PONE-D-21-01012

Psychostimulants and Opioids Differentially Influence the Epigenetic Modification of Histone Acetyltransferase and Histone Deacetylase 

PLOS ONE

Dear Dr. Thangavel

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ACADEMIC EDITOR:

1. The authors did extensive analyses of  HDACs and HATs in the n vitro set up. However, the authors conclusion was oversimplified. The authors need to perform more experiments to validate the conclusion and precise the discussion and conclusion according to the results. 

2. In vivo studies would strengthen the manuscript. Authors are encouraged to do these and to some extent some mechanisitic studies

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PLOS ONE

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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: Partly

Reviewer #2: Partly

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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: Yes

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: The manuscript submitted Mayur Doke et al., entitled “Psychostimulants and Opioids Differentially Influence the Epigenetic Modification of Histone Acetyltransferase and Histone Deacetylase” demonstrated that psychostimulant and opioid exposure significantly modulates the expression of histone acetyl transferases (HDAC) and histone deactylase (HATs) human astrocytes. Further, the authors stated that nootropic drug piracetam prevented psychostimulant effects. The author did extensive analysis HDAC, acetylation, and HATs in in vitro set up. However, the authors conclusion was oversimplified. The authors need to perform more experiments to validate the conclusion and precise the discussion and conclusion according to the results. The following comments are need to be addressed.

Comments

1. The authors analysed well in in vitro, however, It would be more solid conclusion if the author validates these findings in in vivo experimental conditions using rodents’ models.

2. The author also needs to clearly mention about the group comparison in legends, which group compared with which group. Did the author compared cocaine with cocaine+piracetam and similarly for other psychostimulant. If so please mention clearly in legends as well as in results.

3. The authors needs to discuss the roles of other HDACs except HDAC1 in pychostimulant mediated acetylation in astrocytes and its epigenetic modifications. Since most of the HDACs and HAT are not affected by Piracetam treatment.

4. What is the rationale for choosing Piracetam and discuss, how it regulates HDACs in detail.

5. In Figure 3, the results of PCAF, p300 and GCN5 showed no significant difference between psychostimulant with and without piracetam. How the authors concluded piracetam protected psychostimulant mediated effects on astrocytes? This has to be properly mentioned through out the manuscript and has to be discussed.

6. In figure 4, The gene expression analysis of GCN5 was not affected by psychostimulants or piracetam however, the protein expression and ICC in figure 5 showing prominent effects. Please discuss.

7. The authors need to clearly mention in conclusion about the protective effects of piracetam on HDAC and HATs, since most of the modulatory effects of pycostimulants were not prevented by piracetam. However, the author generalized the result conclusion.

8. Did the author expose the cells with all the three psychostimulants together or in two combinations? Does it aggravate the effects on HDAC and HAT as well as its acetylation’s?

9. Please discuss the rationale for choosing the doses of psychostimulants.

10. In Fig 6, the scale bar was missing.

Reviewer #2: In this manuscript, Doke et al. demonstrated the involvement of psychostimulants and opioid-mediated epigenetic changes that occurred at the histone level in human primary astrocytes. The authors also determined the neuroprotective role of piracetam on both the histone deacetylases (HDACs) and histone (HAT) levels in human primary astrocytes exposed to psychostimulants and opioids. Though this study seems very interesting, lack of rigorous experiments and mechanistic approach notably hinders the scientific enthusiasm. Further, most of the experiments were of primitive. Overall, this manuscript needs major revision.

Comments:

1. How does the neuroprotective agent piracetam regulate the expression of HDACs and HATs? Please explain the relevant molecular mechanism.

2. Figure 2A and 2B: missed labeling for the control group.

3. Figure arrangements were not of publication format.

4. More mechanistic studies are warranted in the context of HDACs and HAT inhibitors/activators. Gene silencing approaches would make it more convincing.

5. It would be more compelling if the authors validate these findings in an in vivo model system.

6. Immunostaining data lacks scalebar.

7. It is unclear that piracetam's effects on the combinatorial exposure of all these drugs-cocaine, meth, and morphine.

8. Please provide rationale for choosing the dose concentration of all these drugs.

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Reviewer #1: No

Reviewer #2: No

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Psychostimulants and Opioids Differentially Influence the Epigenetic Modification of Histone Acetyltransferase and Histone Deacetylase

PONE-D-21-01012R1

Dear Dr. Thangavel,

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,

Axel Imhof

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

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

Reviewer #2: Yes

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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

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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 addressed all the comments raised by the reviewer point by point. I recommend to accept and publish the article in the present form.

thank you

Reviewer #2: The authors revised the manuscript satisfactorily. Also they mentioned that due to covid-19 restrictions, they couldn't perform some experiments.

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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