Maintenance of neuronal TDP-43 expression requires axonal lysosome transport

  1. Veronica H. Ryan
  2. Sydney Lawton
  3. Joel F. Reyes
  4. James Hawrot
  5. Ashley M. Frankenfield
  6. Sahba Seddighi
  7. Daniel M. Ramos
  8. Jacob Epstein
  9. Faraz Faghri
  10. Nicholas L. Johnson
  11. Jizhong Zou
  12. Martin Kampmann
  13. John Replogle
  14. Yue A. Qi
  15. Hebao Yuan
  16. Kory R. Johnson
  17. Dragan Maric
  18. Ling Hao
  19. Mike A. Nalls
  20. Michael E. Ward

  • Curated by eLife

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    eLife Assessment

    In this important manuscript, Ryan et al perform a genome-wide CRISPR based screen to identify genes that modulate TDP-43 levels in neurons. They identify a number of genes and pathways and highlight the BORC complex, which is required for anterograde lysosome transport as one such regulator of TDP-43 protein levels. Overall, this is a convincing study, which opens the door for additional future investigations on the regulation of TDP-43.

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Abstract

TDP-43 mislocalization and pathology occurs across a range of neurodegenerative diseases, but the pathways that modulate TDP-43 in neurons are not well understood. We generated a Halo-TDP-43 knock-in iPSC line and performed a genome-wide CRISPR interference FACS-based screen to identify modifiers of TDP-43 levels in neurons. A meta-analysis of our screen and publicly available screens identified both specific hits and pathways present across multiple screens, the latter likely responsible for generic protein level maintenance. We identified BORC, a complex required for anterograde lysosome transport, as a specific modifier of TDP-43 protein, but not mRNA, levels in neurons. BORC loss led to longer half-life of TDP-43 and other proteins, suggesting lysosome location is required for proper protein turnover. As such, lysosome location and function are crucial for maintaining TDP-43 protein levels in neurons.

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  1. Version published to 10.1101/2024.09.30.615241v2 on bioRxiv
  2. PREreview

    This Zenodo record is a permanently preserved version of a Structured PREreview. You can view the complete PREreview at https://prereview.org/reviews/14721199.

    Does the introduction explain the objective of the research presented in the preprint? Yes
    Are the methods well-suited for this research? Highly appropriate
    Are the conclusions supported by the data? Somewhat supported
    Are the data presentations, including visualizations, well-suited to represent the data? Somewhat appropriate and clear
    How clearly do the authors discuss, explain, and interpret their findings and potential next steps for the research? Very clearly
    Is the preprint likely to advance academic knowledge? Highly likely
    Would it benefit from language editing? Yes
    Would you recommend this preprint to others? Yes, it's of high quality
    Is it ready for attention from an editor, publisher or broader audience? Yes, after minor changes

    Competing interests

    The authors declare that they have no competing interests.

  3. Version published to 10.7554/elife.104057.1 on eLife
  4. Version published to 10.7554/elife.104057 on eLife
  5. eLife

    eLife Assessment

    In this important manuscript, Ryan et al perform a genome-wide CRISPR based screen to identify genes that modulate TDP-43 levels in neurons. They identify a number of genes and pathways and highlight the BORC complex, which is required for anterograde lysosome transport as one such regulator of TDP-43 protein levels. Overall, this is a convincing study, which opens the door for additional future investigations on the regulation of TDP-43.

  6. eLife

    Reviewer #1 (Public review):

    Summary:

    As TDP-43 mislocalization is a hallmark of multiple neurodegenerative diseases, the authors seek to identify pathways that modulate TDP-43 levels. To do this, they use a FACS based genome wide CRISPR KD screen in a Halo tagged TDP-43 KI iPSC line. Their screen identifies a number of genetic modulators of TDP-43 expression including BORC which plays a role in lysosome transport.

    Strengths:

    Genome wide CRISPR based screen identifies a number of modulators of TDP-43 expression to generate hypotheses regarding RNA BP regulation and perhaps insights into disease.

    Weaknesses:

    It is unclear how altering TDP-43 levels may relate to disease where TDP-43 is not altered in expression but mislocalized. This is a solid cell biology study, but the relation to disease is not clear without providing evidence of BORC alterations in disease or manipulation of BORC reversing TDP-43 pathology in disease.

    The mechanisms by which BORC and lysosome transport modulate TDP-43 expression are unclear. Presumably, this may be through altered degradation of TDP protein but this is not addressed.

    Previous studies have demonstrated that TDP-43 levels can be modulated by altering lysosomal degradation so the identification of lysosomal pathways is not particularly novel.

    It is unclear whether this finding is specific to TDP-43 levels or whether lysosome localization may more broadly impact proteostasis in particular of other RNA BPs linked to disease.

    Unclear whether BORC depletion alters lysosome function or simply localization.

  7. eLife

    Reviewer #2 (Public review):

    Summary:

    The authors employ a novel CRISPRi FACS screen and uncover the lysosomal transport complex BORC as a regulator of TDP-43 protein levels in iNeurons. They also find that BORC subunit knockouts impair lysosomal function, leading to slower protein turnover and implicating lysosomal activity in the regulation of TDP-43 levels. This is highly significant for the field given that a) other proteins could also be regulated in this way, b) understanding mechanisms that influence TDP-43 levels are significant given that its dysregulation is considered a major driver of several neurodegenerative diseases and c) the novelty of the proposed mechanism.

    Strengths:

    The novelty and information provided by the CRISPRi screen. The authors provide evidence indicating that BORC subunit knockouts impair lysosomal function, leading to slower protein turnover and implicating lysosomal activity in the regulation of TDP-43 levels and show a mechanistic link between lysosome mislocalization and TDP-43 dysregulation. The study highlights the importance of localized lysosome activity in axons and suggests that lysosomal dysfunction could drive TDP-43 pathologies associated with neurodegenerative diseases like FTD/ALS. Further, the methods and concepts will have an impact to the larger community as well. The work also sets up for further work to understand the somewhat paradoxical findings that even though the tagged TDP-43 protein is reduced in the screen, it does not alter cryptic exon splicing and there is a longer TDP-43 half-life with BORC KD.

    Weaknesses:

    While the data is very strong, the work requires some additional clarification.

  8. eLife

    Reviewer #3 (Public review):

    Summary:

    In this work, Ryan et al. have performed a state-of-the-art full genome CRISP-based screen of iNEurons expressing a teggd version of TDP-43 in order to determine expression modifiers of this protein. Unexpectedly, using this approach the authors have uncovered a previously undescribed role of the BORC complex in affecting the levels of TDP-43 protein, but not mRNA expression. Taken together, these findings represent a very solid piece of work that will certainly be important for the field.

    Strengths:

    - BORC is a novel TDP-43 expression modifier that has never been described before and it seemingly acts on regulating protein half life rather than transcriptome level. It has been long known that different labs have reported different half-lives for TDP-43 depending on the experimental system but no work has ever explained these discrepancies. Now, the work of Ryan et al. has for the time identified one of these factors which could account for these differences and play an important role in disease (although this is left to be determined in future studies).
    - The genome wide CRISPR screening has demonstrated to yield novel results with high reproducibility and could eventually be used to search for expression modifiers of many other proteins involved in neurodegeneration or other diseases

    Weaknesses:

    - The fact that TDP-43 mRNA does not change following BORCS6 KD is based on a single qRT-PCR that does not really cover all possibilities. For example, the mRNA total levels may not change but the polyA sites may have switched from the highly efficient pA1 to the less efficient and nuclear retained pA4. There are therefore a few other experiments that could have been performed to make this conclusion more compelling, maybe also performing RNAscope experiments to make sure that no change occurred in TDP-43 mRNA localisation in cells.
    - Even assuming that the mRNA does not change, no explanation for the change in TDP-43 protein half life has been proposed by the authors. This will presumably be addressed in future studies: for example, are mutants that lack different domains of TDP-43 equally affected in their half-lives by BORC KD?. Alternatively, can a mass-spec be attempted to see whether TDP-43 PTMs change following BORCS6 KD?

  9. Version published to 10.1101/2024.09.30.615241v1 on bioRxiv