PONE-D-23-36719Aβ25-35-induced autophagy and apoptosis are inhibited by the CRMP2-derived peptide ST2-104 (R9-CBD3) via a CaMKKβ/AMPK/mTOR signaling hubPLOS ONE

Dear Dr. Sun,

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Qi Chen, PhD

Academic Editor

PLOS ONE

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 [This study was supported by the grant provided by the Major Chronic Disease Program of the Ministry of Science and Technology of China (No. 2018YFC1312301), the General Program of the National Natural Science Foundation of China (No. 82071442), the Jilin Provincial Department of Finance（JLSWSRCZX2021-004）.].  

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Additional Editor Comments:

Fig 1D, as the concentration of ST2-104 increased from 5 uM to 40 uM, the protective effects was not increasing. Instead, it seemed decreasing. The authors should discuss why.

Fig 3. Autophagy flux should be examined with a liposome inhibitor such as chloroquine.

Fig 7 and Fig 8 lacking a STO-609 control.

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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

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

Reviewer #1: 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: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: 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: The authors have evaluated the protective mechanism of ST2-104 in Aβ25-35 peptide induced cytotoxicity in SHY-SY5Y cells. They showed that ST2-104 prevented the neuronal apoptosis by inhibiting autophagy through a CaMKKβ/AMPK/mTOR signaling pathway. Study of the mechanism of Aβ neurotoxicity is invaluable. Overall, there are several concerns regarding the methodology and interpretation of results.

Major Comments:

Title: Aβ25-35-induced autophagy and apoptosis are inhibited by the CRMP2-derived peptide ST2-104 (R9-CBD3) via a CaMKKβ/AMPK/mTOR signaling hub

Comment: The title should accurately describe the contents of the manuscript. Since the authors used the ST2-104 peptide as a pre-treatment, Aβ25-35-induced autophagy and apoptosis are prevented not inhibited .............

Comments (Methodology and results):

Line 133 &139: What was the solvent of Aβ25-35? DMSO or acetic acid?

Control group received no vehicle? These should be addressed in the method section.

In Line 182, the authors mentioned the use of One-way analysis of variance. The authors should provide the F values in the result section.

The authors have stated in line 280, the percentage of cells in gate (M1). They should provide the flow cytometry protocol of gating and quantification, background noise detection, and an unstained sample.

Comments (Discussion):

In the line 349-356 of Discussion,: The sentences are suitable for introduction not discussion.

The SH-SY5Y cells have two phenotypes “N” and “S” types (Encinas et al. J Neurochem 75:991–1003, 2000). These should be discussed in relation to the current results.

In Line 389, the authors stated that “our data supports an anti-autophagic role for ST2-104 in the face of Aβ25-35 induced autophagy. Since the authors performed the pre-treatment, it is true to write ST2-104 prevented Aβ25-35 induced autophagy.

Please omit “we” in Line 399: "We found that when we autophagy was induced with RAPA, the nuclear condensation and the expression of Bax, C-caspase-"

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Reviewer #1: No

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PONE-D-23-36719R1Aβ25-35-induced autophagy and apoptosis are inhibited by the CRMP2-derived peptide ST2-104 (R9-CBD3) via a CaMKKβ/AMPK/mTOR signaling hubPLOS ONE

Dear Dr. Sun,

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 Aug 12 2024 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,

Qi Chen, PhD

Academic Editor

PLOS ONE

Journal Requirements:

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:

Please incorporate changes in response to the previous comments into the appropriate place in the manuscript.

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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

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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

**********

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

Reviewer #1: I Don't Know

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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: 1. The revised manuscript was reviewed. The authors have responded to the most of this reviewer's comments. The author’s explanations to queries must be incorporated in appropriate place in the manuscript. However, they referred to supplementary files. For instance, the discussion about the SH-SY5Y cells phenotypes or the F values.

In my opinion, it is ok if the supplementary files are published with the manuscript.

2. The first sentence of discussion” The ST2-104 peptide represents a novel class of such strategies.” What does it mean? which strategies? It should be corrected.

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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

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[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.

Aβ25-35-induced autophagy and apoptosis are prevented by the CRMP2-derived peptide ST2-104 (R9-CBD3) via a CaMKKβ/AMPK/mTOR signaling hub

PONE-D-23-36719R2

Dear Dr. Sun,

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Kind regards,

Qi Chen, PhD

Academic Editor

PLOS ONE

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