APOE4 and doxorubicin impair inhibitory interneuron function and homeostatic regulation in the entorhinal cortex

Nancy Luo; Harshul Pandit; Shreya Kalra; Erin Tran; Jeanne Mandelblatt; Stefano Vicini; G. William Rebeck

doi:10.1371/journal.pone.0343276

Peer Review History

Original SubmissionNovember 21, 2025
15 Dec 2025 Decision Letter - Ioannis Liampas, Editor Dear Dr. Rebeck, 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 Jan 29 2026 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.. 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. 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[Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: N/A ****** 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 ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: The manuscript presents a relevant topic with potential scientific value; however, several issues require substantial revision to improve the quality of the manuscript. • The abstract reports changes in sERSCs and sIPSCs, PV firing rates, and inactivation block, but provides no numerical value, confidence intervals, or effect sizes. Phrases such as “caused increases” and “significantly lowering firing rates” should be supported with actual measurements. • Overinterpretation of the conclusion in the abstract section for an ex vivo electrophysiology. • The introduction lacks a clear problem statement and does not particularly highlight the importance of this topic. • Some methodological steps, such as instrument settings and replicates, lack detail. • Statistical methodology description is lacking information about the tests applied to compare groups, and how multiple comparisons were corrected. • Ethical approval statements can be improved by adding the committee reference number ID or the protocol number. • The introduction section lacks a critical literature context, citing older references without integrating the latest mechanistic insights. • A graphical workflow is needed to summarise the experimental sequence (genotyping → doxorubicin → slice prep → patch clamp). • For reproducibility, osmolarity, pH, and pipette resistance criteria should be added to the patch clamp internal solution • Typographical errors are present throughout the manuscript, such as in the discussion section PVinactivtion needs space, and eitherGABA or AMPA is missing space. • Provide clearer relevance on developmental neurophysiology examples as they are not directly linked to ex vivo outcomes. • Strengths and limitations of the study are not acknowledged. • Future research direction is not discussed • Conclusion of the manuscript was not mentioned in the end of the manuscript. • Journal formatting guidelines (PLOS ONE style) are not consistently followed. • Some citations are incorrectly formatted. Reviewer #2: This manuscript examines the impact of the APOE4 allele, a major genetic risk factor for Alzheimer’s disease (AD) on synaptic and circuit function in the entorhinal cortex (EC), both at baseline and in response to the chemotherapeutic agent doxorubicin. They used humanized APOE knock-in mice and employed whole-cell patch clamp electrophysiology to measure excitatory and inhibitory neurotransmission in layer 2/3 pyramidal neurons and parvalbumin-positive (PV) interneurons. They examined both baseline genotype effects and responses to doxorubicin, a chemotherapy agent used as an acute stressor to model cancer-related cognitive impairment. The authors conclude that APOE4 impairs circuit flexibility by disrupting PV interneuron function and blocking adaptive responses to stress, potentially explaining heightened vulnerability to cognitive decline in APOE4 carriers facing Alzheimer's pathology or chemotherapy exposure. While the work addresses an important question linking APOE4 to circuit-level dysfunction, but few methodological concerns interpretative issues limit the significance of the study. The authors use different APOE knock-in mouse models across experiments (Sullivan et al. 1997 model in Figures 1-2 vs. Foley et al. 2022 model in Figures 3-4) without adequate justification. While they acknowledge this limitation, it undermines comparisons between baseline genotype effects and doxorubicin responses. Several key findings rely on modest sample size of animals. For example, in Figure 4A APOE4 vehicle group has only 1 mouse. This makes it impossible to determine whether non-significant findings represent true null effects or insufficient statistical power. This is especially problematic for interpreting the lack of doxorubicin effects in APOE4 mice, which is central to the manuscript's conclusions. This study addresses important questions about APOE4-related circuit dysfunction and responses to stress, with potential relevance to both Alzheimer's disease and chemotherapy-related cognitive impairment. However, significant methodological limitations particularly the use of different mouse models, small sample sizes, incomplete mechanistic investigation, and questionable clinical relevance of the doxorubicin paradigm limit the impact of the findings. With revisions, this work could make a valuable contribution to understanding APOE4 effects on neural circuits. ****** what does this mean? ). If published, this will include your full peer review and any attached files.). 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? 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Revision 1
2 Feb 2026 Author Response We appreciate that the reviewers felt that our manuscript “presents a relevant topic with potential scientific value” and “could make a valuable contribution to understanding APOE4 effects on neural circuits.” We agree that the comments and our responses throughout the manuscript have improved the work; we thank the reviewers for their time and insights. Reviewer #1: • The abstract reports changes in sERSCs and sIPSCs, PV firing rates, and inactivation block, but provides no numerical value, confidence intervals, or effect sizes. Phrases such as “caused increases” and “significantly lowering firing rates” should be supported with actual measurements. For space considerations in the Abstract, we could not add as many values as we would like. We now added p-values to support our statement about the effects of APOE genotype on spontaneous excitatory and inhibitory post-synaptic current amplitude. We included actual measurements and confidence intervals for the E/I ratios showing that APOE4 is associated with a higher E/I ratio. Finally, we included the percentages of PV cells that displayed inactivation block. Additional details about important statistical measures are in the text and figure legends. • Overinterpretation of the conclusion in the abstract section for an ex vivo electrophysiology. We edited the Abstract to include our use of ex vivo analyses and removed the last statement about impaired circuit flexibility in APOE4 to narrow the scope. These changes better reflect our data. • The introduction lacks a clear problem statement and does not particularly highlight the importance of this topic. We have consolidated portions of the Introduction and dedicated a specific paragraph to address the unknowns that our study seeks. Among other changes, we expanded some previous research on the effects of APOE genotype on cognitive impairment induced by cancer chemotherapies. • Some methodological steps, such as instrument settings and replicates, lack detail. We went through our methods and believe that the instrument settings are included. For replicates, we have tried to be more transparent in in the numbers of mice and cells used per condition. When possible, we simplified in the Figure Legends the numbers of cells and mice used per data panel to make the sample sizes more apparent. We also added to the Methods that several brain slices from the same mice were used in the electrophysiology experiments to increase the number of cells that could be analyzed to address our research questions. • Statistical methodology description is lacking information about the tests applied to compare groups, and how multiple comparisons were corrected. We have included any post-hoc comparisons tests in our Methods section. We used Bonferonni correction for correction of the alpha-value in the Mann-Whitney U test. Tukey's HSD has inbuilt corrections for multiple comparisons via the studentized range statistic "q" which adjusts the significance threshold for all comparisons. • Ethical approval statements can be improved by adding the committee reference number ID or the protocol number. We have included the IAUCUC protocol number. • The introduction section lacks a critical literature context, citing older references without integrating the latest mechanistic insights. We have expanded the introduction to consider more and newer findings about the effects of APOE and chemotherapy on neuronal functioning. Throughout the manuscript, we have added nineteen new references to incorporate more relevant literature and ideas. We think these changes have helped justify the experiments that we have undertaken. • A graphical workflow is needed to summarise the experimental sequence (genotyping → doxorubicin → slice prep → patch clamp). We have added a graphical workflow as a new Figure 1 that we believe will help readers understand our experiments. We have adjusted the other Figure numbers accordingly. • For reproducibility, osmolarity, pH, and pipette resistance criteria should be added to the patch clamp internal solution. We have added the internal Osm and pH to the description of the patch clamp internal solution, which also contains the pipette resistance. • Typographical errors are present throughout the manuscript, such as in the discussion section PVinactivtion needs space, and eitherGABA or AMPA is missing space. These typographical errors have been fixed. • Provide clearer relevance on developmental neurophysiology examples as they are not directly linked to ex vivo outcomes. We added our consideration of the choice to address our questions in an ex vivo model to the Introduction. We have added a sentence in the Discussion about the relevance of studies in young mice to cognitive impairment related to APOE and chemotherapy with aging. • Strengths and limitations of the study are not acknowledged. We have added a paragraph discussing the strengths and weaknesses of our study. • Future research direction is not discussed A statement of directions has been added to the final paragraph of the Discussion section. • Conclusion of the manuscript was not mentioned in the end of the manuscript. We have edited the conclusion and added it under its own heading after the Discussion. • Journal formatting guidelines (PLOS ONE style) are not consistently followed. We have tried to follow guidelines throughout, making several changes from our original submission. We will work with editors to correct any inconsistencies that we have missed. • Some citations are incorrectly formatted. We have reformatted the manuscript in Vancouver style. There is one paper that is available ahead of print and one that is in BioRxiv, and we would update those as needed. Reviewer #2: This manuscript examines the impact of the APOE4 allele, a major genetic risk factor for Alzheimer’s disease (AD) on synaptic and circuit function in the entorhinal cortex (EC), both at baseline and in response to the chemotherapeutic agent doxorubicin. They used humanized APOE knock-in mice and employed whole-cell patch clamp electrophysiology to measure excitatory and inhibitory neurotransmission in layer 2/3 pyramidal neurons and parvalbumin-positive (PV) interneurons. They examined both baseline genotype effects and responses to doxorubicin, a chemotherapy agent used as an acute stressor to model cancer-related cognitive impairment. The authors conclude that APOE4 impairs circuit flexibility by disrupting PV interneuron function and blocking adaptive responses to stress, potentially explaining heightened vulnerability to cognitive decline in APOE4 carriers facing Alzheimer's pathology or chemotherapy exposure. While the work addresses an important question linking APOE4 to circuit-level dysfunction, but few methodological concerns interpretative issues limit the significance of the study. The authors use different APOE knock-in mouse models across experiments (Sullivan et al. 1997 model in Figures 1-2 vs. Foley et al. 2022 model in Figures 3-4) without adequate justification. While they acknowledge this limitation, it undermines comparisons between baseline genotype effects and doxorubicin responses. The development of a new human APOE knock-in model in 2022 gave us the opportunity to transition to using mice with less “genetic drift” from those developed in 1997. We have now included better justification for this change, including our reference about direct comparisons of these two mouse models, which largely share phenotypes. We also include sentences in the Results and in the Discussion to further identify this limitation and explain our justification. Several key findings rely on modest sample size of animals. For example, in Figure 4A APOE4 vehicle group has only 1 mouse. This makes it impossible to determine whether non-significant findings represent true null effects or insufficient statistical power. This is especially problematic for interpreting the lack of doxorubicin effects in APOE4 mice, which is central to the manuscript's conclusions. We regret having used some data that was insufficient; we removed the comparisons that depended on single mice from the manuscript. In particular, we eliminated Figure 4A, which concerned counts of parvalbumin-positive cells. These data were not important to our main conclusions; they only demonstrated that the chemotherapy did not change the number of PV-positive cells in the EC, which was not expected from a short treatment of our type. Other issues about sample sizes and statistical power are considered above in response to Reviewer 1. This study addresses important questions about APOE4-related circuit dysfunction and responses to stress, with potential relevance to both Alzheimer's disease and chemotherapy-related cognitive impairment. However, significant methodological limitations particularly the use of different mouse models, small sample sizes, incomplete mechanistic investigation, and questionable clinical relevance of the doxorubicin paradigm limit the impact of the findings. As noted above, we have addressed some of these concerns in this revision and added a section about the limitations of the work, to better put into context the importance of the findings. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0343276.r002
3 Feb 2026 Decision Letter - Ioannis Liampas, Editor APOE4 and doxorubicin impair inhibitory interneuron function and homeostatic regulation in the entorhinal cortex PONE-D-25-62615R1 Dear Dr. Rebeck, 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. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support .. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ioannis Liampas, MD. PhD Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0343276.r003
Formally Accepted
Acceptance Letter - Ioannis Liampas, Editor PONE-D-25-62615R1 PLOS One Dear Dr. Rebeck, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ioannis Liampas Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0343276.r004

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