The role of Methyl-CpG binding domain 3 (Mbd3) protein in epileptogenesis

Karolina Nizinska; Maciej Olszewski; Sandra Binias; Dorota Nowicka; Kinga Szydlowska; Kinga Nazaruk; Bartosz Wojtas; Katarzyna Lukasiuk

doi:10.1371/journal.pone.0324580

Peer Review History

Original SubmissionMay 7, 2025
27 Jun 2025 Decision Letter - Giuseppe Biagini, Editor Dear Dr. Lukasiuk, Please submit your revised manuscript by Aug 11 2025 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. 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Kind regards, Giuseppe Biagini, MD Academic Editor PLOS ONE 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-->--> -->-->2. Thank you for stating in your Funding Statement: -->-->This work was supported by an OPUS grant 2015/19/B/NZ4/01401 (KL).  -->--> -->-->Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. -->-->Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.-->?> [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? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: 5. Please add the age and number of rats in the animals’ subheading of the methods section. 6. Please write in vitro in an Italic format in whole parts of the manuscript. 7. Did you check the normality of data? What was the relevant test/s? Please add its detail in the statistical analysis. 8. In the results section, for describing statistical results, please provide details of the statistical test used, and full statistical reporting of the results. Full statistical reporting should include the number of observations, the test statistics (eg t, f values), exact probabilities and degrees of freedom for each of the observations where statistics are provided. 9. The quality of provided Figure 3 is not good and it’s blur. Please provide a better image. In addition, it’s not clear which parts of the brain were evaluated in the figure. For better clarification, please also add an image with lower magnification. Reviewer #2: Nizinska et al. aimed to investigate the role of Mbd3 (an epigenetic regulator in neuron) in the epileptic animal and cell models by overexpression of this protein. They found that overexpression of Mbd3 could decrease anxiety, increase excitability and accelerate epileptogenesis in the PTZ-kindling rats. And then they profiled the mRNA transcripts in cultured cells with magnesium deficiency-induced epileptiform discharges and identified complex and orchestrated time- and state-specific changes of a subset of genes which are involved in various pathways implicating in contribution to epileptogenesis. This work has potential values in the field of epilepsy research. However, I have some concerns as follows: 1) The anayses of transcriptome data seem to be preliminary. Additional bioinformatic analyses help to highlight which enriched functional terms may be more associated with overexpression of Mbd3. 2) Represented genes on transcriptic data should be analysed by qPCR both in vitro and in vivo. 3) Further, the CpG status of some represented genes may also be detected under epileptic and Mbd3 overexpression conditions, which can provide strong evidence for the role of Mbd3 in epileptogenesis. 4) Fig 1E, F and G: Related Western blot images should be present in this figure. 5) Some typo errors need to be revised. ****** 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.] 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.0324580.r001
Revision 1
11 Aug 2025 Author Response POINT-BY-POINT RESPONSE TO ALL REVIEWERS' COMMENTS Reviewer #1: Comment: Please add the age and number of rats in the animals’ subheading of the methods section. Answer: To address this comment, we extracted some information from the "Animal Surgery" chapter and moved it to a newly created "Animals" chapter, and added the required information to that chapter. The added sentence reads: “Altogether, 220 animals were used; two animals died during PTZ kindling. The body weight at the start of the experiment was 280-320 g.” Comment: Please write in vitro in an Italic format in whole parts of the manuscript. Answer: We apologize for this error. It has been corrected throughout the manuscript. Comment: Did you check the normality of data? What was the relevant test/s? Please add its detail in the statistical analysis. Answer: Normality of data distribution was assessed using the Kolmogorov–Smirnov test. This information is now added to the chapter ‘Statistical analysis” Comment: In the results section, for describing statistical results, please provide details of the statistical test used, and full statistical reporting of the results. Full statistical reporting should include the number of observations, the test statistics (eg t, f values), exact probabilities and degrees of freedom for each of the observations where statistics are provided. Answer: To address this comment, we have improved the description of statistics in the “Materials and Methods” section and have rewritten the whole “Results” section to provide the requested information. Comment: The quality of provided Figure 3 is not good and it’s blur. Please provide a better image. In addition, it’s not clear which parts of the brain were evaluated in the figure. For better clarification, please also add an image with lower magnification. Answer: In response to this suggestion, we provide higher-quality images acquired using confocal microscopy. We have also added information on the localization of the presented neurons in the description of the Figure 3 and added relevant information to Materials and Methods section Reviewer #2 Comments: 1) The anayses of transcriptome data seem to be preliminary. Additional bioinformatic analyses help to highlight which enriched functional terms may be more associated with overexpression of Mbd3. In response to this comment we have substantially updated the manuscript. We have performed an additional functional enrichment analysis focusing on Gene Ontology Biological Process (GO BP) terms for differentially expressed genes at each analyzed time point. Additionally we have updated the analysis of Clusters, changing the analysis performed with Reactome to GO BP providing a more accessible for the reader view of biological processes potentially associated with Mbd3 overexpression. The results are now presented in the updated Table 1, with enriched GO BP terms for each time point. Furthermore, we have substantially revised and extended the Discussion section to elaborate on the biological roles of selected genes and pathways identified in the analysis, thereby providing a more comprehensive interpretation of the transcriptomic changes observed. Comments: 2) Represented genes on transcriptic data should be analysed by qPCR both in vitro and in vivo. 3) Further, the CpG status of some represented genes may also be detected under epileptic and Mbd3 overexpression conditions, which can provide strong evidence for the role of Mbd3 in epileptogenesis. Answer: We fully agree that validating transcriptomic data by qPCR both in vitro and in vivo, as well as investigating the CpG methylation status of selected genes under epileptic conditions and Mbd3 overexpression, would provide critical mechanistic insights into the role of Mbd3 in epileptogenesis. At this stage, we are actively seeking funding to pursue these directions, which we intend to address in a follow-up study and subsequent manuscript. Comment: 4) Fig 1E, F and G: Related Western blot images should be present in this figure. Answer: To address this comment, we have added to Figure 1 panel H, presenting the Western Blot image, which shows the only statistically significant difference between protein levels following PTZ injection, that is, the 4h time point in the entorhinal cortex/amygdala. Due to the large number of samples analyzed, the individual time points were run on separate gels. As a result, assembling a single panel with the corresponding Western blot images would require splicing individual lanes from different blots. We believe that such a figure would not be informative and could be potentially misleading. For this reason, we decided to show only representative images of the time point with a statistically significant difference. Comment: 5) Some typo errors need to be revised. Answer: We apologize for the typo errors. The text was checked for spelling, grammatical, and stylistic errors. Attachments Attachment Submitted filename: Nizinska et al rebuttal.odt https://doi.org/10.1371/journal.pone.0324580.r002
27 Aug 2025 Decision Letter - Giuseppe Biagini, Editor Dear Dr. Lukasiuk, 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 Oct 11 2025 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. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Giuseppe Biagini, MD Academic Editor PLOS ONE Journal Requirements: If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 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 Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: (No Response) ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: (No Response) ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: (No Response) ****** Reviewer #1: All concerns have been fully addressed. As a minor issue, please rewrite the conclusion of the abstract. Reviewer #2: The authors have addressed most of my concerns, and this manuscript has been improved upon revision. ****** 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: Yes: Maryam Ghasemi-Kasman 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.] 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 https://doi.org/10.1371/journal.pone.0324580.r003
Revision 2
5 Sep 2025 Author Response Dear Prof Biagini and Reviewers, Thank you for the opportunity to revise and resubmit our manuscript PONE-D-25-22943, entitled “The role of Methyl-CpG binding domain 3 (Mbd3) protein in epileptogenesis. We sincerely appreciate the time and effort that you and the Reviewers have invested in evaluating our work. We have carefully considered all the comments and suggestions, and we have revised the manuscript accordingly. Below, we provide a detailed response to each point raised by the Reviewers. POINT-BY-POINT RESPONSE TO ALL REVIEWERS' COMMENTS Reviewer #1: Comment: All concerns have been fully addressed. As a minor issue, please rewrite the conclusion of the abstract. Answer: The abstract has been rewritten according to the Reviewer's suggestions and now reads: Methyl CpG binding domain 3 (Mbd3) protein belongs to the MBD family of proteins and is responsible for reading the DNA methylation pattern. Our previous study revealed increased levels of Nucleosome Remodeling and Deacetylase (NuRD) complex proteins, including Mbd3, in the brains of epileptic animals. The present study investigated whether the Mbd3 protein level determines the seizure threshold. We demonstrate that seizures induced by pentylenetetrazole (PTZ) cause a transient, brain area-specific increase in Mbd3 protein levels in the entorhinal cortex and amygdala. Overexpression of Mbd3 in the amygdala using AAV decreased anxiety, increased excitability in the open-field test, and accelerated epileptogenesis in the PTZ-kindling model. In vitro, mRNA profiling using RNA-seq in a model of magnesium deficiency-induced epileptiform discharges revealed complex, time- and state-specific changes in gene expression. Genes regulated by Mbd3 overexpression were associated with the Wnt and Notch pathways, potassium channel function, and GABAB receptor signaling. Our findings indicate that increased Mbd3 expression has pro-epileptic properties and contributes to the regulation of multiple pathways potentially involved in seizure development. Significantly, seizures themselves transiently elevate Mbd3 levels, suggesting a potential vicious circle that may aggravate disease progression. Targeting the pro-epileptic effects of Mbd3 could therefore represent a novel therapeutic approach in epilepsy. Attachments Attachment Submitted filename: Nizinska_et_al_rebuttal_auresp_2.odt https://doi.org/10.1371/journal.pone.0324580.r004
16 Sep 2025 Decision Letter - Giuseppe Biagini, Editor The role of Methyl-CpG binding domain 3 (Mbd3) protein in epileptogenesis PONE-D-25-22943R2 Dear Dr. Lukasiuk, 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. For questions related to billing, please contact billing support . 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, Giuseppe Biagini, MD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0324580.r005
Formally Accepted
Acceptance Letter - Giuseppe Biagini, Editor PONE-D-25-22943R2 PLOS ONE Dear Dr. Lukasiuk, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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. Giuseppe Biagini Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0324580.r006

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