Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis

Toni C. Berger; Magnus D. Vigeland; Hanne S. Hjorthaug; Lars Etholm; Cecilie G. Nome; Erik Taubøll; Kjell Heuser; Kaja K. Selmer

doi:10.1371/journal.pone.0226575

Peer Review History

Original SubmissionJuly 18, 2019
10 Sep 2019 Decision Letter - Giuseppe Biagini, Editor PONE-D-19-20265 Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis PLOS ONE Dear Dr. Berger, 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. We would appreciate receiving your revised manuscript by Oct 25 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Giuseppe Biagini, MD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf [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: 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: No ****** 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: Manusript by Berger et al. describes alterations in DNA methylation and gene expression in neurons and glia separately at 24h fllowing status epilepticus. Subject is very timely and of interest for many readers. Data are presented in details which enables readers to dig into particular molecular events of interest for them. I do not have any serious critical comments. Minor issues: - do animals in intracortical KA model develop epilepsy? - tables 1-2 may be misleading since cells in rows do not present the same or similar categories but from different databases as it might be interpreted by reader - does the method used by authors allow maintaining cell integrity during homogenization of frozen tissue, or is cytoplasm lost and only morre resistant nuclei are sorted? Reviewer #2: Review to manuscript PONE-D-19-20265 In the present study, Berger et al. describe cell population specific DNA methylation and gene expression in mice (n=8 per group, KA and sham respectively), 24h following status epilepticus from intracortical (juxta-hippocampal) Kainic acid injection. FANS was used to sort NeuN-positive and NeuN-negative nuclei from pooled HC. DNA and total RNA were extracted and used for sequencing. The experimental design and data are mainly well described, however the following comments remain: 1. Neuronal and non-neuronal nuclei were isolated, not whole cells. NeuN-negative nuclei, do not necessarily have to originate from glial cells. As nuclei were sorted and analyzed not all transcription was studied, but only immediate transcripts that were still present in the nucleus. The preparation method was also not mRNA-specific, but total RNA was extracted. The reviewer is aware of the technical limitations for single cell-sequencing from adult brain tissue and thus only requests that the authors are specific about their methodology to not confuse the reader. 2. p9: „For any gene, its promoter region was defined as the 1 kb segment immediately upstream of the transcription start site and the upstream region from -5 kb to -1 kb, where negative numbers indicate positions upstream of transcription start site.“ Unclear. So the promoter definition was -1kb from TSS and the upstream region is -1kb to -5kb? Please explain why the „upstream region“ was considered interesting? What function it is supposed to have in gene regulation? 3. Authors should make sequencing data (raw files) available through GEO. 4. The authors state “We detected single genetic loci in several epilepsy-related genes, where DNAm and GE changes coincide. These may serve as potential target sites for epigenetic antiepileptogenic therapeutic intervention.” However, the number of differentially methylated CpGs/regions that correlated with gene expression was very low. Also the time point 24h after SE is very short. At this stage the acute response to the KA treatment and status is visible, which has little to do with the later epileptogenic process and questions the suitability of suggested targets for therapeutic interventions in chronic epilepsy. In general, this reviewer feels that the discussion of single genes from the present data overestimates the results. Rather, a discussion of technical limitations should be included. 5. Much information appears to be hidden in the supplements. ****** 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. 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0226575.r001
Revision 1
30 Sep 2019 Author Response Manuscript number: PONE-D-19-20265 Title: Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis Dear Editor Professor Biagini, We thank you for the expert reviews on our paper! Based on the helpful comments of the reviewers, we have now prepared a revised and expanded version of our manuscript. In our opinion, the revision has improved our paper and we trust that you will find the new version suitable for publication in PLOS ONE. Below are our responses to the reviewers’ comments. Reviewer #1: Manuscript by Berger et al. describes alterations in DNA methylation and gene expression in neurons and glia separately at 24h following status epilepticus. Subject is very timely and of interest for many readers. Data are presented in detail which enables readers to dig into particular molecular events of interest for them. I do not have any serious critical comments. Minor issues: - do animals in intracortical KA model develop epilepsy? We want to thank reviewer 1 for the positive review. The answer to the question from reviewer 1 is YES. More than 90% of animals treated with intracortical kainic acid develop epilepsy, as characterized in detail by Bedner P. et al., in the journal Brain in 2015. This is further confirmed by our own experience with the model (Szokol K. et al., Front Exp Neurosci, 2015). Accordingly, a more thorough description of the KA model has been added in the revised manuscript (methods). - tables 1-2 may be misleading since cells in rows do not present the same or similar categories but from different databases as it might be interpreted by reader We agree with reviewer 1 and changed the tables 1 and 2 (now tables 3 and 4) accordingly. We think that they now provide a better overview of GO- and KEGG terms in neuronal and glial up- and downregulated genes. - does the method used by authors allow maintaining cell integrity during homogenization of frozen tissue, or is cytoplasm lost and only more resistant nuclei are sorted? Our method represents a modified variant of Jiang et al., BMC Neurosci, 2008 and is based on sorting of cell nuclei, which has several advantages over cell based sorting (e.g. as stated nuclei are more resistant than whole cells; Grindberg R. et al., PNAS, 2013). Consequently, cytoplasm and its contents are lost. This is already specified in the methods section and has now also been revised in the abstract as well as added to the new technical limitations chapter. Reviewer #2: Review to manuscript PONE-D-19-20265 In the present study, Berger et al. describe cell population specific DNA methylation and gene expression in mice (n=8 per group, KA and sham respectively), 24h following status epilepticus from intracortical (juxta-hippocampal) Kainic acid injection. FANS was used to sort NeuN-positive and NeuN-negative nuclei from pooled HC. DNA and total RNA were extracted and used for sequencing. The experimental design and data are mainly well described, however the following comments remain: 1. Neuronal and non-neuronal nuclei were isolated, not whole cells. NeuN-negative nuclei, do not necessarily have to originate from glial cells. As nuclei were sorted and analyzed not all transcription was studied, but only immediate transcripts that were still present in the nucleus. The preparation method was also not mRNA-specific, but total RNA was extracted. The reviewer is aware of the technical limitations for single cell-sequencing from adult brain tissue and thus only requests that the authors are specific about their methodology to not confuse the reader. First, we want to thank reviewer 2 for the in-depth review. Reviewer 2 is right regarding the NeuN- fraction containing a small number of other cell types than glia. In the revised manuscript, we now have specified this in a new section on technical limitations. Regarding RNA sequencing, we first isolated total RNA from sorted nuclei. The consecutive step of using a oligo(dT) primer for cDNA creation sub selects mRNA transcripts. We agree with reviewer 2 that this has not been pointed out clearly in the article. We have now adjusted nomenclature and added details in the methods section of the revised manuscript for clarification (methods, supplementary methods). Concerning the use of nuclear mRNA, we are aware of technical limitations like enrichment of mRNA coding for proteins with nuclear functions (Barthelson RA et al., BMC Genomics, 2007) and a potentially lower level of immediate early genes (Bakken TE et al., PLOS ONE, 2018) compared to cytosolic mRNA. This has now been added to the discussion (technical limitations). 2. p9: „For any gene, its promoter region was defined as the 1 kb segment immediately upstream of the transcription start site and the upstream region from -5 kb to -1 kb, where negative numbers indicate positions upstream of transcription start site.“ Unclear. So the promoter definition was -1kb from TSS and the upstream region is -1kb to -5kb? Please explain why the „upstream region“ was considered interesting? What function it is supposed to have in gene regulation? We thank the reviewer for the insightful feedback. Regarding the definition of genomic regions, we have modified our formulations in the paper (methods). As correctly stated, the upstream region was defined as from -5kb to -1kb and promoter region from -1kb to TSS. Concerning the use of the upstream region as a separate genomic feature, we used predefined genomic feature types supplied by the annotatr package in R, having no à priori hypothesis of possible importance of genomic feature types per se. 3. Authors should make sequencing data (raw files) available through GEO. RAW files have now been uploaded to GEO (GSE138100). 4. The authors state “We detected single genetic loci in several epilepsy-related genes, where DNAm and GE changes coincide. These may serve as potential target sites for epigenetic antiepileptogenic therapeutic intervention.” However, the number of differentially methylated CpGs/regions that correlated with gene expression was very low. Also the time point 24h after SE is very short. At this stage the acute response to the KA treatment and status is visible, which has little to do with the later epileptogenic process and questions the suitability of suggested targets for therapeutic interventions in chronic epilepsy. Several animal models of temporal lobe epilepsy have shown that important cellular/molecular changes already occur during the first hours and days after status epilepticus (Bedner P. et al., Brain, 2015; Rakhade SN et Jensen FE, Nat Rev Neurol. 2009). In this model, the latent phase of epilepsy starts after around 4 hours (post injection) and lasts 5+-2.9 days (see also figure below). We therefore believe that 24 hours post injection is an appropriate time point for studying early epigenetic mechanisms and gene expression precipitating downstream molecular changes. We agree with reviewer 2 that it also would be of high interest to study DNA methylation and gene expression at a later time point reflecting a chronic stage of epileptogenesis. Figure: Epileptogenesis in the intracortical kainic acid model of mTLE (modified from Bedner P. et al., Brain, 2015) In general, this reviewer feels that the discussion of single genes from the present data overestimates the results. Rather, a discussion of technical limitations should be included. We are aware of the limitations and restricted interpretability of a few genomic loci at one time point and have revised the discussion accordingly as well as added a new section on technical limitations. We want to thank reviewer 2 for the critical feedback, as this surely will increase the quality of this paper. 5. Much information appears to be hidden in the supplements. We have integrated the list of differentially expressed genes (up- and downregulated genes) as Tables 1 and 2 into the revised manuscript (numbers of the previous tables have been adjusted). With kind regards, Toni Berger Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0226575.r002
20 Nov 2019 Decision Letter - Giuseppe Biagini, Editor PONE-D-19-20265R1 Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis PLOS ONE Dear Dr. Berger, 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 following points: - Indicate how animals were killed. - Provide a section describing statistics in methods. - Check tables for values' unit (thousands). We would appreciate receiving your revised manuscript by Jan 04 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Giuseppe Biagini, MD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 Reviewer #2: 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 Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: no further comments Reviewer #2: (No Response) ****** 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 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. 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0226575.r003
Revision 2
25 Nov 2019 Author Response Dear Editor Professor Biagini, We thank you for your feedback regarding our paper: - Indicate how animals were killed. - Provide a section describing statistics in methods. - Check tables for values' unit (thousands). All three points have been addressed and the manuscript adjusted accordingly. We hope that you will find the new version suitable for publication in PLOS ONE. With kind regards, Toni Berger Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0226575.r004
3 Dec 2019 Decision Letter - Giuseppe Biagini, Editor Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis PONE-D-19-20265R2 Dear Dr. Berger, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Giuseppe Biagini, MD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0226575.r005
Formally Accepted
10 Dec 2019 Acceptance Letter - Giuseppe Biagini, Editor PONE-D-19-20265R2 Neuronal and glial DNA methylation and gene expression changes in early epileptogenesis Dear Dr. Berger: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Giuseppe Biagini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226575.r006

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