Deciphering the interactome of Ataxin-2 and TDP-43 in iPSC-derived neurons for potential ALS targets

Yuan Tian; Nicolette Heinsinger; Yinghui Hu; U-Ming Lim; Yi Wang; Aaron Zefrin Fernandis; Sophie Parmentier-Batteur; Becky Klein; Jason M. Uslaner; Sean M. Smith

doi:10.1371/journal.pone.0308428

Peer Review History

Original SubmissionJuly 26, 2024
11 Nov 2024 Decision Letter - Asif Ali, Editor PONE-D-24-30547Deciphering the interactome of Ataxin-2 and TDP-43 in iPSC-derived neurons for potential ALS targetsPLOS ONE Dear Dr. Tian, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec 26 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Additional Editor Comments: The manuscript by Tian et al. offers a comprehensive investigation into the interaction between Ataxin-2 and TDP-43, two key proteins implicated in Amyotrophic Lateral Sclerosis (ALS). Using iPSC-derived GABA neurons, the authors convincingly demonstrate that the interaction between Ataxin-2 and TDP-43 is mediated by the RNA Recognition Motif (RRM) of Ataxin-2. They further highlight the role of Ataxin-2 in stress granule dynamics and its influence on TDP-43-associated neurotoxicity. However, Reviewer #1 notes the need to address the possibility of RNA-mediated interactions and suggests RNase treatment as a control. Additionally, Figure 7's relevance is questioned due to the minimal effect observed with TAF-15 knockdown. Minor clarifications, such as defining TARDBP, detailing stress duration, and improving figure clarity, are also recommended. Reviewer #2 finds the study impactful and suggests including raw data and analysis details for enhanced transparency. Overall, this work provides new insights into ALS pathology and lays the groundwork for potential therapeutic targets. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The paper investigates the interaction between Ataxin-2 and TDP-43, two proteins implicated in Amyotrophic Lateral Sclerosis (ALS). Primarily using human iPSC-derived GABA neurons, the authors show that Ataxin-2 and TDP-43 interact in cells and that this interaction is dependent on the RRM of Ataxin-2. They also show that Ataxin-2 depletion decrease stress granule formation and decreases neuron toxicity associated with TDP-43 overexpression. Finally, they perform Ataxin-2 IP-MS in the neuronal culture to identify interaction partners. Overall, the paper provides a comprehensive analysis of the Ataxin-2 and TDP-43 interactome, offering new insights into the molecular mechanisms underlying ALS and identifying potential targets for future therapeutic development. The experiments are carefully performed and well described. Major Comments: 1. The authors clearly demonstrate that mutations in the RRM of TDP-43 reduce the pulldown of Ataxin-2, however they do not show that this interaction is direct. As the RRM of TDP-43 presumably binds RNA, this interaction may be mediated through RNA. The authors should either make this caveat clear, or even better, try treating their lysates with RNase prior to pulldown to rule out an RNA-mediated interaction. 2. I do not think that Figure 7 adds much scientifically. The TAF-15 knockdown does lead to slight neuronal death, but this effect is not amplified in the context of TDP-43 overexpression. Thus, there is no evidence that TAF15 is modulating TDP-43 mediated toxicity as the authors suggest it may be. Minor Comments: 340: Clarify that TARDBP encodes TDP-43 375: How long was the stress? Also mention in figure legend Figure 2G: please make clear what the SG marker is Figure 3B: Do you think this interaction is mediated by RNA (RRM being the RNA-binding domain). Can you do an RNase treatment before the IP? 404: HEK293T? Fig. 5C/D: label X-axis directly with FC compared to IgG control Fig. 7B: Coloring is confusing (looks the same as the AAV-syn-TDP-43) Reviewer #2: The manuscript titled "Deciphering the Interactome of Ataxin-2 and TDP-43 in iPSC-Derived Neurons for Potential ALS Targets" by Tian et al. presents a valuable study on the interactome of TDP-43 and Ataxin-2. This research is of considerable interest to those studying ALS protein characterization, neurobiology, and related fields. I anticipate that this article will be widely cited. The manuscript is well-written, and I commend the authors for their efforts. I have only one minor suggestion for improvement: I recommend that the authors provide all raw data in a supplementary file along with a detailed description of their data analysis process, as this would be beneficial for readers. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Pawan Kumar ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0308428.r001
Revision 1
27 Nov 2024 Author Response November 25th, 2024 Re: PLOS ONE Decision: Revision required [PONE-D-24-30547] - [EMID:43544307ce9c1c20] Asif Ali Academic Editor PLOS ONE Dear Editor Asif, Thank you for considering the publication of our manuscript "Deciphering the interactome of Ataxin-2 and TDP-43 in iPSC-derived neurons for potential ALS targets". The authors thank the reviewers for their enthusiastic response to our work and insightful comments for improvement. We are excited that the reviewers feel our work will be “of considerable interest” to the field, that “our experiments are carefully performed and well described”, and they “anticipate that this article will be widely cited”. We addressed the reviewers’ comments and are excited to present this revised and significantly strengthened manuscript for you to review. Below you will find an itemized list of the reviewers' comments and our response describing revisions made. The authors’ responses are in blue, and changes made in the manuscript are labeled with page and line numbers and in blue italic. Reviewer #1: The paper investigates the interaction between Ataxin-2 and TDP-43, two proteins implicated in Amyotrophic Lateral Sclerosis (ALS). Primarily using human iPSC-derived GABA neurons, the authors show that Ataxin-2 and TDP-43 interact in cells and that this interaction is dependent on the RRM of Ataxin-2. They also show that Ataxin-2 depletion decrease stress granule formation and decreases neuron toxicity associated with TDP-43 overexpression. Finally, they perform Ataxin-2 IP-MS in the neuronal culture to identify interaction partners. Overall, the paper provides a comprehensive analysis of the Ataxin-2 and TDP-43 interactome, offering new insights into the molecular mechanisms underlying ALS and identifying potential targets for future therapeutic development. The experiments are carefully performed and well described. Major Comments: 1. The authors clearly demonstrate that mutations in the RRM of TDP-43 reduce the pulldown of Ataxin-2, however they do not show that this interaction is direct. As the RRM of TDP-43 presumably binds RNA, this interaction may be mediated through RNA. The authors should either make this caveat clear, or even better, try treating their lysates with RNase prior to pulldown to rule out an RNA-mediated interaction. We agree with the reviewer that perhaps this interaction is mediated by RNA, and we carefully considered their suggestion. However, the IP methodology that we used here may not be sensitive enough to pick up on small changes that may result from using RNase treatment as the reviewer suggests. We acknowledge the possibility that using RNase treatment before IP may cause the protein-protein interactions to be weaker; however, we feel that this experiment would require more sensitive methodology and much further experimentation. This experiment would be very interesting for follow up studies in the future. Additionally, we added the following text to the discussion on line 656 to make this caveat clear as the reviewer suggests: “Additionally, the RRM domain of the TDP-43 protein notably binds RNA, and therefore, it is a possibility that the Ataxin-2 and TDP-43 interaction may also be mediated through RNA. Future studies may seek to explore the contribution of RNA to this interaction.” 2. I do not think that Figure 7 adds much scientifically. The TAF-15 knockdown does lead to slight neuronal death, but this effect is not amplified in the context of TDP-43 overexpression. Thus, there is no evidence that TAF15 is modulating TDP-43 mediated toxicity as the authors suggest it may be. We agree with the reviewer that the TAF15 story needs more experimentation that would be out of scope of this manuscript. Therefore, we decided to remove Figure 7 from the paper because we agree that as it stands alone now is a bit confusing to the reader. However, we did leave some speculation about the effect of TAF 15 in our discussion because it was an interesting finding from our Mass Spectrometry analysis that has been supported by recent literature, and we wanted to highlight its interest to the field. Minor Comments: 340: Clarify that TARDBP encodes TDP-43 We added this clarification on line 347. The texts now reads: Tar6/Tar6 is a transgenic mouse line that overexpresses human wild-type TARDBP (encodes for TDP43) at about 1.2-fold over endogenous protein level(16). 375: How long was the stress? Also mention in figure legend We added that the stress was for 2 hours to both the results on line 413 and figure legend on line 389. Figure 2G: please make clear what the SG marker is We added this clarification to the 2G figure legend. It now reads: High-content imaging quantification of eIFh+ stress granule numbers per neuron... Figure 3B: Do you think this interaction is mediated by RNA (RRM being the RNA-binding domain). Can you do an RNase treatment before the IP? We agree with the reviewer that it is possible that this interaction may be mediated by RNA, though fully exploring this possibility would require more sensitive methodology that is outside the scope of this paper. Please see our comments above for a more complete explanation. 404: HEK293T? HEK293T cells are a derivative of HEK293 cells, that have been transformed with the SV40 large T antigen. This mutation results in faster growth and higher transfection efficiency rates compared to HEK293 cells, which is why we chose this cell line. Fig. 5C/D: label X-axis directly with FC compared to IgG control We modified the x-axis on Figure 5 to clearly state it is compared to IgG control. Fig. 7B: Coloring is confusing (looks the same as the AAV-syn-TDP-43) We decided to remove this figure in agreement with the reviewer’s comment above. Please see our response above for further explanation. Reviewer #2: The manuscript titled "Deciphering the Interactome of Ataxin-2 and TDP-43 in iPSC-Derived Neurons for Potential ALS Targets" by Tian et al. presents a valuable study on the interactome of TDP-43 and Ataxin-2. This research is of considerable interest to those studying ALS protein characterization, neurobiology, and related fields. I anticipate that this article will be widely cited. The manuscript is well-written, and I commend the authors for their efforts. I have only one minor suggestion for improvement: I recommend that the authors provide all raw data in a supplementary file along with a detailed description of their data analysis process, as this would be beneficial for readers. We agree that providing raw data would add value to readers. We uploaded an Excel file of the raw data for the Mass Spectrometry experiment and have included a detailed methods section describing the analysis process. We also included a supplemental document showing uncropped Western blot data. Finally, all graphs in our manuscript have individual data points to represent the data. We hope that these efforts improve data transparency for our readers. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0308428.r002
3 Dec 2024 Decision Letter - Asif Ali, Editor Deciphering the interactome of Ataxin-2 and TDP-43 in iPSC-derived neurons for potential ALS targets PONE-D-24-30547R1 Dear Dr. Tian, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Asif Ali Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0308428.r003
Formally Accepted
9 Dec 2024 Acceptance Letter - Asif Ali, Editor PONE-D-24-30547R1 PLOS ONE Dear Dr. Tian, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Asif Ali Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0308428.r004

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