TET3 is a positive regulator of mitochondrial respiration in Neuro2A cells

Valeria Leon Kropf; Caraugh J. Albany; Anna Zoccarato; Hannah L. H. Green; Youwen Yang; Alison C. Brewer

doi:10.1371/journal.pone.0294187

Peer Review History

Original SubmissionAugust 19, 2023
21 Sep 2023 Decision Letter - Jyotshna Kanungo, Editor PONE-D-23-26086TET3 is a positive regulator of mitochondrial respiration in neuronal cellsPLOS ONE Dear Dr. Brewer, 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. Mostly positive, the reviewers have raised a few concerns, especially on the presentation and interpretation of some data. A reviewer has concerns regarding the title of the manuscript. The authors are advised to address those concerns in their revised manuscript. Please submit your revised manuscript by Nov 05 2023 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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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: Partly 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: This study used pharmacological and genetic manipulations, as well as transcriptomic and biochemical analysis to investigate the role and targets of TET3 in N2A cell line. The result from RNA seq suggested that oxidative phosphorylation was identified as the most significantly down-regulated pathway by Tet3-silencing, consisted with the downregulation of oxygen consumption rate, the mRNA levels of nuclear- and mitochondrial encoded oxidative phosphorylation, and mitochondrial quality control genes. Among these genes, EndoG was identified as a direct target of TET3-catalytic activity at non-CpG methylated sites within its gene body. The authors indicated that the decrease in OxPhos may be due to dysfunctional mitochondria rather than direct transcriptional regulation of OxPhos genes by TET3. This work represents enriched data and a series of interesting observations. However, there are some major concerns regarding the interpretation of the results. Major 1. The protein levels of each of the components assessed (ATP5A, UQCRC2, mtCO1, SDHB and NDFUB8) were found to be slightly increased upon Tet3-silencing, in contrast to the transcriptome data and the Q-PCR verification. Does Tet3-silencing enhance the protein synthesis or decrease the protein degradation? It is easy to assess by different inhibitors. 2. Figure 4 Restoring the full-length Tet3 or the kinase-dead mutants should be included to elucidate the specific role of TET3 on the EndoG regulation in N2a cells. 3. What is the functional relationship of TET3 and EndoG in regulating the mitochondrial respiration or oxidative phosphorylation in N2A cell? Synergistically or not? Does restore EndoG rescue the impaired phenotype by TET3 silencing? 4. The title is misleading because N2A is neuroblastoma cell line. 5 “The decrease in OxPhos may be due to dysfunctional mitochondria rather than direct transcriptional regulation of OxPhos genes by TET3” is overstated. Reviewer #2: This is a well written manuscript with some minor issues: 1. Please define "low glucose" in the methods, with mM concentration for the N2a cells. 2. Please define the concentrations of drugs used in the seahorse assay (oligomycin, FCCP, and Rotenone/Antimycin A) 3. In the results section page 6, 2nd paragraph there is a highlighted XXX which needs to be filled in with the correct information for the GEO accession number. ****** 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
20 Oct 2023 Author Response We would like to thank the reviewers for taking the time to review our manuscript and for their helpful suggestions. We have now amended our manuscript as detailed below. Reviewer #1: This study used pharmacological and genetic manipulations, as well as transcriptomic and biochemical analysis to investigate the role and targets of TET3 in N2A cell line. The result from RNA seq suggested that oxidative phosphorylation was identified as the most significantly down-regulated pathway by Tet3-silencing, consisted with the downregulation of oxygen consumption rate, the mRNA levels of nuclear- and mitochondrial encoded oxidative phosphorylation, and mitochondrial quality control genes. Among these genes, EndoG was identified as a direct target of TET3-catalytic activity at non-CpG methylated sites within its gene body. The authors indicated that the decrease in OxPhos may be due to dysfunctional mitochondria rather than direct transcriptional regulation of OxPhos genes by TET3. This work represents enriched data and a series of interesting observations. However, there are some major concerns regarding the interpretation of the results. Major 1. The protein levels of each of the components assessed (ATP5A, UQCRC2, mtCO1, SDHB and NDFUB8) were found to be slightly increased upon Tet3-silencing, in contrast to the transcriptome data and the Q-PCR verification. Does Tet3-silencing enhance the protein synthesis or decrease the protein degradation? It is easy to assess by different inhibitors. Response: We fully concur that both enhanced (aberrant) protein synthesis and reduced protein degradation could result in the discrepancy between the changes in mRNA and protein levels of the OxPhos that we observed upon TET3 downregulation together with the decrease in mitochondrial bioenergetics. This point is now discussed (highlighted in the 4th para of discussion). However, we have not, as yet, carried out any further experiments (using inhibitors) to investigate this further. We believe that due to the complex nature of the different pathways which regulate mitochondrial function this would prove quite challenging and time consuming. We believe that this kind of mechanistic study (based on our findings here) might be more informative (and potentially clinically relevant) if performed in primary neurons, which is beyond the scope of this paper. 2. Figure 4 Restoring the full-length Tet3 or the kinase-dead mutants should be included to elucidate the specific role of TET3 on the EndoG regulation in N2a cells. Response: We identify EndoG as a catalytic target of TET3 as we show it to be upregulated by 5-azaC (suggesting it is regulated by methylation), to be downregulated by DMOG (indicating it to be regulated by the catalytic activity of a 2-OGDD enzyme), and to exhibit changes in 5-hmC upon TET3 downregulation. The rescue experiments proposed, using wild-type and kinase (catalytic?)-dead mutants would be inappropriate in our system, since we deplete TET3 expression using siRNAs, which would also degrade the “rescuing” gene expression. In addition, TET3 in mouse has both long and short isoforms, and it is not clear which would be the relevant isoform to test functionally in the rescue experiment. However, this is, of course a very interesting question with respect to the biochemistry of TET3, and determining which isoform is responsible for the catalytic activity on the EndoG promoter in a neuronal-like cell is something that we are currently aiming to determine. 3. What is the functional relationship of TET3 and EndoG in regulating the mitochondrial respiration or oxidative phosphorylation in N2A cell? Synergistically or not? Does restore EndoG rescue the impaired phenotype by TET3 silencing? Response: The silencing of TET3 resulted in the significant downregulation of a number of genes, known to be involved in mitochondrial function (Fig 1E &F). One purpose of our study was to determine which genes might be regulated by the catalytic activity of TET3, and only EndoG fell into that category. However, the other genes regulated by other (non-catalytic/more downstream) mechanisms would also contribute to the functional dysregulation of the mitochondria. Thus, we do not suggest that EndoG misexpression alone is responsible for the loss of mitochondrial bioenergetics, or that Endo G expression alone would rescue the phenotype. Rather, we suggest that it may be involved. However, we now realise that this was not made sufficiently clear and we apologise. We have now included a paragraph at the end of the discussion to clarify this point. 4. The title is misleading because N2A is neuroblastoma cell line. Response: We have now changed the title of the manuscript to make it clear that the study was performed in Neuro2A cells. 5 “The decrease in OxPhos may be due to dysfunctional mitochondria rather than direct transcriptional regulation of OxPhos genes by TET3” is overstated. Response: We have changed this sentence (at the end of the abstract) to “Accordingly, we propose that aberrant mitochondrial homeostasis may contribute to the decrease in OxPhos, observed upon Tet3-downregulation in Neuro2A cells”. We believe that this makes it clearer that the changes in OxPhos observed may in part also result from the changes in OxPhos gene expression. Reviewer #2: This is a well written manuscript with some minor issues: 1. Please define "low glucose" in the methods, with mM concentration for the N2a cells. Response: This information is now included in the methods section. 2. Please define the concentrations of drugs used in the seahorse assay (oligomycin, FCCP, and Rotenone/Antimycin A) Response: This information is now included in the methods section. 3. In the results section page 6, 2nd paragraph there is a highlighted XXX which needs to be filled in with the correct information for the GEO accession number. Response: If our manuscript is accepted, we will provide the repository information. We have also informed the editor of this. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0294187.r002
27 Oct 2023 Decision Letter - Jyotshna Kanungo, Editor TET3 is a positive regulator of mitochondrial respiration in Neuro2A cells PONE-D-23-26086R1 Dear Dr. Brewer, 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, Jyotshna Kanungo, Ph.D. 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: My questions have been addressed appropriately by the authors in the revision. I have no additional comments. ****** 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.0294187.r003
Formally Accepted
21 Nov 2023 Acceptance Letter - Jyotshna Kanungo, Editor PONE-D-23-26086R1 TET3 is a positive regulator of mitochondrial respiration in Neuro2A cells Dear Dr. Brewer: 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 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. Jyotshna Kanungo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0294187.r004

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