Drug concentrations are provided in Figure 2A, 4A, C, E, F, 5A-D, F, Figure 6F, G, Figure 7A)
We explain the STRING analysis in more detail now. Basically, STRING is a protein-protein interaction network that reports all potential interactions between any proteins in human proteome. Given the potential off-target effect of siRNA, we assume that if the screen identifies multiple components of a protein interaction network or pathway, the result is more likely to be real.
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 of them. 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 control transfection efficiency. We would only process the data if siRNAdeath control killed > 90% of the cells. Lastly, the XPO1 knockdown result was independently validated by small molecule inhibitors. For TDP-43 overexpression, the study by Yu and colleagues suggested that the expression is more than 20-fold higher than endogenous TDP-43, but they showed that anisosome formation is not an artifact of protein overexpression. When the expression level was titrated down, they could still detect anisosomes.
(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. What we showed before was averaged expression levels. We pooled the organoids of the same treatment from the two batches to reduce the impact of batch variation.
Given the criticisms from both reviewers 1 and 2 on the limited interpretation power of the RNAseq study, we have removed 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 cytotoxicity. Notably, the processes identified by our screens are all house-keeping pathways that are conserved in neurons. Thus, we believe that the reported findings are likely applicable to neurons. That being said, we have revised our paper to ensure 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.
We thank the reviewer for these critical points. 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 they are investigational drugs, but Leptomycin B was not in our library. In the follow-up studies, we switched to Leptomycin B because 1) it is highly potent and specific; 2) it was better characterized and 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 probably insignificant. Given that this negative data is not conclusive, we have removed 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 24 h of induction. Prolonged expression up to 48 h did not alter the number of anisosome, nor does it change TDP-43 phase behavior. We now clarify this point on page 4.
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 previous manuscript did not address how nuclear export inhibition affect TDP-43 phase behavior. As discussed in our 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 spectrometry to identify proteins that are differentially present in the nucleus. However, knockdown of the identified top candidates did not abolish LMB-induced phase alteration (not shown). Considering our observation that RNA splicing is another modulator of TDP-43 phase behavior, we reasoned that it is possible that it is the combined change of RNA and protein composition in the nucleus that alters TDP-43 phase behavior. In new experiments presented in Figure 6, we now used a semi-permeabilized in vitro system to demonstrate that LMB treatment stabilized anisosomes in an RNA-dependent manner (see response to point 4 by reviewer 1). This new data allows us to propose a new model that link RNA splicing and nuclear export in TDP-43 phase regulation (Discussion).
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors):
(1) Include appropriate controls for all overexpression experiments. In particular, overexpression of WT TDP-43 alone and suitable tag-only controls (e.g., mCherry alone or mCherry fused to a protein unrelated to TDP-43/XPO1) should be included to control for aggregation driven by non-physiological protein levels.
In Supplemental Figure S4, we included a tag-only control, which shows that mCherry alone does not affect the localization of XPO1, neither did we see mCherry co-localizes with TDP-43.
Since WT TDP-43 itself does not form anisosome and because the goal of the study was to test how anisosome dynamics is affected by various conditions, we did not repeat our experiments with WT TDP-43.
(2) Address whether TDP-43 anisosomes form under endogenous or near-physiological expression levels. If possible, include experiments using lower expression systems or endogenous tagging to demonstrate that anisosome formation is not solely an overexpression artifact.
As mentioned above, in a titration experiment done by Yu et al. 2020 (PMID: 33335017), they showed that ectopic TDP-43 undergoes demixing even at concentrations lower than endogenous TDP-43, although the demixed puncta are small. Their result suggested that overexpression per se does not change TDP-43 phase behavior. Instead, it only enlarges the demixed TDP-43 structures, which is necessary for our screen and imaging-based characterization.
(3) Clearly define biological versus technical replicates throughout the manuscript and report exact n-numbers for all experiments in figure legends and/or methods. Resolve discrepancies between stated and displayed n-numbers (e.g., figures showing more data points than the number of independent experiments reported). Further, include how data points were defined (e.g., cells, fields of view, wells).
We now state clearly the biological repeats in figure legends. We did not use N number to specify technical replicate. The discrepancy between the stated N number (biological repeats) and the data points is because for imaging experiments, data points usually represent single cells collected from 2-3 biological replicates (N=2 or 3). Data points are now clearly defined in the figure legends (anisosome, cell, imaging field, or independent experiment).
(4) The authors state that they identified a list of compounds that reduced anisosomes. Please clarify how the threshold was determined: Was this a statistical analysis or a specific threshold that has been used?
For both siRNA screen and chemical genetic screen, we calculated the Z-score and used Z-score>2 as a cutoff. This is mentioned in the method.
(5) Provide a complete list of compounds used in the chemical screen, including concentrations tested and whether multiple doses were evaluated.
As mentioned above, the initial screen was done with just one concentration (10 mM). Identified positive hits were re-tested with multiple doses as shown in Figure 1. The compounds are from a commercial library (LOPAC R1280, Sigma #LO4200). The list of compounds can be found at vender’s website.
(6) Clearly explain the criteria used to reduce the initial 1,533 screening hits to 211 candidates following STRING analysis, including cutoffs and prioritization logic.
We now explain that the Z-score was used to further narrow down the hit (page 6). Additionally, we provide an explanation on how we use STRING to further narrow down the list. The sentence reads as “To further narrow down the list, we performed a STRING protein network analysis based on the assumption that a protein interaction network bearing multiple positive hits would be more likely to be a true effector.”
(7) Report knockdown and overexpression efficiencies for all genetic perturbations used in the study.
For TDP-43 overexpression, the study by Yu and colleagues suggested that the stable cell line expresses 20-fold more TDP-43 than endogenous one, but they showed that anisosome formation is not an artifact of protein overexpression. When the expression level was titrated down, they could still detect anisosomes (Yu, H. et al., Science 2021). For knockdown efficiency, since the screen used 6 different siRNAs for each identified target (a few hundred), it is technically challenging to validate the knockdown efficiency of each siRNA by conventional qRT-PCR. To control knockdown efficiency, we transfected cells in parallel with siRNA-death that contains a mixture of siRNAs targeting several essential genes (Qiangen, #1027299). We would only process the data if siRNAdeath control killed > 90% of the cells, indicating good knockdown efficiency.
(8) Clarify the biological interpretation of changes in anisosome size versus number, particularly in conditions where fewer but larger anisosomes are observed. Discuss whether larger assemblies are hypothesized to be protective, neutral, or deleterious.
Live cell imaging was used to dissect why cells treated with certain drugs such as XPO1 inhibitors have fewer but larger anisosome. Figure 5F shows that this is caused by the fusion of small anisosomes. Our data does not suggest that the size of anisosomes can differentiate between protective or deleterious state, but rather it is the LLPS state and subcellular localization of these assemblies that may play a more critical role in determining whether TDP-43 forms deleterious protein aggregates. The discussion is on page 10.
(9) Specify whether all anisosomes induced by XPO1 overexpression were gel-like or whether this applied only to a subset. If only a subset was affected, please provide quantifications, otherwise state clearly that all anisosomes in XPO1 overexpression were gel-like.
All TDP-43 puncta mislocalized to the cytoplasm in XPO1-overexpressing cells are gel-like because the FRAP experiment in Figure 5I was done with randomly selected TDP-43 puncta mislocalized to the cytoplasm.
(10) Clarify which anisosomes (nuclear vs cytosolic; gel-like vs non-gel-like) were selected for FRAP analyses in Figure 5I.
For Figure 5I, the control anisosomes in untreated cells are nuclear while under mCh-XPO1 expressing condition, only those in the cytoplasm were randomly selected for photobleaching.
(11) The translatability of the conclusion based on cancer cell lines to brain organoids is not convincingly shown and could be strengthened by including additional assessment of anisosomes. While this might not be feasible in 3D cultures, the authors could alternatively use 2D cultured neurons to perform the same assays as performed in the cancer cell line. Additionally, the same treatment strategy should be applied. The reasoning for increasing treatment to 35 days in the organoids is unclear.
In another manuscript that is currently under revision, we compared 2D iNeuron culture with 3D organoids. A pre-print is available at https://www.biorxiv.org/content/10.1101/2025.11.09.687455v1.full. In this study, we found that endogenous TDP-43 K181E mutant do not undergo phosphorylation-dependent transition to aggregate in 2D cultures. Only when these cells were grown into 3-D organoids, TDP-43 phosphorylation could be detected. (see supplemental Fig. S1c, d in https://www.biorxiv.org/content/10.1101/2025.11.09.687455v1.full). Thus, it is not possible to repeat the experiments in this study in 2D iNeuron cultures. We agree with the review that there is a gap between the study using the cancer cell line and the use of K181E iPSC-derived 3D organoids. We have toned down our conclusions throughout the text.
(12) Address neuronal vulnerability explicitly by assessing toxicity, viability, or functional neuronal readouts, particularly given prior reports that WT TDP-43 overexpression alone is neurotoxic.
We agree that this is an important point, but the main goal of this study was to dissect the cellular pathways/mechanisms that govern TDP-43 phase separation. We feel that the requested experiments are beyond the scope of the current study.
(13) Clearly state the number of biological replicates used for each RNA-seq condition. Establish baseline transcriptional differences between WT and mutant TDP-43 prior to assessing the effects of nuclear export inhibition. Include PCA plots and heatmaps, including all samples.
As mentioned above, we have decided to remove the RNAseq data from the manuscript to save room for new results.
(14) Given the role of TDP-43 as an RNA-binding protein, consider including splicing analyses to assess whether nuclear export inhibition preserves or disrupts TDP-43-dependent RNA processing.
We thank the reviewer for this suggestion. However, we feel that the proposed experiments are beyond the scope of the current study.
(15) Improve clarity of transcriptomic visualizations (e.g., GO-term plots) and explicitly define all group labels used (e.g., Group A vs Group B).
We have removed the RNAseq data.
(16) Ensure consistent use of disease terminology (ALS vs FTD) throughout the manuscript, e.g., lines 222 and 244.
We have checked the usage of these terms to make sure they are accurately used.
(17) Correct figure and axis labeling errors (e.g., Figure 3A x-axis range).
Figure 3A indicates the Z score distribution of the entire human genome. As stated on page 6, 21,404 genes were targeted.
(18) Avoid overstatements in the Discussion that are not directly supported by the presented data, particularly regarding the interpretation of proteasome inhibition and gel-like anisosome states.
We have revised our discussion substantially to tone down our conclusions.
(19) Clarify the rationale for switching between different nuclear export inhibitors across experiments and discuss whether results were consistent across compounds.
In the acute experiments down with the cancer cell line, we used LMB because it is potent and well characterized. In organoid experiment, we switched to KPT-276 because it is better tolerated by organoids, especially during longer treatment.
Reviewer #3 (Recommendations for the authors):
Major concerns that require clarification or further strengthening:
(1) The connection between nuclear export and liquid-solid phase transition is not clear. The 2KQ mutant forms nuclear anisosomes. The manuscript does not provide data about its nuclear-cytoplasmic distribution normally, nor how the distribution is changed upon nuclear export inhibition or enhancement. In Figure 5I, it is unclear whether the anisosomes are in the nucleus or cytoplasm. The dynamics of nuclear vs cytoplasmic anisosomes should be measured separately. What is the mechanism that promotes nuclear export and changes the dynamics, especially nuclear anisosomes?
As mentioned by the reviewer, the 2KQ mutant forms anisosomes only in the nucleus. This was documented in Yu, H. et al., Science 371 (2021), and also shown in our Figure 4A, F, Figure 5A. Figure 5A also shows that nuclear export inhibition does not change anisosome localization, only making them bigger while reducing the numbers. For Figure 5I, the control anisosomes in untreated cells are nuclear while under mCh-XPO1 expressing condition, only those present in the cytoplasm were randomly selected for bleaching.
(2) Figure 5J, no obvious XPO1 is sequestered to anisosomes, as described in lines 208-209.
Unlike Figure 5G, this experiment studied the localization of endogenous XPO-1 by immunostaining. As discussed in Yu et al., Science 371 (2021), proteins inside anisosomes could not be stained by antibodies due to an accessibility problem. This explains why we could only detect reduced XPO1 after anisosome induction.
(3) Figure 6A, the localization of phosphor-TDP-43 is not clear. And it is not clear what cell types contain the aggregates. Higher-resolution images need to be included. The mechanism by which XPO1 inhibition reduces TDP-43 aggregation requires further validation. It remains unclear whether it is directly mediated through altered nucleocytoplasmic transport of TDP-43.
We agree that it is technically challenging to visualize the precise subcellular localization of p-TDP-43 in 3D organoids. In the manuscript that reports the characterization of the 3D organoids, we dissociated cells from the 3D organoids by trypsin digestion and plated them out in 2D before immunostaining and imaging. We could clearly see p-TDP-43 co-localizes with the neuronal marker TUJ1 and is localized outside of nucleus (see figure 1 of https://www.biorxiv.org/content/10.1101/2025.11.09.687455v1.full)
In the newly added Figure 6, we used a semi-permeabilized cell system to dissect the phase separation dynamics of TDP-43 2KQ in cells treated with the nuclear export inhibitor LMB. Our data suggests that nuclear export inhibition alters the nuclear environment, making it more favorable for the liquid phase of TDP-43. This is dependent on nuclear RNA.
(4) XPO1 controls the export of numerous essential proteins, and its inhibition can produce broad, potentially toxic effects unrelated to TDP-43. The manuscript should include a discussion of these off-target consequences.
We thank the reviewer for this point. Given the new data in Figure 6, we now add some more discussion on the potential mechanism by which nuclear export inhibition modulates TDP-43 phase separation. This can be found on page 10.
References:
Zhang, Q. et al. A human forebrain organoid model phenocopies dysregulated RNA and protein homeostasis in ALS/FTD-associated TDP-43 proteinopathies. bioRxiv (2025). (https://www.biorxiv.org/content/10.1101/2025.11.09.687455v1.full
