Tau phosphorylation suppresses oxidative stress-induced mitophagy via FKBP8 receptor modulation

Michael O. Isei; Meredith Crockett; Emily Chen; Joel Rodwell-Bullock; Trae Carroll; Peter A. Girardi; Keith Nehrke; Gail V. W. Johnson

doi:10.1371/journal.pone.0307358

Peer Review History

Original SubmissionJuly 3, 2024
17 Sep 2024 Decision Letter - Vladimir Trajkovic, Editor PONE-D-24-27298Tau phosphorylation suppresses oxidative stress-induced mitophagy via FKBP8 receptor modulationPLOS ONE Dear Dr. Johnson, 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. ============================== Reviewer 1: In this work, Isei et al. have shown that phosphorylated tau at specific residues decreases oxidative stress-induced mitophagy. In previous studies, the group described that mutated tau (Thr231E) inhibits mitophagy, now they found that also tau phosphorylation at other residues suppresses oxidative-induced mitophagy, through a novel mechanism, the interaction of phospho tau to FKBP8 receptor. The work is sound but some specific points should be discussed. - There are several factors, like phosphorylated tau, parkin or FKBP8 receptor that could affect mitochondrial function. About tau, it should be indicated not only the gain of (dys)function by phosphor tau, but also that the absence of tau could facilitate mitochondrial action (see for example Front. Neurosc. 2021, 14: 586710). Also, it was shown that modified tau impairs mitophagy via parkin inhibition (see for example reference 9). Now, in this work, a new role for tau in mitophagy based on its binding to FKBP8 receptor has been clearly indicated. In summary, it should be focused the role of tau by different ways on mitophagy regulation. The roles of tau or parkin have been extensively shown. However, it is reported that FKBP8 knock down does not affect to mitophagy. It is suggested that a compensatory mechanism could upregulate mitophagy through other pathways. Perhaps, some of those pathways should be commented. Also, k.o. of FKBP8 does not have effect in CCCP-induced mitophagy in HEK 293 cells and it was suggested that it may be due to the stressor type used. In this way, for some experiments, it could be advisable to use two different stressor types. - The data of Figure S3B may be complemented with immunochemistry analysis if a suitable antibody is available. Minor point: Eight Figures are many figures, perhaps Figure 1 could be a supplementary Figure and Figures 6 and 7 may be joined in a single one. ============================== Please submit your revised manuscript by Nov 01 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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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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. 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In previous studies, the group described that mutated tau (Thr231E) inhibits mitophagy, now they found that also tau phosphorylation at other residues suppresses oxidative-induced mitophagy, through a novel mechanism, the interaction of phospho tau to FKBP8 receptor. The work is sound but some specific points should be discussed. - There are several factors, like phosphorylated tau, parkin or FKBP8 receptor that could affect mitochondrial function. About tau, it should be indicated not only the gain of (dys)function by phosphor tau, but also that the absence of tau could facilitate mitochondrial action (see for example Front. Neurosc. 2021, 14: 586710). Also, it was shown that modified tau impairs mitophagy via parkin inhibition (see for example reference 9). Now, in this work, a new role for tau in mitophagy based on its binding to FKBP8 receptor has been clearly indicated. In summary, it should be focused the role of tau by different ways on mitophagy regulation. The roles of tau or parkin have been extensively shown. However, it is reported that FKBP8 knock down does not affect to mitophagy. It is suggested that a compensatory mechanism could upregulate mitophagy through other pathways. Perhaps, some of those pathways should be commented. Also, k.o. of FKBP8 does not have effect in CCCP-induced mitophagy in HEK 293 cells and it was suggested that it may be due to the stressor type used. In this way, for some experiments, it could be advisable to use two different stressor types. - The data of Figure S3B may be complemented with immunochemistry analysis if a suitable antibody is available. Minor point: Eight Figures are many figures, perhaps Figure 1 could be a supplementary Figure and Figures 6 and 7 may be joined in a single one. ****** 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. 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Revision 1
9 Oct 2024 Author Response PONE-D-24-27298 “Tau phosphorylation suppresses oxidative stress-induced mitophagy via FKBP8 receptor modulation” To the Editor: We thank the editor for overseeing the review of our manuscript and the opportunity to revise it. The reviewer’s comments are in italics and our responses in regular font. To reviewer 1: We thank the reviewer for their careful review and appreciation of our work. Their comments and suggestions are very thoughtful and useful, and we have addressed them on a point-by-point basis as outlined below. 1. “About tau, it should be indicated not only the gain of (dys)function by phosphor tau, but also that the absence of tau could facilitate mitochondrial action (see for example Front. Neurosc. 2021, 14: 586710)”. Response: We appreciate the reviewer for bringing this important point to our attention and believe this addition will strengthen our manuscript. We agree that it is important to expand our manuscript to address that coupled with the gain of (dys)function imposed by phosphorylated tau, the absence of tau could facilitate mitochondrial action. We have now discussed our previous work and other findings indicating that tau depletion/knockout may have neuroprotective effects, particularly on mitochondrial function and aging (Pallas-Bazarra et al., 2019, PMID:31235881; Pallo & Johnson, 2015, PMID:25888814; Jara et al., 2020, PMID:33679286; Jara et al., 2018, PMID:30077079). We also highlighted that tau depletion may prevent aging-related accumulation of phosphorylated tau and mitophagy dysregulation (page 24, line 520-529 in the manuscript). We also added a paper that explored the interplay between tau phosphorylation and Alzheimer’s disease (Avila, 2006, PMID:16529745) 2. “The roles of tau or parkin have been extensively shown. However, it is reported that FKBP8 knock down does not affect to mitophagy. It is suggested that a compensatory mechanism could upregulate mitophagy through other pathways. Perhaps, some of those pathways should be commented” Response: We thank the reviewer for the suggestion. We have now included a discussion on relevant studies highlighting such compensatory mechanisms. For example, studies in Drosophila and HeLa cells (Yun et al., 2014, PMID:24898855) demonstrated that MUL1-mediated mitophagy can compensate for the loss of PINK1/Parkin by regulating mitofusin levels. Similarly, in C. elegans, knockdown of DCT-1 (a mitophagy receptor; BNIP3 in vertebrates) or PINK1 results in upregulation of SKN-1 (Nrf2 in vertebrates)-mediated mitophagy (Palikaras et al., 2015, PMID:25896323). These examples provide insights into how alternative mitophagy pathways may be activated when FKBP8 is knocked down, ensuring mitochondrial quality control. (Page 23, line 488-497 in the manuscript) 3. Also, k.o. of FKBP8 does not have effect in CCCP-induced mitophagy in HEK 293 cells and it was suggested that it may be due to the stressor type used. In this way, for some experiments, it could be advisable to use two different stressor types. Response: We thank the reviewer for the insightful comment. In this study, besides Paraquat, we also investigated the effect of Urolithin A on mitophagy in our model, and unexpectedly, it did not significantly stimulate mitophagy. The insignificant mitophagy induction observed with Urolithin A in our cell line underscores the complexity and context-dependency of mitophagy regulation. This result, while initially surprising, aligns with the growing body of evidence suggesting that mitophagy induction is highly dependent on the specific stressor, cell type, and organism involved. Furthermore, the efficacy of Urolithin A may depend on specific genetic factors or signaling pathways present in certain cell types but not others. Our cell line may lack certain receptors or signaling components necessary for Urolithin A-mediated mitophagy activation. Our group is currently exploring the effects of three additional stressors, each with distinct mechanisms of action, on mitophagy in C. elegans. These studies aim to understand how different stressors induce mitophagy and the potential compensatory pathways involved. We are also extending this investigation to our newly acquired immortalized human neural progenitor cell line (ReN cells), which can differentiate into neurons and glial cells, allowing us to test these stressors in a more relevant human model. This approach will provide deeper insights into the variability of mitophagy responses across different cell types and stressor conditions. 4. The data of Figure S3B may be complemented with immunochemistry analysis if a suitable antibody is available. Response: We appreciate the reviewer's suggestion. Previously, we have tried to assess microtubule dynamics upon treatment with nocodazole but encountered some challenges. We attempted to use a suitable antibody for immunochemistry analysis of microtubules upon exposure to nocodazole. We found that our antibodies lack the specificity and sensitivity required for reliable immunochemistry analysis in our experimental setup. To overcome this limitation, we developed an alternative approach. We generated a BFP:FKBP8:Tau plasmid, which will allows us to monitor FKBP8 dynamics along the microtubules through dynamic fluorescent imaging in real-time using confocal microscopy. However, we encountered a significant challenge with this approach. Due to the spectral properties of the fluorophores involved in our experimental setup (BFP:CFP), we observed considerable spectral bleed-through leading to false positive signals and reduced data reliability. 5. Minor point: Eight Figures are many figures, perhaps Figure 1 could be a supplementary Figure and Figures 6 and 7 may be joined in a single one Response: We thank the reviewer for this suggestion. Figure 1 has been moved to the supplementary materials as Supplementary Figure S1. As suggested, we have merged Figures 6 and 7, now labeled as Figure 5 in the revised manuscript. With these changes, we have reduced the total number of main figures from eight to six. We have updated all figure references throughout the text to reflect these changes. The figure legends have been revised to accommodate the merged figure, ensuring clear explanation of all data presented. Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. Response: We have revised the manuscript to align with the style requirements and templates. Specifically, we adjusted the font sizes of the major sections and subsections, added asterisks to indicate the corresponding authors, and removed physical addresses, leaving only their email addresses. The changes have been highlighted for your reference. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. Response: Thank you for the observation. The funding information is NIH R01 AG067617. 3. Thank you for stating the following financial disclosure: Response: Thank you. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Response: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: [We extend our gratitude to Dr. Hemachandra Reddy from Texas Tech University for generously providing the HT22 cells, and to Dr. Christoph Pröschel from the University of Rochester for supplying the lentiviral vectors. We also thank Dr. Peter Davies for the phospho-Ser396/404 antibody. This work was supported by the National Institutes of Health (NIH) R01 AG067617. The diagram in this study was prepared by BioRender (https://www.biorender.com/).] We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [NIH R01 AG067617]. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: Thank you for appreciating our acknowledgement. We have now removed the funding information from the acknowledgement section and other area in the manuscript. The funding statement is, NIH R01 AG067617. 5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Response: All uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files are included in Supporting Information as: S1_raw_images. 6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Response: We have now included title and captions of the supporting information files at the end of the manuscript, and update in-text citations accordingly. 7. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Response: We have reviewed the reference list and highlighted the six newly added references. Thank you very much for considering our submission. We believe that these revisions have substantially improved the manuscript, and we hope that it is now acceptable for publication in PLOS ONE journal. Sincerely, Gail VW Johnson Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0307358.r002
21 Oct 2024 Decision Letter - Vladimir Trajkovic, Editor Tau phosphorylation suppresses oxidative stress-induced mitophagy via FKBP8 receptor modulation PONE-D-24-27298R1 Dear Dr. Johnson, 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, Vladimir Trajkovic Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. 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 ****** 5. 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 ****** 6. 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: This revised manuscript has been clearly improved and all the points raised by this reviewer have been properly answered. ****** 7. 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 ******** https://doi.org/10.1371/journal.pone.0307358.r003
Formally Accepted
1 Nov 2024 Acceptance Letter - Vladimir Trajkovic, Editor PONE-D-24-27298R1 PLOS ONE Dear Dr. Johnson, 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 Prof. Vladimir Trajkovic Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307358.r004

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