PONE-D-24-53655Proliferative arrest induces neuronal differentiation and innate immune responses in normal and Creutzfeldt-Jakob Disease agent infected rat septal neuronsPLOS ONE

Dear Dr. Manuelidis,

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.

==============================

The reviews for the above manuscript have been received. The reviewers' comments are included along with this letter.

Two independent reviewers have assessed the manuscript's suitability for publication and solicit additional clarification on certain outstanding issues.

Based on the reports of the reviewers and my own assessment, I recommend that the authors revise the manuscript to address the concerns raised.

==============================

Please submit your revised manuscript by Apr 14 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.

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). 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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,

Abhinava Kumar Mishra, PhD

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 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. 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.

3. 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

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: General Comments

This study investigates the impact of proliferative arrest on neuronal differentiation and innate immune responses in rat septal neurons, comparing normal cells to those infected with the Creutzfeldt-Jakob Disease (CJD) agent. It employs transcriptomic analysis to uncover changes associated with latent and productive CJD infection states. The findings highlight that proliferative arrest in normal neurons leads to upregulation of neuronal differentiation markers, anti-proliferative genes, and innate immune pathway genes.

In contrast, neurons with latent (CJ-) infection exhibit epigenetic imprinting without significant transcriptional differences from normal neurons. However, productive (CJ+) arrested neurons show suppressed neuronal differentiation, heightened interferon and cytokine responses, partial escape from proliferative control, and persistent high infectivity. The study proposes that latent CJD infections can cause lasting transcriptional and epigenetic changes, contributing to the silent persistence of infectious agents in peripheral tissues.

While the study is intriguing and relevant, it requires additional validation to strengthen its conclusions and enhance its suitability for publication.

Major Comments

1. Lack of Functional Validation for Transcriptomic Findings

The manuscript provides valuable transcriptomic data but lacks functional or biochemical validation of key findings. While reproducibility across biological replicates is mentioned, RNA-seq results need to be validated using methods such as RT-qPCR or Western blot. RNA-level changes do not always correlate with protein-level changes or cellular phenotypes, making functional validation essential. To strengthen the robustness of the bioinformatics conclusions, the authors should validate the expression levels of selected genes with significant fold changes using RT-qPCR or similar methods.

2. Restricted Focus on a Single CJD Strain and In Vitro Model

The study's focus on a single CJD strain (FU-CJD) limits the generalizability of its findings. Including additional CJD strains in the analysis would provide broader insights and enhance the study's applicability. Similarly, reliance solely on an in vitro cell culture model may not fully recapitulate in vivo conditions. Expanding the scope of the study to include other models or strains is recommended.

Minor Comments

1. Abstract Revision

The abstract should be concise and easily understandable. The current abstract is overly complex and includes unnecessary details, such as specific candidate gene names, which are better suited for the main text. Simplifying the abstract will make it more accessible to readers.

2. Discussion Length

The discussion section is excessively long (six pages), with repetitive elaboration on results. A discussion should provide a concise summary of the findings, their implications, and their alignment with existing literature. The authors should restructure this section to avoid redundancy and improve clarity.

Reviewer #2: This manuscript by Pagano et al. investigated how RNA expression varies in rat post-mitotic septal neuronal cell lines at arrested versus proliferative states in both infected and uninfected conditions. The study identifies several pathways that are associated with specific states and conditions which can provide the basis for future studies into the mechanisms of neural differentiation as well immune responses in neurodegenerative diseases. The study was rigorously performed and the findings are well presented.

I suggest some minor modifications:

1. The authors need to always provide the full form of terms while introducing them for the first time

2. Th authors need to provide a protocol for qPCRs in the materials and methods section and provide a list of all the primers used along with their sequences

3. The figures have been written as figure.tif at multiple occasions. The authors need to correct that. Also, the authors often tended to start sentences by 'The figure shows' which is a rather uncommon practice. Its better to just state the findings and mention the relevant figure in bracket.

4. The introduction requires some rewriting to improve clarity about the necessity of this endeavor. It might be better if the authors start with the disease aspect of this study and make a logical progression to justify the usage of SEP cells for this study. Then they can provide information about relevant studies in these cells and then proceed to the next section.

**********

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: Yes:  Avinash Chandel

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.

Attachments

Attachment

Submitted filename: Manuscript Number PONE-D-24-53655.docx

Proliferative arrest induces neuronal differentiation and innate immune responses in normal and Creutzfeldt-Jakob Disease agent (CJ) infected rat septal neurons

PONE-D-24-53655R1

Dear Dr. Manuelidis,

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,

Abhinava Kumar Mishra, PhD

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

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: The authors have effectively addressed the concerns raised and have also cited relevant previous studies to support their responses to some of the issues highlighted. The current version of the abstract and discussion has been significantly improved Thus, I find the current version of the manuscript to be well-prepared and suitable for publication.

Reviewer #2: The authors have satisfactorily addressed all my comments. I am satisfied with the current version of the manuscript and recommend it being accepted without further changes.

**********

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

**********