PONE-D-24-18366Identifying putative substrates of Calpain-15 in neurodevelopmentPLOS ONE

Dear Dr. Sossin,

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Academic Editor

PLOS ONE

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Additional Editor Comments:

While both reviewers commented favorably on your manuscript, some concerns need to be addressed. These include validation that the different sex of the mice did not affect the results and validation of more hits. The authors do not need to address the suggestion that other ages or specific regions of the brain should be investigated (concern #5 of one of the reviewers).

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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: No

Reviewer #2: Yes

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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: I think this research paper is technically sound and appropriately analyzed. The one minor issue I noticed during my read-through, I noticed on line 338, that the paper says "moues brain" instead of "mouse brain."

Reviewer #2: This manuscript by Zha et al. examined the putative substrates of Calpain 15 (Capn15) by using Capn15 KO P2 mice of both sexes. The authors used RNA-seq proteomics, and N-terminomics/terminal amino isotopic labelling of substrates (TAILS) to elucidate the probable substrates of capn15. This manuscript aimed to provides some insights into the unidentified Capn 15 substrates. However, the manuscript can be strengthened by further addressing the points below.

1. The authors stated using both sexes of CapN15 KO mice. While it might be difficult to correctly identify the mice sex at P2, does the author have any evidence that there are no sex differences in these KO mice and if that influences the 38 mRNAs found to have significant differential gene expression changes? Please address this possibility.

2. Figure 1A and 1B: It is definitely critical to represent the up- and down-regulated genes in a figure rather than a table since it is being shown as a figure. The current version can be included as a table by itself.

3. The authors tested only two of the hits from the LISA analysis screen: Pax and Smarca4. Pax2 was shown to be significantly increased. I suggest the authors test and validate more than those two. Furthermore, additional work such as investigating effect of Pax2 inhibition can be performed. The same can be performed for Dcx that was suggested to be a putative cleavage product of Capn15.

4. The study will benefit from employing a computational approach that can help in labeling calpain substrate cleavage sites from amino acid sequence using conditional random fields or any computational technique using the sequences of peptides obtained from the TAILS experiment.

5. Since no substrate was validated and found in this study I suggest using other ages and not just P2 as well as focusing on a particular area of the brain such as cerebral cortex as opposed to using whole brain.

6. I believe this current manuscript can be really strengthened by performing further in vivo validation experiments.

Minor:

7. Figure 4: The images in figure 4 are not well aligned and seem scattered around.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-18366R1Identifying putative substrates of Calpain-15 in neurodevelopmentPLOS ONE

Dear Dr. Sossin,

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 manuscript has been evaluated by two reviewers, and their comments are available below. There are some remaining requests for the underlying data and further discussion. Could you please carefully revise the manuscript to address all comments raised?

Please submit your revised manuscript by Feb 24 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,

Helen Howard

Staff Editor

PLOS ONE

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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.

Additional Editor Comments (if provided):

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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

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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: All comments to the author has been addressed. I believe that the manuscript is technically sound and the data does support the conclusion.

Reviewer #2: The authors have partly addressed comments as it seems more work would be needed to strengthen this study.

1. However, I think it's advisable for the authors to make the raw and processed RNA-seq data available by depositing in the GEO database. The proteomics data should also be deposited in a public repository database such as Global Proteome Machine Database (GPMDB), PeptideAtlas, the PRoteomics IDEntifications database (PRIDE), Tranche, and NCBI Peptidome.

2. Additionally, the authors should discuss the strengths and limitations of the analysis from their study.

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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.]

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.

Identifying putative substrates of Calpain-15 in neurodevelopment

PONE-D-24-18366R2

Dear Dr. Sossin,

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.

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Kind regards,

Patrick Goymer

Staff Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: