Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Comments

Manuscript Title: From Evasion to Collapse: The Kinetic Cascade of TDP-43 and the Failure of Proteostasis

Journal: International Journal of Molecular Sciences

Manuscript ID: ijms-4073165

Article Type: Review

Summary

This review manuscript presents a comprehensive overview of TDP-43 proteinopathy in the context of ALS and FTD, with particular focus on how TDP-43 evades cellular clearance mechanisms and drives proteostatic collapse. The authors propose a unified "kinetic cascade" model that integrates intrinsic protein properties, post-translational modifications, and the failure of both the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP). The manuscript is well-written and logically structured. However, several aspects require attention before publication.

Overall Assessment

Recommendation: Major Revision

The manuscript provides a valuable synthesis of current knowledge on TDP-43 pathology. The conceptual framework of a "kinetic cascade" is novel and potentially useful for understanding disease progression. However, the therapeutic implications section needs substantial expansion, and several scientific claims require additional support or clarification.

Strengths

The manuscript has several notable strengths. The integration of diverse literature into a coherent kinetic cascade model provides readers with a useful conceptual framework for understanding TDP-43 pathogenesis. The discussion of the "second hit" hypothesis is well-articulated and clinically relevant. The figures, particularly Figure 3 depicting the kinetic cascade, effectively communicate complex concepts. The coverage of both UPS and ALP dysfunction is balanced and thorough. Additionally, the discussion of PTMs and their role in aggregation is comprehensive and up-to-date.

Major Comments

1. Therapeutic Implications: The conclusion mentions that "therapeutic strategies must move beyond simple clearance" and target "kinetic inflection points," but this is not adequately developed. Given the clinical relevance of this topic, the authors should expand this section significantly. Please include a dedicated discussion of current therapeutic approaches targeting different stages of the cascade (e.g., antisense oligonucleotides, small molecule stabilizers, autophagy modulators) and their clinical status. A summary table of therapeutic strategies mapped to the kinetic cascade stages would be valuable.
2. Species-Specific Considerations: The review draws heavily from both in vitro studies, cell culture models, and animal studies without adequately addressing species-specific differences in TDP-43 biology. The authors should discuss the limitations of current animal models (mice, zebrafish, C. elegans) in recapitulating human TDP-43 pathology. How do findings from these models translate to human disease? Are there known species-specific differences in TDP-43 sequence, expression, or regulation that might affect the kinetic cascade?
3. Biomarker Discussion: Given the emphasis on kinetic inflection points, the authors should discuss the potential for biomarker development at different stages of the cascade. Are there measurable indicators of UPS impairment, oligomer formation, or ALP failure that could serve as diagnostic or prognostic markers? This would strengthen the translational relevance of the review.
4. Cell Type Specificity: The review does not adequately address why motor neurons and specific neuronal populations are preferentially vulnerable to TDP-43 pathology. What makes these cells more susceptible to the kinetic cascade? Is it related to their proteostatic capacity, metabolic demands, or other cell-intrinsic factors? This is a critical question that deserves discussion.
5. LATE Discussion: The authors briefly mention LATE (limbic-predominant age-related TDP-43 encephalopathy) in the introduction but do not discuss how the kinetic cascade model applies to this condition. Given that LATE affects a different brain region and age group compared to ALS/FTD, does the same cascade mechanism apply? This would broaden the impact of the review.
6. Missing Recent Literature: Several important recent publications appear to be missing from the discussion. The authors should conduct an updated literature search to include studies from 2024-2025 on TDP-43 structure (cryo-EM findings), novel therapeutic targets, and clinical trial results. Specifically, recent work on TDP-43 liquid-liquid phase separation kinetics and the role of RNA in preventing aggregation should be more thoroughly incorporated.

Minor Comments

1. Summary Table: The manuscript would benefit from a summary table listing the key factors contributing to TDP-43 aggregation and evasion, categorized by intrinsic vs. extrinsic factors. This would help readers quickly grasp the main points.
2. Figure Quality: While the figures are informative, the resolution and labeling could be improved. In Figure 1, the font size for some labels is small and may be difficult to read. In Figure 2, the legend should be expanded to explain all abbreviations used. Figure 3 would benefit from clearer demarcation of the three phases.
3. Abbreviations: Please provide a list of abbreviations. The manuscript uses numerous acronyms (UPS, ALP, CTD, NTD, RRM, LLPS, SG, etc.) that may be unfamiliar to readers outside the immediate field.
4. Section Length Balance: Section 3.4 (Proteasomal Evasion) is considerably longer than other sections and could be condensed. Conversely, Section 4.2.2 on nucleation barrier is relatively brief given its importance to the kinetic cascade model and could be expanded.
5. Graphical Abstract: The graphical abstract is informative but somewhat complex. Consider simplifying it to focus on the key message of the "second hit" model and kinetic cascade.

Specific Comments by Section

Abstract

1. Line 9-10: The statement that ALS and FTD are "currently untreatable" is not entirely accurate. FDA-approved treatments exist for ALS (riluzole, edaravone, AMX0035, tofersen for SOD1-ALS). Please revise to "lack disease-modifying treatments that halt progression" or similar.

Introduction

2. Line 33-34: The description of TDP-43 functions could be expanded to include its role in stress granule dynamics, which is discussed later but not introduced here.
3. Line 57-58: The statement about LATE affecting "ages beyond 80" should include prevalence data to contextualize its clinical significance.

Section 2

4. Line 86-87: When referring to the CTD as the "low-complexity domain," please clarify the relationship between glycine-rich content and low sequence complexity.
5. Line 101-110: The comparison between in vitro and patient-derived TDP-43 core sequences is interesting but could be strengthened by discussing what might account for the structural differences (e.g., cofactors, cellular environment).

Section 3

6. Line 191-192: The claim that "cellular macroaggregates of TDP-43 are reversible when both systems are functional" is a strong statement. Please clarify the experimental evidence supporting this and any limitations.
7. Line 289-290: The statement that D169G "is cleaved by caspase-3 more efficiently" needs clarification. What is the fold-change in cleavage efficiency?
8. Line 368-389: The discussion of proteasome inhibition by oligomers is well-referenced for Aβ, α-synuclein, and huntingtin, but the direct evidence for TDP-43 oligomers is acknowledged to be limited. Consider moving the speculative statement to a "Future Directions" section.

Section 4

9. Line 563-571: The discussion of stress granule demixing is important but somewhat technical. Consider adding a brief explanation of what "demixing" means in this context for readers unfamiliar with phase separation biology.
10. Line 657-667: The range of reported TDP-43 half-lives (4-34 hours) is quite broad. Please discuss what factors might account for this variability and which values are most physiologically relevant.

Section 5 (Conclusions)

11. Line 730-732: The statement about whether aggregates are "the primary toxic driver" or "loss of protein function is the principal culprit" deserves more discussion. What evidence supports each view? This is central to therapeutic strategy selection.

Language and Style

The manuscript is generally well-written, but the following issues should be addressed:

12. Line 148: "counterbalancing" might be better as "counteracts" for clarity.
13. Line 405-407: "mild oxidative stress can transiently enhance UPS activity; however, chronic or severe oxidative stress can compromise" - consider revising for parallel construction.
14. Line 694: "sealing the cell's fate" is somewhat colloquial for a scientific review. Consider "committing the cell to an irreversible pathological state" or similar.
15. Throughout: Please ensure consistent use of "TDP-43" vs. "TDP43" (without hyphen). The hyphenated form appears standard.

References

The reference list is comprehensive (143 citations). However, please verify the following: Reference 9 appears to cite a bioRxiv preprint - has this been published in a peer-reviewed journal? If so, please update. Several key reviews on TDP-43 proteostasis from 2023-2024 appear to be missing. Please ensure the reference formatting is consistent throughout (some entries have inconsistent author name formatting).

Final Remarks

This review provides a valuable synthesis of current understanding of TDP-43 pathobiology and introduces a useful conceptual framework. With appropriate revisions addressing the therapeutic implications, cell type specificity, and translational relevance, this manuscript will make a significant contribution to the field. I look forward to reviewing the revised version.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English language needs to be improved by professionals.

Author Response

We thank the reviewer for taking their time in evaluating our manuscript. Please refer to the attachment for our responses. For easier viewing, we indicated all responses in blue text.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the authors have complied an extensive review of the literature regarding TDP-43. The language is clear and the English is of high quality. There are some terms I would have chosen differently (import instead of importation, degradation pathways instead of degradative pathways) but these are details that do not hinder the readers understanding of the content.

Many interesting, thought-provoking insights were discussed and based on the review content I would have wished for a deeper discussion about the fact that TDP-43 aggregation is cytosolic even though its nuclear concentration is multiple times higher and anisomes are observed. Also, I missed a discussion of what species/principle causes toxicity. Toxicity due to sequestration of TDP43 and toxicity of oligomers were mentioned in a half-sentence, but not further discussed. 

And lastly, I do not agree with the claim, made in the abstract, that "a unified kinetic cascade model was synthesized" in this review, considering that no quantitative or qualitative modeling of any kind was done. I would describe it as "the multitude of disease contributing factors was listed in a well organized and logical way". 

All in all I recommend the manuscript for publication and suggest the authors address the couple of minor points mentioned above.

Author Response

In this manuscript, the authors have complied an extensive review of the literature regarding TDP-43. The language is clear and the English is of high quality. There are some terms I would have chosen differently (import instead of importation, degradation pathways instead of degradative pathways) but these are details that do not hinder the readers understanding of the content.

Response: We appreciate your comments. Upon review, we agree that the terms suggested fit better and were incorporated into the manuscript. The updated text is highlighted in green.

Many interesting, thought-provoking insights were discussed and based on the review content I would have wished for a deeper discussion about the fact that TDP-43 aggregation is cytosolic even though its nuclear concentration is multiple times higher and anisomes are observed. Also, I missed a discussion of what species/principle causes toxicity. Toxicity due to sequestration of TDP43 and toxicity of oligomers were mentioned in a half-sentence, but not further discussed.

Response: We appreciate these points raised, completely agreeing that these are critical areas that should be expounded on. Please refer to the sections below that we have inserted in the manuscript:

 4.2. Toxic Species That Drive TDP-43 Pathology

       The propensity of TDP-43 to naturally form aggregates has been described, with its early phase characterized as an oligomeric species that is stable, spherical, and amyloid-like, featuring exposed hydrophobic surfaces [35,75]. These oligomers were reported to have reduced nucleic-acid binding and direct neurotoxicity in neuronal models [75]. Regions in TDP-43 that facilitate its self-aggregation were identified, and candidate peptides targeting these regions were designed to reduce TDP-43 aggregation. Despite identifying two constructs that successfully reduced aggregation in a dose-dependent manner, the reduction in aggregation did not prevent cell death [Liu, 2013]. However, it must be noted that the authors considered only observable aggregates; thus, it is possible that cell death could mainly stem from the toxicity of soluble oligomers, which have much more exposed hydrophobic regions that can interact with functional cellular proteins relative to stable aggregates [Diociaiuti, 2021]. Moreover, liquid-like RNP granules in axons become more viscous and less dynamic with ALS-linked mutations, disrupting granule transport and inducing the toxic gain-of-function effect [Gopal, 2017]. Finally, cytoplasmic liquid droplets that formed independently of classical SGs were reported to gel up or solidify under stress, which is enough to disrupt nuclear import and deplete nuclear TDP-43, resulting in cell death [Streit, 2022; Gasset-Rosa, 2019]. Given these findings, primary toxic TDP-43 species appear to arise from oligomeric and gel-like assemblies rather than end-stage fibrils, thereby combining direct proteotoxicity with the disruption of RNA metabolism and nucleocytoplasmic transport.

CTFs are also species that play a significant role in TDP-43 toxicity, as they have been characterized as able to seed full-length TDP-43. On their own, they are capable of inducing cell death through a toxic gain-of-function without sequestering their full-length counterpart [59], as well as disrupting proper proteasomal assembly [76]. Together, these findings support a conceptual framework in which soluble oligomers, viscous RNP granules, and CTFs act as the principal toxic species. At the same time, large aggregates may be later consequences that correlate with, but do not strictly determine, neuronal death. Simply put, pathogenicity may be best explained as a combination of misfolded assembly-mediated gain of function and depletion of functional TDP-43 within the nucleus.

4.4. Buffered Nucleus, Toxic Cytoplasm: Resolving the TDP-43 Aggregation Paradox

The apparent paradox, where TDP-43 aggregates are predominantly cytosolic despite their steady-state nuclear concentration being much higher, can be clarified by considering the different buffering capacities of these compartments. In the nucleus, high-affinity RNA binding and the formation of large ribonucleoprotein assemblies, nuclear bodies, and anisotropic intranuclear droplets (anisomes) maintain TDP-43 in dynamic, liquid-like states, and keep its concentration of free, aggregate-competent monomers low [121][Gasset-Rosa, 2019]. By contrast, once monomerization and RNA disengagement enable TDP-43 entry into the cytoplasm, the protein reaches an environment with weaker RNA buffering, fewer dedicated chaperone elements, and more chronic stressors, driving phase separation in stress granules and demixed cytoplasmic droplets that could rapidly solidify into irreversible aggregates [Loganathan, 2019].

In this view, intranuclear anisomes represent a high-density but still regulated ‘buffer’ for RNA-free TDP-43, whereas the cytoplasm functions like a sink where the quality-control capacity is lower, nucleocytoplasmic transport is progressively impaired, and aggregates become trapped as hyperphosphorylated, ubiquitinated inclusions [Gasset-Rosa, 2019].

And lastly, I do not agree with the claim, made in the abstract, that "a unified kinetic cascade model was synthesized" in this review, considering that no quantitative or qualitative modeling of any kind was done. I would describe it as "the multitude of disease contributing factors was listed in a well organized and logical way". 

Response: We appreciate the reviewer for pointing out that our phrasing overstated the degree of modeling performed. We did not carry out quantitative or formal qualitative modeling. To avoid this implication, we have revised it to the following: "Here, we organize these factors into a conceptual kinetic cascade that links TDP-43 misfolding, phase separation, and clearance failure." 

We feel that this now better reflects that our review assembled published mechanisms into a logical, stepwise "framework" rather than deriving a formal kinetic model.

All in all I recommend the manuscript for publication and suggest the authors address the couple of minor points mentioned above.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors are to be congratulated for an excellent manuscript without serious weaknesses in this reviewer's opinion. Several minor weaknesses could be corrected:

1. In abstract and else where, "The histopathological hallmark..." should be "A histopathological hallmark..."
2. In the abstract and elsewhere, it should be clear that "FTD" actually includes two main types: FTLD-TDP and FTLD-tau, in which FTLD-TDP is TDP-43 proteinopathy. 
3. The Figure Abstract leaves the impression that the proteosome is only in the nucleus, which is not correct -- particularly important in TDP-43 proteinopathy. 
4. In Introduction "regulation of translation for other proteins [1–3]." is not really discussed in the 3 references (only splicing, stress granules, PTMs, etc) -- if this is what the authors intend, then this should be clarified. 
5. In section 2.2, "Specifically, Tan et al. found TDP-43 inclusions in the brains and spinal cords of sporadic ALS (sALS) and non-mutant superoxide dismutase 1 (SOD1) familial ALS (fALS), but not in two mutant SOD1 cases [32]." should be expanded to include mutant FUS-ALS, which also obviates TDP-43 inclusions.
6. In section 3.4.1 "A study by Qin et al. (2014), through the use of NMR spectroscopy and CS-Rosetta modeling (supported by 78 unambiguous long-range NOE constraints), revealed that the NTD of TDP-43 adopts a previously unknown ubiquitin-like fold despite not bearing any  sequence homology with ubiquitin [69]." However in FEBS 2016, Mompeán et al. found that the NTD adopts a stable fold that is reminiscent of the axin-1 family, not ubiquitin. It is believed by many in the field that this structure supersedes the Qin structure. 
7. Figure 3: what are the wavy lines supposed to indicate? Only the nucleus role is clear -- suggest omission of the non-essential lines. 

Author Response

The authors are to be congratulated for an excellent manuscript without serious weaknesses in this reviewer's opinion. Several minor weaknesses could be corrected:

Response: We thank you for your kind comments. Below are our responses to each point. Revisions per your comments are indicated in light blue text.

1. In abstract and else where, "The histopathological hallmark..." should be "A histopathological hallmark..."
   
   Response: Changes were made as suggested.
2. In the abstract and elsewhere, it should be clear that "FTD" actually includes two main types: FTLD-TDP and FTLD-tau, in which FTLD-TDP is TDP-43 proteinopathy. 
   
   Response: We thank you for raising this important distinction. We reviewed the reference and it indeed specified FTLD-TDP as the specific subtype. We have revised it to the following: "Notably, this core sequence resembled that described by Arseni et al. when they isolated TDP-43 pathological filaments from the brains of ALS/FTLD-TDP (type A) patients, a subtype of FTLD (the other subtype is FTLD-tau) [28]. 
   
   
3. The Figure Abstract leaves the impression that the proteosome is only in the nucleus, which is not correct -- particularly important in TDP-43 proteinopathy. 
   
   Response: We agree and have revised the figure to include extranuclear proteasomal processing. 
   
4. In Introduction "regulation of translation for other proteins [1–3]." is not really discussed in the 3 references (only splicing, stress granules, PTMs, etc) -- if this is what the authors intend, then this should be clarified. 
   
   Response: We appreciated that this was pointed out. The original wording overstated what is directly supported by the references. The sentence was appropriately revised to the following: "regulation of translation of other proteins" >> "the regulation of mRNA metabolism within stress granules, which are sites of translational repression under stress conditions."
   
   
5. In section 2.2, "Specifically, Tan et al. found TDP-43 inclusions in the brains and spinal cords of sporadic ALS (sALS) and non-mutant superoxide dismutase 1 (SOD1) familial ALS (fALS), but not in two mutant SOD1 cases [32]." should be expanded to include mutant FUS-ALS, which also obviates TDP-43 inclusions.
   
   Response: Thank you for this suggestion. We have expanded the sentence to include that, in addition to mutant SOD1-ALS, FUS-ALS represents another exception where TDP-43 inclusions are absent, and the pathology observed is characterized only by FUS-positive inclusions. The following clause was added: and subsequent clinicopathological studies have shown that ALS resulting from FUS mutations likewise exhibits FUS-positive, TDP-43-negative inclusions [1-2].
   
   References added were the following:
   
   
   1. King, A., Troakes, C., Smith, B., Nolan, M., Curran, O., Vance, C., Shaw, C., & Al-Sarraj, S. ALS-FUS pathology revisited: singleton FUS mutations and an unusual case with both a FUS and TARDBP mutation. Acta Neuropathologica Communications. 2015; 3. https://doi.org/10.1186/s40478-015-0235-x.
   2. Blair, I., Williams, K., Warraich, S., Durnall, J., Thoeng, A., Manavis, J., Blumbergs, P., Vucic, S., Kiernan, M., & Nicholson, G. FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis. Journal of Neurology, Neurosurgery & Psychiatry. 2009; 81. https://doi.org/10.1136/jnnp.2009.194399.
   
   
   
6. In section 3.4.1 "A study by Qin et al. (2014), through the use of NMR spectroscopy and CS-Rosetta modeling (supported by 78 unambiguous long-range NOE constraints), revealed that the NTD of TDP-43 adopts a previously unknown ubiquitin-like fold despite not bearing any  sequence homology with ubiquitin [69]." However in FEBS 2016, Mompeán et al. found that the NTD adopts a stable fold that is reminiscent of the axin-1 family, not ubiquitin. It is believed by many in the field that this structure supersedes the Qin structure.
    
   Response: We thank you for raising this critical point. We have revised the entire section to include this reference:
   "Early NMR and CS-Rosetta modeling by Qin et al. (2014) suggested that the NTD of TDP-43 can adopt a ubiquitin-like fold in slow equilibrium with a highly disordered state, and that binding to single-stranded DNA (ssDNA) favors the shift toward the folded conformation, leading to the proposal that nucleic-acid binding stabilizes the NTD, protecting against aggregation. Subsequent high-resolution NMR analysis by Mompeán et al. (2016), which incorporated far more extensive NOE and dynamics data, showed that the isolated NTD (residues 1–77) forms a stably folded domain whose topology more closely represents the axin-1 Dix domain, with no unfolded population under a broad range of conditions [REF]. Here, the disordered 78–102 segment, rather than the folded NTD core, was enough for binding DNA oligonucleotides, leading the authors to argue that nucleic acid binding is unlikely needed to stabilize the NTD fold per se and that the physiological relevance of folding NTD dynamics remains unclear."
   
   Reference: Mompeán M, Romano V, Pantoja-Uceda D, Stuani C, Baralle FE, Buratti E, et al. The TDP-43 N-terminal domain structure at high resolution. FEBS J. 2016 Apr;283(7):1242–60. DOI: 10.1111/febs.13651
   
   
7. Figure 3: what are the wavy lines supposed to indicate? Only the nucleus role is clear -- suggest omission of the non-essential lines. 
   
   Response: The wavy lines represent the cell membrane. We agree that these add clutter to the figure and have been removed. Thank you.
   
   

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No further comments