The PINK1—Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells

Aaron V. Bradshaw; Philip Campbell; Anthony H. V. Schapira; Huw R. Morris; Jan-Willem Taanman

doi:10.1371/journal.pone.0259903

Peer Review History

Original SubmissionSeptember 15, 2021
15 Sep 2021 Author Response Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0259903.r001
22 Oct 2021 Decision Letter - Ivan R. Nabi, Editor PONE-D-21-29921The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear celPLOS ONE Dear Dr. Taanman, 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. With respect to Reviewer 2 comments: 1. if Ub blots are available I strongly recommend that they be included to strengthen the argument that the upper Mfn band is ubiquitinated. 2. I agree that while your data do show that Pink-Parkin signaling is not active in PBMCs, your results more specifically show that PINK1 is absent. This is in and of itself interesting as Pink1 mediates mitophagy by other Ub ligases other than Parkin. While I recognize that the focus of the paper is to show that PBMC screening would not be appropriate for PD, I would recommend that the manuscript mention the existence of Pink/Parkin independent mitophagy pathways. Indeed, a number of studies have shown that Pink is dispensable for basal mitophagy - McWilliams TG, Prescott AR, Montava-Garriga L, Ball G, Singh F, Barini E, et al. Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand. Cell Metab 2018; 27:439-49 e5. - Lee JJ, Sanchez-Martinez A, Zarate AM, Beninca C, Mayor U, Clague MJ, et al. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. J Cell Biol 2018; 217:1613-22. 3. an interesting suggestion but not necessary. Please submit your revised manuscript by Dec 06 2021 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. 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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: In this revision, the authors have responded to both reviewers comments very well and the manuscript is much improved. They have added additional data where required which has more strongly supported their conclusions. Reviewer #2: The work on "The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells" by Jan-Willem Taanman et al is very interesting. Here I have some suggestions: (1) The detection of ubiquitination of mitofusins can be more convincing if anti-ubiquitin WB is shown side by side (2)The statement "The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells" is too strong. The work shows the PINK1 – Parkin mitophagy signalling pathway is very limited due to the low level of PINK1 (3)Different cells may have different level responses to the stress (4 hours to 24 hours) or different treatments (CCCP, antimycin A, valinomycin, and rotenone). Have the authors tested them? ****** 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: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. 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Revision 1
25 Oct 2021 Author Response REBUTTAL PONE-D-21-29921 The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells PLOS ONE Dear Dr. Taanman, 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. With respect to Reviewer 2 comments: 1. if Ub blots are available I strongly recommend that they be included to strengthen the argument that the upper Mfn band is ubiquitinated. We agree with Reviewer #2 that it is important to demonstrate that the slower migrating bands recognised by anti-MFN1 and anti-MFN2 antibodies in CCCP-treated samples represent ubiquitinated mitofusin. We were the first to demonstrate that MFN1 and MFN2 are ubiquitinated in in a PINK1/Parkin-dependent manner upon induction of mitophagy [Gegg et al. Hum Mol Genet. 2010;19:4861-70; Ref. 10]. In our 2010 paper, we demonstrated that these slower migrating bands in CCCP-treated cells represent ubiquitinated mitofusin by means of anti-mitofusin pull-down immunoprecipitation experiments with an anti-ubiquitin antibody (Fig 4C and D in Gegg et al., 2010). The current manuscript builds on our previous observations. We have indicated this in the revised manuscript more clearly (line 102─104): “This size change is consistent with mono-ubiquitination and our previous work anti-mitofusin immunoprecipitation experiments [10] indicated that the extra MFN1 and MFN2 bands detected post-CCCP treatment are ubiquitin positive”. 2. I agree that while your data do show that Pink-Parkin signaling is not active in PBMCs, your results more specifically show that PINK1 is absent. This is in and of itself interesting as Pink1 mediates mitophagy by other Ub ligases other than Parkin. While I recognize that the focus of the paper is to show that PBMC screening would not be appropriate for PD, I would recommend that the manuscript mention the existence of Pink/Parkin independent mitophagy pathways. Indeed, a number of studies have shown that Pink is dispensable for basal mitophagy - McWilliams TG, Prescott AR, Montava-Garriga L, Ball G, Singh F, Barini E, et al. Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand. Cell Metab 2018; 27:439-49 e5. - Lee JJ, Sanchez-Martinez A, Zarate AM, Beninca C, Mayor U, Clague MJ, et al. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. J Cell Biol 2018; 217:1613-22. We have included a paragraph mentioning the existence of Pink/Parkin independent mitophagy pathways in the revised manuscript (lines 328─334): “There are multiple pathways orchestrating mitophagy of mammalian mitochondria [33]. Some involve mitophagy receptors expressed on the OMM that directly interact with the autophagy machinery. Others function indirectly. Of these, the ubiquitin-dependent PINK1 – Parkin pathway is the best studied. The fact that PINK1 and PRKN mutations are causative of early-onset PD underscores the importance of the PINK1 – Parkin mitophagy pathway in the brain but it has been demonstrated that PINK1 is dispensable for basal mitophagy [34, 35]. Our observations suggest that PBMCs employ PINK1-independent mitophagy pathways to clear defective mitochondria”. 3. an interesting suggestion but not necessary. See below. We look forward to receiving your revised manuscript. Kind regards, Ivan R. Nabi, Ph.D. Academic Editor PLOS ONE 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: Partly ________________________________________ 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: In this revision, the authors have responded to both reviewers comments very well and the manuscript is much improved. They have added additional data where required which has more strongly supported their conclusions. We thank Reviewer #1 for her/his kind words. Reviewer #2: The work on "The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells" by Jan-Willem Taanman et al is very interesting. Here I have some suggestions: (1) The detection of ubiquitination of mitofusins can be more convincing if anti-ubiquitin WB is shown side by side See above. (2) The statement "The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells" is too strong. The work shows the PINK1 – Parkin mitophagy signalling pathway is very limited due to the low level of PINK1 See above. (3) Different cells may have different level responses to the stress (4 hours to 24 hours) or different treatments (CCCP, antimycin A, valinomycin, and rotenone). Have the authors tested them? Our standard treatment to induce acute mitophagy in PBMCs was 2 h, 20 �M CCCP. However, we have tested a range of CCCP treatment times (0─24 h) and CCCP concentrations (0─100 �M). In addition, we have stressed PBMCs prior and during CCCP treatment by culturing for up to 8 h in Hank’s balanced salt solution or by culturing for up to 7 d in medium is which glucose was substituted by gala Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0259903.r003
29 Oct 2021 Decision Letter - Ivan R. Nabi, Editor The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells PONE-D-21-29921R1 Dear Dr. Taanman, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Ivan R. Nabi, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0259903.r004
Formally Accepted
2 Nov 2021 Acceptance Letter - Ivan R. Nabi, Editor PONE-D-21-29921R1 The PINK1 – Parkin mitophagy signalling pathway is not functional in peripheral blood mononuclear cells Dear Dr. Taanman: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Ivan R. Nabi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0259903.r005

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