Review Reports - Phenylketonuria Alters the Prefrontal Cortex Genome-Wide Expression Profile Regardless of the Mouse Genetic Background

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Cells-4028236 PKU mice + prefrontal cortex gene expression

Reviewer comments

This paper compares gene expression profiles of the pre-frontal cortex between two background strains of PKU-model mice which have been bred with an introduced (and presumably identical) PKU mutation and have a similar level of hyperphenylalaninemia and related biochemical profile. The two PKU-induced mouse strains are known from previous work to show characteristic differences in response in certain behavioural tests, suggesting differences between the two strains in the effect of the mutations on brain function, despite apparently similar blood biochemistry. The aim of this paper seems to be to assess whether that difference may be reflected in gene expression differences (differently-expressed genes, or DEGs) between the two strains and compared with controls in the prefrontal cortex that could give information on target pathways for research into reduction of long term associated effects of PKU in humans, which seem to occur irrespective of the strictness of control of Phe levels.

The results do suggest that compared with controls from both strains, there are some minor changes in behaviour detectable in the more mildly affected mouse strain (in similar test domains to those previously reported in the more severely affected strain), and that the pre-frontal cortex gene-expression profiles show that between 20-25% of DEGs (versus controls) do show similar profiles of up- and down-regulation of genes in both strains, but notably also with one or two opposite-direction exceptions.

The investigation seems well-designed and performed, and the paper is well-written, including good English language. It would seem to be a useful report for promoting further studies.

There are one or two points which require clarification through some additional explanation or information in the text, as indicated in the Table below, as well as some more trivial corrections required.

List of Points requiring further explanation in the text, or trivial correction.

1. Page 2, Line 76

Current Text: Black and Ban Brachyury (BTBR)

Suggested text: Black and Tan Brachyury (BTBR)

Comments: Typo error

2. Page 2, Line 78-9 and 84-5

Current Text: (ENU)-induced mutation in the gene encoding for PAH (C57enu2 and BTBRenu2 mice).
… and: Genetic characterization was performed as previously described [4].

Suggested text:

Comments: The authors need to state here whether the ENU induced a single same mutation(s) in both strains, - and what the mutation(s) is (/are) - or whether it produced several different mutations. (In Reference 11 it is implied that it is the same mutation).

3. P3, L109-112

Current Text: Libraries were prepared for sequencing and sequenced on paired-end 150 bp mode on NovaSeq 6000 (Illumina, San Diego, CA). Libraries were prepared for sequencing and sequenced on paired-end 150bp mode on NovaSeq 6000 (Illumina, San Diego, CA) at a depth of 30 million reads.

Suggested text: Libraries were prepared for sequencing and sequenced on paired-end 150bp mode on NovaSeq 6000 (Illumina, San Diego, CA) at a depth of 30 million reads.

Comments: Word processing error. The majority of this sentence is duplicated.

4. P3, L124-5

Current Text: Behavioral assessment was performed on C57enu2 mice compared with the control group.

Suggested text:

Comments:         -i) The authors need to clarify whether these were the same mice that were sacrificed subsequently, or other mice from the same strain.
                            -ii) Also, the authors need to give the age of the mice.
                           -iii) Why was behavioural assessment not performed in this study on BTBRenu2 mice and BTBR controls as well ?. Is ref. [11] sufficient for this ?. This needs to be explained in more detail, as otherwise the absence of BTBR-enu2 behavioural testing using exactly the same apparatus and contemporaneous conditions as for C57enu2 mice may undermine the behavioural finding in these latter mice, and the interpretation of any differences in the prefrontal cortex DEGs.

5. P5, L179

Current: Colouring of Fig. 1A and 1B

Suggested revision: Please choose very obviously different colours between the two diagrams – ie. for Fig.1A so that Fig.1B still matches the colours of Fig.2.

Comments: It is confusing to have almost identical colours for C57 versus BTBR in Fig. 1A; and downregulated versus upregulated in Fig.1B.

6. P10, L277-9

Current Text: while adult BTBRenu2 mice do not discriminate between an object explored 24 hrs before and a novel object, adult C57enu2 mice do discriminate between the two [12].

Suggested text:

Comments: The evidence (and reference citation) for the behavioural difference between BTBRenu2 and C57enu2 mice should be introduced in the Introduction, rather than late in the Discussion, as this impacts on the reason for not, in this paper, testing behaviour in BTBRenu2 mice versus BTBR controls – as mentioned in Point 4. above.

7. P10 ,L280

Current Text: but not in mice PKU mice with C57 genetic background.

Suggested text: but not in PKU mice with C57 genetic background.

Comments: Word processing error: duplication of ‘mice’

8. Supplementary Fig.S1 Legend

Current Text: Figure S1. No differences between C57 and C57enu2 were find

Suggested text: Figure S1. No differences between C57 and C57enu2 were found

Comments: Grammar error

Author Response

We wish to thank Reviewer 1 for all its valuable suggestions

Comment 1. Page 2, Line 76. Typo error

Response 1. The typo has been fixed.

Comment 2. Page 2, Line 78-9 and 84-5. The authors need to state here whether the ENU induced a single same mutation(s) in both strains, - and what the mutation(s) is (/are) - or whether it produced several different mutations. (In Reference 11 it is implied that it is the same mutation).

Response 2. The statement has been better explained.

Comment 3. P3, L109-112. Word processing error. The majority of this sentence is duplicated.

Response 3. The error has been fixed

Comment 4. P3, L124-5. -i) The authors need to clarify whether these were the same mice that were sacrificed subsequently, or other mice from the same strain. -ii) Also, the authors need to give the age of the mice. -iii) Why was behavioural assessment not performed in this study on BTBRenu2 mice and BTBR controls as well?

Response 4. According to the principles of the 3Rs (in particular with Reduction, requiring to minimise the number of animals used consistent with scientific aims), BTBR^enu2 mice have not been further tested in the object recognition test (being well-known their inability to recognise novelty in the presence of only two objects). This choice has been detailed in the last sentence in the Introduction, and more references have been added.

Comment 5. P5, L179. It is confusing to have almost identical colours for C57 versus BTBR in Fig. 1A; and downregulated versus upregulated in Fig.1B.

Response 5. Colour has been modified in order to increase clarity. Figure 1A has been revised using a distinct colour scheme for strain identity, while the colour coding of Figure 1B was preserved to maintain consistency with Figure 2. This resolves the potential ambiguity highlighted by the reviewer.

Comment 6. P10, L277-9. The evidence (and reference citation) for the behavioural difference between BTBRenu2 and C57enu2 mice should be introduced in the Introduction, rather than late in the Discussion, as this impacts on the reason for not, in this paper, testing behaviour in BTBRenu2 mice versus BTBR controls – as mentioned in Point 4. above.

Response 6. The evidence (and reference citation) for the behavioural difference between BTBR^enu2and C57^enu2 mice has been introduced in the Introduction.

Comment 7. P10 ,L280. Word processing error: duplication of ‘mice’

Response 7. Error has been fixed

Comment 8. Supplementary Fig.S1 Legend. Grammar error

Response 8. Fig.S1 Legend Error has been fixed

Reviewer 2 Report

Comments and Suggestions for Authors

General Comment: The manuscript “Phenylketonuria alters the prefrontal cortex genome-wide expression profile regardless of the mouse genetic background”, by Fiori et al addresses a relevant question and presents novel findings on PKU-related transcriptomic and cognitive alterations. The study is well-structured and potentially impactful, but several aspects require clarification and refinement to strengthen methodological transparency, data interpretation, and translational relevance. With these revisions, the work could make a valuable contribution to the field.

Minor Comments:

Comment 1: Abstract: The abstract would benefit from a stronger concluding statement. I recommend that the authors explicitly highlight the translational relevance of their findings, emphasizing how the observed transcriptomic and cognitive alterations in PKU mouse models may inform future research directions or therapeutic strategies.

Comment 2: The authors refer to mouse genes. Formatting should be checked throughout the text, as genes should be in lowercase italics.

Comment 3: Please ensure all acronyms are introduced by writing the full term followed by the abbreviation in parentheses the first time they appear in the text. Thereafter, only the acronym should be used consistently throughout the manuscript. Please review your formatting accordingly.

Comment 4: Introduction: The rationale is compelling, but authors should better highlight the specific knowledge gap addressed compared to prior PKU mouse studies.

Comment 5: P3 line 109-113. There is a typo. The authors should fix the duplicate text.

Comment 6: Methods: The description of animal housing conditions is clear, but authors should indicate whether environmental enrichment was provided, as this may influence cognitive outcomes.

Comment 7: Methods: The Methods section would benefit from a more explicit statement on randomization and blinding procedures during behavioral testing and data analysis.

Comment 8: Methods: In the RNA extraction subsection, the coordinates for pFC punches are reported, but a schematic or reference figure would improve reproducibility.

Comment 9: Methods: The description of statistical software (GraphPad Prism v.10) is adequate, but authors should also report the version of DeSeq2 used for transcriptomic analysis.

Comment 10: Methods: The manuscript should clarify whether both sexes were equally represented across groups in behavioral tasks, and whether sex was considered as a biological variable in transcriptomic analyses.

Comment 11: Results: Figures are informative, but legends should provide clearer explanations of abbreviations and statistical thresholds. Authors should ensure that all p-values, fold changes, and FDR corrections are consistently reported.

Comment 12: Results, Fig.3: Panel D is not cited in the figure legend.

Comment 13: Results: Figures and text should consistently report exact statistical values (F, p, n) rather than summarizing significance levels only. This would improve transparency and reproducibility.

Major Comments:

Comment 14: The introduction provides a solid clinical background and cites classical studies on PKU outcomes. However, it does not include more recent neuroimaging or longitudinal evidence documenting prefrontal cortex dysfunctions in early-treated adult PKU patients. Incorporating such references would strengthen the rationale and better justify the focus on transcriptomic and cognitive analyses in mouse models.

Comment 15: Methods: The Methods section specifies that mice exploring for less than 5 seconds were excluded from behavioral analyses. However, no additional inclusion/exclusion criteria are reported. Authors should clarify whether further criteria were applied (e.g., health status, outliers in transcriptomic data) to ensure transparency and reproducibility.

Comment 16: Methods: Sample sizes are relatively small across groups. A power analysis or justification of group sizes would strengthen confidence in the robustness of the findings.

Comment 17: Methods: The RNA-seq workflow is described in detail, but the manuscript does not clarify whether sequencing runs were randomized or whether batch effects were assessed and corrected. Given the relatively small sample sizes, addressing batch variability is critical to ensure that the reported differential expression patterns reflect biological rather than technical differences. Authors should provide information on how batch effects were controlled or demonstrate that they did not significantly impact the results.

Comment 18: Methods: The behavioral assessment appropriately employs the Identical Object Task (IOT). However, the rationale for progressively increasing the number of objects (from 4 to 6) as a measure of prefrontal cortex-dependent working memory is not fully explained. Authors should clarify whether this progression is based on established literature or pilot data, and discuss how object number specifically relates to pFC load and cognitive span. This would strengthen the methodological justification and translational relevance of the behavioral findings.

Comment 19: Methods: The Statistical Analysis section does not specify whether assumptions of normality and homogeneity of variance were tested prior to applying parametric tests (t-test, ANOVA). Given the relatively small sample sizes, authors should clarify how these assumptions were assessed and whether non-parametric alternatives were considered when appropriate.

Comment 20: Results: The Results section reports that only 62 genes overlap between the two genetic backgrounds, corresponding to 16–20% of the DEGs identified. While this overlap is noteworthy, it represents a relatively limited fraction of the transcriptomic changes. The authors should temper the claim of a ‘largely conserved’ transcriptional response and provide a more nuanced interpretation. In particular, they should discuss whether the shared pathways, rather than the absolute number of overlapping genes, better support the idea of conserved molecular mechanisms. This clarification would strengthen the validity of the conclusion and align the interpretation more closely with the presented data.

Comment 21: Results: Behavioral results show significant differences only under high load (6-IOT). Authors should discuss the variability observed in C57enu2 mice at 4-IOT and whether some individuals display resilience. This would add nuance to the interpretation of cognitive deficits.

Comment 22: Resuts: Figures and text should consistently report exact statistical values (F, p, n) rather than summarizing significance levels only. This would improve transparency and reproducibility.

Comment 23: Discussion: The Discussion occasionally over-interprets transcriptomic findings, particularly regarding compensatory mechanisms in protein synthesis. Authors should temper these claims or support them with functional assays or references.

Comment 24: Discussion: The downregulation of myelination-related programs is an important finding, but the Discussion would benefit from integration with histological or imaging evidence, or at least a more detailed reference to recent literature.

Comment 25: Discussions: The Discussion does not sufficiently address study limitations. Authors should acknowledge the relatively small sample sizes, lack of protein-level validation, and absence of direct clinical correlations. This would provide a more balanced perspective.

Comment 26: Discussion: The Discussion would benefit from a stronger translational conclusion. Authors should explicitly link the transcriptomic and behavioral findings in PKU mouse models to the variability observed in patients, particularly regarding prefrontal cortex-dependent cognitive deficits. Highlighting how these models capture both vulnerability and resilience would underscore the clinical relevance of the study and its potential to inform therapeutic strategies.

Comments for author File: Comments.pdf

Author Response

We wish to thank Reviewer 2 for all its valuable suggestions

Response 1. The following concluding statement has been reported in the abstract. “These findings identify convergent pFC molecular and cognitive alterations induced by HPA across distinct genetic backgrounds, providing clinically relevant insights into mechanisms that may contribute to executive dysfunctions in PKU”.

Comment 2: The authors refer to mouse genes. Formatting should be checked throughout the text, as genes should be in lowercase italics.

Response 2. Formatting has been fixed

Response 3. Formatting has been fixed

Comment 4: Introduction: The rationale is compelling, but authors should better highlight the specific knowledge gap addressed compared to prior PKU mouse studies.

Response: The state of the art and the knowledge acquired through our study have been highlighted in the introduction

Comment 5: P3 line 109-113. There is a typo. The authors should fix the duplicate text.

Response 5. Typo has been fixed

Comment 6: Methods: The description of animal housing conditions is clear, but authors should indicate whether environmental enrichment was provided, as this may influence cognitive outcomes.

Response 6. The description of the environmental enrichment has been clarified.

Comment 7: Methods: The Methods section would benefit from a more explicit statement on randomization and blinding procedures during behavioral testing and data analysis.

Response 7. The randomization and blinding methods have been clarified.

Comment 8: Methods: In the RNA extraction subsection, the coordinates for pFC punches are reported, but a schematic or reference figure would improve reproducibility.

Response 8. A graphical representation of the anatomical coordinates and the punching procedure has now been added to the graphical abstract. In addition, the corresponding methodological description has been clarified in the Materials and Methods section as follows: brain tissue micro-punches of the prefrontal cortex (pFC) were obtained from two coronal brain sections (300 μm thickness) using a stainless-steel tube with a 1 mm internal diameter and stored at −80 °C until the day of the assay. The anatomical coordinates were determined according to the Franklin and Paxinos mouse brain atlas (+1.50 to +2.10 mm from bregma).

Comment 9: Methods: The description of statistical software (GraphPad Prism v.10) is adequate, but authors should also report the version of DeSeq2 used for transcriptomic analysis.

Response 9. The version of DESeq2 used for transcriptomic analysis (v1.34.0) has now been specified in the Materials and Methods section, specifically in Section 2.3 (Library Preparation, mRNA Sequencing and Analysis).

Response 10. Both sexes were used for behavioral tasks. Both male and female mice were included in all transcriptomic analyses, and samples from both sexes were analyzed together. Given the limited sample size, the study was not powered to detect sex-specific effects; therefore, sex was not included as an independent biological variable. However, in our behavioural experiment, no significant main effects or interactions involving sex were detected in the variables analyzed.

Response 11. Figure legends have been revised to provide clearer explanations of all abbreviations and to consistently report statistical thresholds, including p-values, fold-change criteria, and FDR corrections. These clarifications have been specifically added to the legends of Figures 1 and 2.

Comment 12: Results, Fig.3: Panel D is not cited in the figure legend.

Response 12. Error in Figure 3 legend has been fixed.

Response 13. F, p, n have been added to figure legends

Response 14. Two more recent references are incorporated in the MS (https://doi.org/10.1186/s11689-025-09622-8; https://doi.org/10.1093/brain/awae139)

Response 15. No exclusion criteria other the exploration time have been applied, as better stated in pg.4 l.148.

Comment 16: Methods: Sample sizes are relatively small across groups. A power analysis or justification of group sizes would strengthen confidence in the robustness of the findings.

Response 16. Based on the reference paper Olivito et al 2014, the G Power analysis for C57 mice revealed that 10 mice/group is a suitable number on 6-IOT. Since no data are available on the same transgenic model, we used 10 mice for ENU2 group as well. For 4-IOT, no data are available to perform G Power analysis, especially for this transgenic model. Since previous studies have demonstrated that 7 mice is a sufficient number to successfully perform behavioural characterisation (Pascucci 2018), we decided to settle for this number, according to the Reduction principle.

Response 17. We thank the Reviewer for raising this important point. All RNA-seq libraries were prepared using the same protocol and reagents and subsequently pooled into a single library pool. The pooled libraries were then sequenced across two independent sequencing runs with a balanced distribution of reads (millions of reads per sample) between runs. Because all samples were included in the same pool and sequenced with balanced output across runs, potential batch effects related to library preparation or sequencing were minimized. In addition, exploratory data analyses, including principal component analysis, did not reveal clustering of samples according to sequencing run; therefore, no batch correction was applied. This information has now been clarified in the Materials and Methods section, specifically in Section 2.3 (Library Preparation, mRNA Sequencing and Analysis).

Response 18. The progressively increasing number of objects (i.e., 3, 6 or 8) as a measure of prefrontal cortex-dependent working memory is based on the study of Sannino et al., 2012, demonstrating that the progressive load of details to discriminate can load the working memory system. This test offers important advantages for studying cognitive functions regulated by the fronto-striatal dopamine system.

Response 19. Normality test has been performed using Kolmogorov-Smirnov test. One group did not pass the test, non parametric statistics have therefore been carried out, using Kruskal-Wallisand Friedman test. The Statistical Analysis, Results and figure legends have been corrected accordingly.

Response 20. We thank the Reviewer for this insightful comment. We have now tempered the interpretation of the overlap between differentially expressed genes and provided a more nuanced discussion in the Results section. Specifically, immediately after reporting that 62 genes are shared between the two genetic backgrounds, we added a clarification emphasizing that, although the proportion of overlapping DEGs represents a limited fraction of the total transcriptomic changes, these genes are among the most significantly altered and show a high degree of directional consistency. Moreover, we now highlight that gene ontology analysis of all DEGs reveals convergence on common functional pathways, supporting the presence of conserved molecular mechanisms. These additions have been included in Section 3.1 of the Results.

Response 21. We discuss in more detail the response of C57^enu2 mice at 4-IOT and 6-IOT .

Response 22. Statistical values (F, p, n) have been added in figures legends a better stated in the results sections.

Response 23. The Discussion has been revised to temper the interpretation of transcriptomic findings and to avoid causal claims regarding compensatory mechanisms, aligning conclusions more closely with the presented data.

Response 24: Two more recent references are incorporated in the MS (https://doi.org/10.1186/s11689-025-09622-8; https://doi.org/10.1093/brain/awae139)

Response 25. Study limitations have been addressed.

Response 26. A stronger translational conclusion has been discussed.

Reviewer 3 Report

Comments and Suggestions for Authors

This is a well written paper with transcriptomic analysis accompanied by behavioral analysis. The findings of alteration of pathways involved in protein translation and protein levels are consistent with past research and suggest future directions for the research. The comparison of the two different mouse strains and models for PKU research are very helpful for those using mice in the PKU field. While the paper can stand on its own, there are several suggestions that could improve the overall findings and conclusions. Most importantly, one additional experiment using new biological replicates to confirm the differentially expressed genes should be included.

The authors could include more information on the specific variants in each strain in their introduction, as the information is published, but still pertinent when comparing two mouse strains induced with emu.
Could the authors include the time of day for collection of the brain tissues? Brain circadian rhythms could confound the study if different mice were collected at different times of day.
Was a comparison of only the WT strains done for the transcriptomic analysis? I ask because of the differences in the behavioral phenotypes shown for the two strains that was noted in the introduction (although not specifically tested in this manuscript—see comment #3 below). There are known QTL for different behaviors between different strains in mice and it would be interesting to know if any gene loci correlate to these between strains.
It is unclear why the BRBR strain was not also used in the behavioral assays so that a direct comparison between the two strains for these tests could be considered.
For the three genes that are differentially-regulated between the four models (strains by variant)--Nup62, Psph, and Arhgef2—the authors could consider a QPCR analysis on new biological replicates for each mouse model to confirm this finding. This additional experiment is standard practice for RNA-seq identified transcripts.
1. The authors should also include more on the function of the protein products for these genes in their discussion. For example, Nup62 is part of the nuclear pore complex and could affect transport of mRNAs out of the nucleus for protein translation. Psph variants are linked to Williams Syndrome, which manifests with some behavioral characteristics which could be discussed with references to the differences in the mouse models, and patients with PKU. Variants in Arhgef2 can lead to mid-brain and hindbrain abnormalities, and the protein product of this gene is involved in G-protein coupled signaling.
2. Were there sex differences in the expression patterns?

Author Response

We wish to thank Reviewer 3 for all its valuable suggestions

Comment 1. The authors could include more information on the specific variants in each strain in their introduction, as the information is published, but still pertinent when comparing two mouse strains induced with emu.

Response 1. More information on the differences between strains has been added in the introduction.

Comment 2. Could the authors include the time of day for collection of the brain tissues? Brain circadian rhythms could confound the study if different mice were collected at different times of day.

Response 2. The time of day for collection of the brain tissues has been added.

Comment 3. Was a comparison of only the WT strains done for the transcriptomic analysis? I ask because of the differences in the behavioral phenotypes shown for the two strains that was noted in the introduction (although not specifically tested in this manuscript—see comment #3 below). There are known QTL for different behaviors between different strains in mice and it would be interesting to know if any gene loci correlate to these between strains.

Response 3. We thank the reviewer for the comment. However, the transcriptomic analysis in this study was specifically designed to compare enu2 mutants with their respective WT controls within each genetic background, in order to identify molecular changes associated with hyperphenylalaninemia while controlling for strain-specific baseline differences. Thus, we did perform a direct comparison between WT C57 and WT BTBR mouse prefrontal cortex. However, this analysis was not included in the present manuscript, as it falls outside the scope of this work, reflecting inherent genetic background differences rather than PKU-related alterations. In a subsequent study, we intend to associate QTLs with a battery of behavioral tests to be conducted in both strains.

Comment 4. It is unclear why the BRBR strain was not also used in the behavioral assays so that a direct comparison between the two strains for these tests could be considered.

Response 4. According to the principles of the 3Rs (in particular with Reduction, requiring to minimise the number of animals used consistent with scientific aims), BTBRenu2 mice have not been further tested in the object recognition test (being well-known their inability to recognise novelty in the presence of only two objects). This choice has been detailed in the last sentence in the Introduction.

Comment 5. For the three genes that are differentially-regulated between the four models (strains by variant)--Nup62, Psph, and Arhgef2—the authors could consider a QPCR analysis on new biological replicates for each mouse model to confirm this finding. This additional experiment is standard practice for RNA-seq identified transcripts.

Response 5. Although additional validation would strengthen the finding, we do not perform further qPCR analysis on new biological replicated. The advances in RNA-Sequencing technologies have led to a highly accurate and reproducible quantitative method, allowing for the minimisation of the use of animals. Moreover, the consistency of these genes across two independent comparisons between mouse models increases internal validation, reducing the need for additional technical confirmation.

Comment 6. The authors should also include more on the function of the protein products for these genes in their discussion. For example, Nup62 is part of the nuclear pore complex and could affect transport of mRNAs out of the nucleus for protein translation. Psph variants are linked to Williams Syndrome, which manifests with some behavioral characteristics which could be discussed with references to the differences in the mouse models, and patients with PKU. Variants in Arhgef2 can lead to mid-brain and hindbrain abnormalities, and the protein product of this gene is involved in G-protein coupled signaling.

Response 6. We have added informations on the function of these proteins as the reviewer suggested.

Comment 7. Were there sex differences in the expression patterns?

Response 7. Both sexes were used for behavioral tasks and included in all transcriptomic analyses, and samples from both sexes were analyzed together. Given the limited sample size, the study was not powered to detect sex-specific effects; therefore, sex was not included as an independent biological variable. However, in our behavioural experiment, no significant main effects or interactions involving sex were detected in the variables analyzed.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the careful and coherent revisions. You have addressed all my comments appropriately, and the manuscript is now clearer, more rigorous, and substantially improved. I find the topic highly relevant and of strong interest to the scientific community.

Author Response

Comment 1: Thank you for the careful and coherent revisions. You have addressed all my comments appropriately, and the manuscript is now clearer, more rigorous, and substantially improved. I find the topic highly relevant and of strong interest to the scientific community.

Response 1: Thanks for the helpful review and kind final feedback.

Reviewer 3 Report

Comments and Suggestions for Authors

This is a revision to a previously submitted and reviewed manuscript. The authors have responded to most of the comments adequately, improving the overall content of the article. However, several items should still be addressed from the original review.

In response to my second comment: "Could the authors include the time of day for collection of the brain tissues? Brain circadian rhythms could confound the study if different mice were collected at different times of day." The authors responded "The time of day for collection of the brain tissues has been added.". However, the authors only note that the animals were collected at the same time of day--not detailing the actual time of day that this occurred, relative to the light-dark cycle. This detail is needed for any reproducibility.
The authors note that secondary analysis of RNA-seq is no longer necessary, but do not provide any citation for this, or statement on this in the manuscript methods. That would be a minimal addition to the manuscript. However, in my mind, it is still standard to perform analyses--perhaps other than QPCR such as RNAScope or protein analysis to confirm and follow up on RNA seq data for the major genes/transcripts identified. The authors have stated this in the conclusions, which is good but the lack of these data reduces the overall impact of the paper.
In my comment on whether there were sex differences, the authors responded that the study was under powered to detect differences. I would like to know further if the females were taken only in a specific phase of the estrous cycle (i.e. dietrous), or if females were not measured with respect to stage of cycle--if this was not done then the information should be added to the methods, and as a confounder in the discussion/conclusion.

Author Response

We wish to thank Reviewer 3 for all its valuable comments

Comment 1. In response to my second comment: "Could the authors include the time of day for collection of the brain tissues? Brain circadian rhythms could confound the study if different mice were collected at different times of day." The authors responded "The time of day for collection of the brain tissues has been added.". However, the authors only note that the animals were collected at the same time of day--not detailing the actual time of day that this occurred, relative to the light-dark cycle. This detail is needed for any reproducibility.

Response 1. Animals were sacrificed between 12:00 and 13:00 during the light phase of the light–dark cycle (ZT5–ZT6). The information has been added to the manuscript.

Comment 2. The authors note that secondary analysis of RNA-seq is no longer necessary, but do not provide any citation for this, or statement on this in the manuscript methods. That would be a minimal addition to the manuscript. However, in my mind, it is still standard to perform analyses--perhaps other than QPCR such as RNAScope or protein analysis to confirm and follow up on RNA seq data for the major genes/transcripts identified. The authors have stated this in the conclusions, which is good but the lack of these data reduces the overall impact of the paper.

Response 2. We thank the Reviewer for the comment. The reliability of RNA-Seq technology (DOI: 10.1038/s41598-017-01617-3; DOI: 10.1038/nbt.2957) has recently reduced the need of validation with alternative in methods, in many recent publications (i.e. doi.org/10.3389/frnar.2024.1419833). Moreover, in our study, the consistency of gene expression changes observed across two independent genetic backgrounds provides an internal validation, as it was for “Lo Iacono et al. DOI: 10.1016/j.ynstr.2021.100406. Nevertheless, we acknowledge that further functional or molecular validation would strengthen these findings and will be addressed in future studies.

Comment 3. In my comment on whether there were sex differences, the authors responded that the study was under powered to detect differences. I would like to know further if the females were taken only in a specific phase of the estrous cycle (i.e. dietrous), or if females were not measured with respect to stage of cycle--if this was not done then the information should be added to the methods, and as a confounder in the discussion/conclusion.

Response 3. Females were not measured in relation to the stage of their cycle. Females were housed in the same cages and in the presence of males in nearby cages (increasing the likelihood of synchronizing the female cycle). The information has been added to the method section.