PONE-D-22-26036Heterozygous deletion of Gpr55 does not affect a hyperthermia-induced seizure, spontaneous seizures or survival in the Scn1a+/- mouse model of Dravet syndromePLOS ONE

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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: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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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 article is of interest. However, in my opinion, some improvements could be made. For instance, I think you could deeply discuss your results. Indeed, you should also take into consideration that perhaps other molecular pathways could be involved in the effects of CBD. Notably, the antiseizure effects of CBD (120 mg/kg i.p.) were associated with upregulation of PPARγ in the hippocampus of a rat model of temporal lobe epilepsy (Costa et al., 2022). Like CBD, cannabigerol (CBG) and tetrahydrocannabinolic acid (Δ9-THCA) have been reported to mediate their anti-inflammatory effects through PPARγ (Stone et al., 2020; Valdeolivas et al., 2015). In addition to this, the upregulation of PPARγ in the hippocampus was not only related to the antiseizure effects of CBD but also to those of EP-80317, a ghrelin receptor antagonist (Lucchi et al., 2017).

Other comments:

1. “…that co-administration of CID16020064 with CBD occluded the effects of CBD (3).” This sentence is unclear. Please rephrase.

2. The company purchasing “CID16020064” and specific information about this compound should be provided.

3. The total number of animals (and the total number of animals per group) should be clarified.

4. The total number of male and female mice per group should be clarified.

5. The statistical test used in the manuscript should be summarized in a specific paragraph.

References:

Lucchi, C.; Costa, A.M.; Giordano, C.; Curia, G.; Piat, M.; Leo, G.; Vinet, J.; Brunel, L.; Fehrentz, J.-A.; Martinez, J.; et al. Involvement of PPARγ in the Anticonvulsant Activity of EP-80317, a Ghrelin Receptor Antagonist. Front. Pharmacol. 2017, 8, 676.

Costa, A.-M.; Russo, F.; Senn, L.; Ibatici, D.; Cannazza, G.; Biagini, G. Antiseizure Effects of Cannabidiol Leading to Increased Peroxisome Proliferator-Activated Receptor Gamma Levels in the Hippocampal CA3 Subfield of Epileptic Rats. Pharmaceuticals 2022, 15, 495. https://doi.org/10.3390/ph15050495

Stone, N. L., Murphy, A. J., England, T. J., & O’Sullivan, S. E. (2020). A systematic review of minor phytocannabinoids with promising neuroprotective potential. British Journal of Pharmacology, bph.15185. https://doi.org/10.1111/bph.15185

Valdeolivas, S., Navarrete, C., Cantarero, I., Bellido, M. L., Muñoz, E., & Sagredo, O. (2015). Neuroprotective properties of cannabigerol in Huntington’s disease: Studies in R6/2 mice and 3-nitropropionate-lesioned mice. Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics, 12(1), 185–199. https://doi.org/10.1007/s13311-014-0304-z

Reviewer #2: Anderson et al report the effects of a heterozygous deletion of Gpr55 on hyperthermia-induced and spontaneous seizures in the Scn1a+/- mouse model of Dravet syndrome. The experiments are very well performed, and the results are important and interesting. I Have a few minor comments to the authors:

Introduction/Discussion.

The last two sentences of the first paragraph are somewhat redundant.

Although the Scn1a+/- mice show a severe Dravet syndrome phenotype, the effect is strain-specific. Thus, it is likely that it is species-specific too, meaning that this model may not reflect real mechanisms in humans (different genetic modifiers in different species and strains).

Deletion of Gpr55 is not the same as its inhibition, which can be partial, that is maintaining the interaction at a “normal”, non-epileptic level.

These caveats are common for most of studies, but I suggest discussing them. Taking them into account, I also suggest to moderate the main conclusion of the study concerning the antiepileptic mechanisms of cannabinoids (“Our study suggests developing GPR55 antagonists might not be a viable strategy for advancing new treatments”). “to unequivocally rule out GPR55 as a new drug target for treating Dravet syndrome” may not be a goal for future studies. Might be on the contrary?

Methods/Results.

CID2921524: Please, indicate the volume of injected solution per body weight.

This compound reaches high brain levels 15 min after administration. It is not clear at which time point the seizure thresholds were measured. In the description we can read: “mice received a single i.p. injection of vehicle or CID2921524 following the 5 min acclimation period”. Just two or four minutes could be insufficient to penetrate; please explain.

How clonic convulsions were defined?

During GTCS monitoring, were animals housed in groups or individually?

Could the seizure severity be compared by using nonparametric statistics?

How the brains were dissected: frozen, on ice, etc?

“tissue from 3-4 mice (at least one from each sex) were combined into pooled samples (n = 7-11 pools per group) to isolate total RNA” – this description is not entirely clear.

Where the rabbit anti-CB1 polyclonal antibody was obtained?

“CID2921524 is an antagonist of GPR55 antagonist” – please correct.

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Reviewer #1: No

Reviewer #2: No

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Heterozygous deletion of Gpr55 does not affect a hyperthermia-induced seizure, spontaneous seizures or survival in the Scn1a+/- mouse model of Dravet syndrome

PONE-D-22-26036R1

Dear Dr. Arnold,

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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Giuseppe Biagini, MD

Academic Editor

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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: (No Response)

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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: (No Response)

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

Reviewer #1: Yes

Reviewer #2: (No Response)

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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: (No Response)

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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: (No Response)

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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: All comments have been addressed.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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