Autophagic flux is impaired in the brain tissue of Tay-Sachs disease mouse model

Tugce Sengul; Melike Can; Nurselin Ateş; Volkan Seyrantepe

doi:10.1371/journal.pone.0280650

Peer Review History

Original SubmissionApril 3, 2022
20 Jun 2022 Decision Letter - Vladimir Trajkovic, Editor PONE-D-22-09806AUTOPHAGIC FLUX IS IMPAIRED IN BRAIN TISSUE OF TAY-SACHS DISEASE MOUSE MODELPLOS ONE Dear Dr. Seyrantepe, 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. In some of the LC3 blots (e.g. Fig. 4J) there are three clearly visible bands. The authors should explain which of the two lower bands was considered as LC3-II. If possible, a higher quality LC3 blots should be presented in Fig. 4J. Reviewer 1: This study aims to investigate alterations in autophagic flux in the new mouse model of Tay-Sachs disease, which should provide information for future mechanistic and therapeutic studies in this field. 1. However, this study only includes 3 mice each groups, lacking statistical rigor. 2. The implications of alterations of those gene expression are not well-explained. The rationale why the authors are looking at a certain gene is not fully explained. It is difficult to follow why change of one gene in a certain brain region means something for a better understanding of pathogenesis mechanisms of this mouse model. Moreover, how the information collected in this study will contribute to a better understanding of Tay-Sachs disease and this mouse model is not clear. Basically, changes of several genes in some brain regions (but not others) at a certain time is only an indicator (weak evidence) of something. This study lacks a clear logic, strong evidence, and a meaningful conclusion. 3. The reviewer is not convinced that targeting autophagic flux could be potentially therapeutic, which seems to be a conclusion of this study. 4. There are many grammar mistakes. Reviewer 2: The authors examined autophagic flux initiation, autophagosome number in the elongation and p62/SQSTM1 in termination in 2 moth and 5 month TSD mice model cortex, cerebellum, thalamus and hippocampus by qRT-PCR, Western Blot and imminohistochemical techniques. The data revealed accumulated autophagosomes, indicating impairment in autophagic flux in the brain. This work elucidates the contribution of altered autophagy to TSD pathology. I would like to see the following points addressed in the manuscript before its publication. 1. Hexa-/-Neu3-/- undergo progressive neurodegeneration and survived up to 5 months. However, no significant difference in the protein level of Beclin-1 was detected in 2-month-old WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice. Thus, more time point data is necessary to determine when autophagic flux alteration in those gene KO mice. 2. LC3 protein level in cortex and cerebellum regions of 2-month-old Hexa-/-Neu3-/- mice was notably elevated compared to WT. It is necessary to analyse the number of LC3(+) vesicles in cortex and cerebellum using immunohistochemistry. 3. P62 protein level was significantly elevated shown in both cortex and hippocampus of 2-month-old Hexa-/-Neu3-/- mice compared to WT. It is necessary to analyse the P62 in cortex and hippocampus using immunohistochemistry. Please submit your revised manuscript by Jul 28 2022 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, Vladimir Trajkovic Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following financial disclosure: “This study is funded by TUBİTAK Grant No: 215Z083” Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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? 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: Yes ****** 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: No ****** 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: No Reviewer #2: Yes ****** 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: This study aims to investigate alterations in autophagic flux in the new mouse model of Tay-Sachs disease, which should provide information for future mechanistic and therapeutic studies in this field. 1. However, this study only includes 3 mice each groups, lacking statistical rigor. 2. The implications of alterations of those gene expression are not well-explained. The rationale why the authors are looking at a certain gene is not fully explained. It is difficult to follow why change of one gene in a certain brain region means something for a better understanding of pathogenesis mechanisms of this mouse model. Moreover, how the information collected in this study will contribute to a better understanding of Tay-Sachs disease and this mouse model is not clear. Basically, changes of several genes in some brain regions (but not others) at a certain time is only an indicator (weak evidence) of something. This study lacks a clear logic, strong evidence, and a meaningful conclusion. 3. The reviewer is not convinced that targeting autophagic flux could be potentially therapeutic, which seems to be a conclusion of this study. 4. There are many grammar mistakes. Reviewer #2: The authors examined autophagic flux initiation, autophagosome number in the elongation and p62/SQSTM1 in termination in 2 moth and 5 month TSD mice model cortex, cerebellum, thalamus and hippocampus by qRT-PCR, Western Blot and imminohistochemical techniques. The data revealed accumulated autophagosomes, indicating impairment in autophagic flux in the brain. This work elucidates the contribution of altered autophagy to TSD pathology. I would like to see the following points addressed in the manuscript before its publication. 1. Hexa-/-Neu3-/- undergo progressive neurodegeneration and survived up to 5 months. However, No significant difference in the protein level of Beclin-1 was detected in 2-month-old WT, Hexa-/-, Neu3-/- and Hexa-/-Neu3-/- mice. Thus, more time point data is necessary to determine when autophagic flux alteration in those gene KO mice. 2. LC3 protein level in cortex and cerebellum regions of 2-month-old Hexa-/-Neu3-/- mice was notably elevated compared to WT. It is necessary to analysis the number of LC3(+) vesicles in cortex and cerebellum using immunohistochemical. 3. P62 protein level was significantly elevated shown in both cortex and hippocampus of 2-month-old Hexa-/-Neu3-/- mice compared to WT. It is necessary to analysis the P62 in cortex and hippocampus using immunohistochemical. ****** 6. 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 ******** [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. https://doi.org/10.1371/journal.pone.0280650.r001
Revision 1
19 Sep 2022 Author Response Response to reviewers Reviewer 1: This study aims to investigate alterations in autophagic flux in the new mouse model of Tay-Sachs disease, which should provide information for future mechanistic and therapeutic studies in this field. 1. However, this study only includes 3 mice each groups, lacking statistical rigor. All data presented here were replicated in at least three mice with all histochemical staining data quantified by experimenters who were blind to genotypes/treatments. Animals were randomly allocated into control and experimental groups. 2. The implications of alterations of those gene expression are not well-explained. The rationale why the authors are looking at a certain gene is not fully explained. The rationale for why looking at a certain gene expression is explained in the introduction section of the manuscript as below In mammalian cells, two major mechanisms are carried out for the degradation of intracellular proteins which are the ubiquitin-proteasome system (UPS) and autophagy [6]. In particular, autophagy is involved in lysosome-dependent pathways for damaged organelles, unfolded proteins, and accumulated cellular components to maintain cellular homeostasis [7]. Autophagic flux includes vesicle trafficking and a network in which newly produced autophagosomes (double-membrane vesicles) are fused with lysosomes to degrade autophagic cargo. In this process, autophagore formation, autophagosome completion with the closure of membrane, and autophagosome-lysosome fusion (autolysosome) take place respectively [8]. Each step of autophagic flux is finely regulated by specific protein complexes and the initiation step, the sequestering of autophagic cargo within an isolation membrane (phagophore), is controlled by the Beclin-1. Beclin-1 consists of three distinct structural domains interacting with proteins that regulate autophagy machinery. In particular, Beclin-1, which can interact with several cofactors, promotes the formation of the Beclin-1-Vps34-Vps15 core body, in this way induces the initiation of autophagic flux [9]. Autophagy-related (Atg) proteins generate phagophore assembly sites and enable the envelopment of cytoplasmic material. In particular, the Atg9 protein is a key regulator of autophagy induction. During the maturation of autophagosome, Atg7 has involved the conversion of the cytosolic form of microtubule-associated protein 1 light chain 3 (LC3-I) to LC3-II which is located on both inner and outer autophagosomal membranes [9]. LC3 is involved in phagophore edge folding that resulted in autophagosome formation. After the formation of autolysosomes formed by the combination of autophagosomes and lysosomes, LC3-II on the outer membrane is converted back to LC3-I and then intra-autophagosomal LC3-II is degraded by lysosomal hydrolyses. Therefore, the level of LC3-II as a marker of dynamic autophagosomal membranes is generally studied to monitor autophagic activity [10]. In addition, the level of ubiquitin-binding scaffold protein p62 (aggregated endogenous substrates) which know to be associated with LC3-II in the autophagosome is mostly evaluated as a marker of termination of autophagy [9,11]. A bunch of studies also showed a defect in autophagic flux and secondary accumulation of autophagic substrates such as autophagosomes in several LSDs [6,12–14]. It is difficult to follow why change of one gene in a certain brain region means something for a better understanding of pathogenesis mechanisms of this mouse model. Moreover, how the information collected in this study will contribute to a better understanding of Tay-Sachs disease and this mouse model is not clear. Basically, changes of several genes in some brain regions (but not others) at a certain time is only an indicator (weak evidence) of something. This study lacks a clear logic, strong evidence, and a meaningful conclusion. In this study, we show that abnormal GM2 accumulation in the different region of brain tissue from TSD mouse model leads to impaired autophagy evident from attenuated expression of some components of the autophagy network, decreased autophagosome formation, and reduced autophagy flux. We for the first time study the expression of some fundamental autophagic markers (LC3, p62, and Beclin-1) in a TSD murine model by q-RT-PCR, immunohistochemistry and Western blot. 3. The reviewer is not convinced that targeting autophagic flux could be potentially therapeutic, which seems to be a conclusion of this study. We are sorry for not convincing the reviewer that targeting autophagic flux could be potentially therapeutic in this manuscript. However, we have some preliminary in vitro data indicating lithium as a therapeutic agent could be used to restore dysregulated autophagy in Tay-Sachs cells. That data is not completed yet to add to the manuscript. Although additional studies are required to fully characterize the autophagy machinery in the recently generated TSD murine model, this paper suggests impaired autophagy as a new aspect involved in the molecular pathogenesis of TSD. Despite important advances made in the last years in TSD research, the principally studied therapies such as gene and anti-inflammatory therapy and their combination can increase the life expectancy of the treated TSD mice (unpublished data yet) autophagy modulation could be a promising option to be tested in combination therapies to help to achieve a complete TSD phenotype rescue. 4. There are many grammar mistakes. In this final version of the manuscript, we used a free online writing assistant called Grammarly Reviewer 2: The authors examined autophagic flux initiation, autophagosome number in the elongation, and p62/SQSTM1 in termination in 2-month and 5-month TSD mice model cortex, cerebellum, thalamus and hippocampus by qRT-PCR, Western Blot, and immunohistochemical techniques. The data revealed accumulated autophagosomes, indicating impairment in autophagic flux in the brain. This work elucidates the contribution of altered autophagy to TSD pathology. I would like to see the following points addressed in the manuscript before its publication. 1. Hexa-/-Neu3-/- undergo progressive neurodegeneration and survived up to 5 months. However, no significant difference in the protein level of Beclin-1 was detected in 2-month-old WT, Hexa-/-, Neu3-/-, and Hexa-/-Neu3-/- mice. Thus, more time point data is necessary to determine when autophagic flux alteration in those gene KO mice. We added one more time point (3-months old mice) to determine when autophagic flux alterations (Fig Supp 1 and Fig Supp 2) 2. LC3 protein level in cortex and cerebellum regions of 2-month-old Hexa-/-Neu3-/- mice was notably elevated compared to WT. It is necessary to analyze the number of LC3(+) vesicles in cortex and cerebellum using immunohistochemistry. We added new data showing LC3 protein levels in cortex and cerebellum regions of 2-month-old mice using immunohistochemistry (Fig Supp 3) 3. P62 protein level was significantly elevated shown in both cortex and hippocampus of 2-month-old Hexa-/-Neu3-/- mice compared to WT. It is necessary to analyze the P62 in the cortex and hippocampus using immunohistochemistry. We added new data showing p62 protein levels in cortex and cerebellum regions of 2-month-old mice using immunohistochemistry (Fig Supp 4) Attachments Attachment Submitted filename: PONE Response to reviewers.docx https://doi.org/10.1371/journal.pone.0280650.r002
19 Oct 2022 Decision Letter - Vladimir Trajkovic, Editor PONE-D-22-09806R1AUTOPHAGIC FLUX IS IMPAIRED IN BRAIN TISSUE OF TAY-SACHS DISEASE MOUSE MODELPLOS ONE Dear Dr. Seyrantepe, Thank you for submitting your manuscript to PLOS ONE. Before your manuscript could be further evaluated, please submit the response to the issues raised by the Academic Editor in the previous decision letter. Please submit your revised manuscript by Dec 03 2022 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, Vladimir Trajkovic Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 #2: All comments have been addressed ******** 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 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 #2: 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 #2: 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 #2: The authors have addressed all my concerns. I suggest this manuscript is acceptable for publication. ****** 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 #2: Yes: Zhanjun Li ******** [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. https://doi.org/10.1371/journal.pone.0280650.r003
Revision 2
6 Dec 2022 Author Response We did not see the comments from the reviewer 1. Attachments Attachment Submitted filename: PONE Response to reviewers.docx https://doi.org/10.1371/journal.pone.0280650.r004
20 Dec 2022 Decision Letter - Vladimir Trajkovic, Editor PONE-D-22-09806R2AUTOPHAGIC FLUX IS IMPAIRED IN BRAIN TISSUE OF TAY-SACHS DISEASE MOUSE MODEL PLOS ONE Dear Dr. Seyrantepe, Thank you for submitting your manuscript to PLOS ONE. Before your manuscript could be further evaluated, please submit the response to the issues raised by the Academic Editor in the first decision letter (please see below): In some of the LC3 blots (e.g. Fig. 4J) there are three clearly visible bands. The authors should explain which of the two lower bands was considered as LC3-II. If possible, the higher quality LC3 blots should be presented in Fig. 4J. Also, the LC3-II levels should be expressed relative to loading control (actin), not LC3-I, as recommended in the last edition of autophagy guidelines (Autophagy 2021;17:1-382. doi: 10.1080/15548627.2020.1797280). Please submit your revised manuscript by Feb 03 2023 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, Vladimir Trajkovic Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: [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. https://doi.org/10.1371/journal.pone.0280650.r005
Revision 3
4 Jan 2023 Author Response Response to reviewers Reviewer 1: This study aims to investigate alterations in autophagic flux in the new mouse model of Tay-Sachs disease, which should provide information for future mechanistic and therapeutic studies in this field. 1. However, this study only includes 3 mice each groups, lacking statistical rigor. All data presented here were replicated in at least three mice with all histochemical staining data quantified by experimenters who were blind to genotypes/treatments. Animals were randomly allocated into control and experimental groups. 2. The implications of alterations of those gene expression are not well-explained. The rationale why the authors are looking at a certain gene is not fully explained. The rationale for why looking at a certain gene expression is explained in the introduction section of the manuscript as below In mammalian cells, two major mechanisms are carried out for the degradation of intracellular proteins which are the ubiquitin-proteasome system (UPS) and autophagy [6]. In particular, autophagy is involved in lysosome-dependent pathways for damaged organelles, unfolded proteins, and accumulated cellular components to maintain cellular homeostasis [7]. Autophagic flux includes vesicle trafficking and a network in which newly produced autophagosomes (double-membrane vesicles) are fused with lysosomes to degrade autophagic cargo. In this process, autophagore formation, autophagosome completion with the closure of membrane, and autophagosome-lysosome fusion (autolysosome) take place respectively [8]. Each step of autophagic flux is finely regulated by specific protein complexes and the initiation step, the sequestering of autophagic cargo within an isolation membrane (phagophore), is controlled by the Beclin-1. Beclin-1 consists of three distinct structural domains interacting with proteins that regulate autophagy machinery. In particular, Beclin-1, which can interact with several cofactors, promotes the formation of the Beclin-1-Vps34-Vps15 core body, in this way induces the initiation of autophagic flux [9]. Autophagy-related (Atg) proteins generate phagophore assembly sites and enable the envelopment of cytoplasmic material. In particular, the Atg9 protein is a key regulator of autophagy induction. During the maturation of autophagosome, Atg7 has involved the conversion of the cytosolic form of microtubule-associated protein 1 light chain 3 (LC3-I) to LC3-II which is located on both inner and outer autophagosomal membranes [9]. LC3 is involved in phagophore edge folding that resulted in autophagosome formation. After the formation of autolysosomes formed by the combination of autophagosomes and lysosomes, LC3-II on the outer membrane is converted back to LC3-I and then intra-autophagosomal LC3-II is degraded by lysosomal hydrolyses. Therefore, the level of LC3-II as a marker of dynamic autophagosomal membranes is generally studied to monitor autophagic activity [10]. In addition, the level of ubiquitin-binding scaffold protein p62 (aggregated endogenous substrates) which know to be associated with LC3-II in the autophagosome is mostly evaluated as a marker of termination of autophagy [9,11]. A bunch of studies also showed a defect in autophagic flux and secondary accumulation of autophagic substrates such as autophagosomes in several LSDs [6,12–14]. It is difficult to follow why change of one gene in a certain brain region means something for a better understanding of pathogenesis mechanisms of this mouse model. Moreover, how the information collected in this study will contribute to a better understanding of Tay-Sachs disease and this mouse model is not clear. Basically, changes of several genes in some brain regions (but not others) at a certain time is only an indicator (weak evidence) of something. This study lacks a clear logic, strong evidence, and a meaningful conclusion. In this study, we show that abnormal GM2 accumulation in the different region of brain tissue from TSD mouse model leads to impaired autophagy evident from attenuated expression of some components of the autophagy network, decreased autophagosome formation, and reduced autophagy flux. We for the first time study the expression of some fundamental autophagic markers (LC3, p62, and Beclin-1) in a TSD murine model by q-RT-PCR, immunohistochemistry and Western blot. 3. The reviewer is not convinced that targeting autophagic flux could be potentially therapeutic, which seems to be a conclusion of this study. We are sorry for not convincing the reviewer that targeting autophagic flux could be potentially therapeutic in this manuscript. However, we have some preliminary in vitro data indicating lithium as a therapeutic agent could be used to restore dysregulated autophagy in Tay-Sachs cells. That data is not completed yet to add to the manuscript. Although additional studies are required to fully characterize the autophagy machinery in the recently generated TSD murine model, this paper suggests impaired autophagy as a new aspect involved in the molecular pathogenesis of TSD. Despite important advances made in the last years in TSD research, the principally studied therapies such as gene and anti-inflammatory therapy and their combination can increase the life expectancy of the treated TSD mice (unpublished data yet) autophagy modulation could be a promising option to be tested in combination therapies to help to achieve a complete TSD phenotype rescue. 4. There are many grammar mistakes. In this final version of the manuscript, we used a free online writing assistant called Grammarly Reviewer 2: The authors examined autophagic flux initiation, autophagosome number in the elongation, and p62/SQSTM1 in termination in 2-month and 5-month TSD mice model cortex, cerebellum, thalamus and hippocampus by qRT-PCR, Western Blot, and immunohistochemical techniques. The data revealed accumulated autophagosomes, indicating impairment in autophagic flux in the brain. This work elucidates the contribution of altered autophagy to TSD pathology. I would like to see the following points addressed in the manuscript before its publication. 1. Hexa-/-Neu3-/- undergo progressive neurodegeneration and survived up to 5 months. However, no significant difference in the protein level of Beclin-1 was detected in 2-month-old WT, Hexa-/-, Neu3-/-, and Hexa-/-Neu3-/- mice. Thus, more time point data is necessary to determine when autophagic flux alteration in those gene KO mice. We added one more time point (3-months old mice) to determine when autophagic flux alterations (Fig Supp 1 and Fig Supp 2) 2. LC3 protein level in cortex and cerebellum regions of 2-month-old Hexa-/-Neu3-/- mice was notably elevated compared to WT. It is necessary to analyze the number of LC3(+) vesicles in cortex and cerebellum using immunohistochemistry. We added new data showing LC3 protein levels in cortex and cerebellum regions of 2-month-old mice using immunohistochemistry (Fig Supp 3) 3. P62 protein level was significantly elevated shown in both cortex and hippocampus of 2-month-old Hexa-/-Neu3-/- mice compared to WT. It is necessary to analyze the P62 in the cortex and hippocampus using immunohistochemistry. We added new data showing p62 protein levels in cortex and cerebellum regions of 2-month-old mice using immunohistochemistry (Fig Supp 4) Attachments Attachment Submitted filename: PONE Response to reviewers.docx https://doi.org/10.1371/journal.pone.0280650.r006
5 Jan 2023 Decision Letter - Vladimir Trajkovic, Editor AUTOPHAGIC FLUX IS IMPAIRED IN BRAIN TISSUE OF TAY-SACHS DISEASE MOUSE MODEL PONE-D-22-09806R3 Dear Dr. Seyrantepe, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. 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, Vladimir Trajkovic Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0280650.r007
Formally Accepted
7 Mar 2023 Acceptance Letter - Vladimir Trajkovic, Editor PONE-D-22-09806R3 AUTOPHAGIC FLUX IS IMPAIRED IN THE BRAIN TISSUE OF TAY-SACHS DISEASE MOUSE MODEL Dear Dr. Seyrantepe: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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. If we can help with anything else, please email us at plosone@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 Prof. Vladimir Trajkovic Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0280650.r008

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .