PLOS ONE

Dear Dr. Sargeant,

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.

==============================

Reviewer 1

The authors report aging effects on the aging effects on autophagy flux across three tissues (peripheral blood monocytes, heart, and motor cortex neurons) by examining a tandem-fluorescent LC3B mouse at 6, 12 and 18 months of age. In addition, mice fed a high-fat diet were also evaluated at each group. Both sexes were studied.

The study addresses a significant gap in the field using a novel mouse model and a naturally aging model.

MAJOR COMMENTS:

1. The presentation of the mouse phenotype is incomplete. This aspect is particularly important given the naturally aging model. Specifically, information regarding appearance and weight gain is in Suppl. Fig. 1, but additional data seem necessary (e.g., survival curves or tumor free survival) to better understand the model (some of this data appears to have been collected given the n of animals indicated in Suppl. Fig. 1). Other behavioral and motor performance characteristics would be desirable. Similarly, information on non-transgenic mouse of the same background seems important to understand possible differences in aging effects.

2. The high fat diet mice did not develop fasting hyperglycemia (suppl. Fig. 1) despite apparently greater weight gain at all ages

3. The aging phenotype of many mouse models is very limited by 18 months of age (survival rates are usually >75%) and the lack of an older group with lower survival rates is not justified in the manuscript.

4. The characterization of autophagy flux is limited to the GFP:RFP ratio in cells of interest, but other measurements (e.g., puncta per cell, LC3 abundance are not presented).

5. The presumed DAPI+ of lipofuscin granules is intriguing. It would be expected that pigment granules containing lipofuscin would be cytosolic (mostly lysosomes) and lacking DNA (e.g., PLoS ONE 2024; 19(7): e0306275. https://doi.org/10.1371/journal.pone.0306275). This aspect of the work is insufficiently presented to provide confidence in this methodological approach to address autofluorescence in the brain, particularly in older animals.

6. Many essential methodological aspects of confocal fluorescence imaging (e.g., image acquisition, selection of dynamic range, quantification of puncta) are not provided. These aspects are particularly important for the model characterization and the tissue-based measures. For example, sarcomeres appear visible on some of the GFP images of the heart.

7. Corroborative information would be useful in comparing the measured changes in autophagy flux with other measures of autophagy. For instance, there appears to be very low levels of GFP detected suggesting the near absence of autophagosomes. Whether there are global changes in LC3 protein is not clear.

8. The analyses of GFP:RFP ratio presented in figs. 2-4 only reflect mean values per cell/field, clustered across animals, and there is no information presented on the variability across cells within animals, which may be of interest given expected increases in heterogeneity with older age.

9. Concluding statements regarding aging effects on autophagy flux in the heart and brain (results sections and first paragraph of the Discussion) do not seem to be supported by the figures. There are no significant post-hoc differences highlighted in Fig 3B or 4B/D, even if a main age effect is reported. None of the diet effects were significant in the 2-way ANOVA and a significant interaction with age was only reported in the male heart.

MINOR COMMENTS:

1. The quantification of GFP:RFP ratios is insufficiently presented in the results and figure legends to facilitate interpretation (Fig. 2 uses “median fluorescence intensity”, Figs. 3 and 4 “puncta per field”.

2. Post hoc analysis in suppl. Fig. 1B are not presented despite a significant age effect.

3. Statements in the text regarding tumor frequency seem to be based on only 8 animals per age/diet/sex group (suppl. Fig. 1D, 1E). A tumor-free survival curve is likely obtainable for the aging colony.

4. The section on motor cortex neurons suggests that p62/SQSTM1 puncta were identified, but there is no such data in any of the figures (including supplemental).

Reviewer 2

In their manuscript, Carosi et al. analyze autophagy in PBMCs, heart and motor cortex neurons during aging and high-fat diet. For that, they use a transgenic mouse expressing a tandem- fluorescent LC3 reporter (RFP-GFP-LC3), which allows the quantification of autophagic activity. They show that autophagy changes differently across the different tissues analyzed, with differences in response to aging and HFD, and between males and females. The manuscript addresses an interesting topic, and tries to elucidate how autophagy changes during aging and HFD in different tissues. However, there are some concerns the authors should address to add strength to their conclusions.

1. The main issue with the data shown in this manuscript is that the authors have only used the transgenic mouse model expressing RFP-GFP-LC3 reporter. Although very useful, it has some limitations (as the authors state in their conclusions). The activity and acidity of the lysosome are two key aspects in the interpretation of the results obtained using this technique. For example, changes in lysosomal degradation without changes in the pH would lead to the misinterpretation of the results. In this regard, I suggest the authors to evaluate lysosomal activity and pH. Although challenging in tissues in vivo, they can assess it easily in PBMCs using lysosensor and other probes that can be used for flow cytometry.

2. In Figure 2, the authors show autophagic flux in PBMCs isolated from male and female mice at different ages and diet. Although they show the data in PBMCs in general, it would be interesting to show autophagic flux in the different main cell populations ((T cells, B cells, monocytes, etc).

3. The addition of some data regarding the expression of the main autophagy regulators and proteins would add important information and would help to strengthen the conclusions of the study.

==============================

Please submit your revised manuscript by Jan 21 2025 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.

• 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 investigation was supported by Lysosomal Health in Ageing at SAHMRI, and an Ideas Grant from the National Health and Medical Research Council (GNT2002608) awarded to JB and TJS. JMC is supported by an EMCR Fellowship from The Hospital Research Foundation Group (2022-CF-EMCR-007). The authors acknowledge Microscopy Australia (ROR: 042mm0k03) resources at the Future Industries Institute, University of South Australia, enabled by NCRIS.  

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. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

This investigation was supported by Lysosomal Health in Ageing at SAHMRI, and an Ideas

Grant from the National Health and Medical Research Council (GNT2002608) awarded to JB

and TJS. JMC is supported by an EMCR Fellowship from The Hospital Research Foundation

Group (2022-CF-EMCR-007). The authors acknowledge Microscopy Australia (ROR:

042mm0k03) resources at the Future Industries Institute, University of South Australia,

enabled by NCRIS.

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

This investigation was supported by Lysosomal Health in Ageing at SAHMRI, and an Ideas Grant from the National Health and Medical Research Council (GNT2002608) awarded to JB and TJS. JMC is supported by an EMCR Fellowship from The Hospital Research Foundation Group (2022-CF-EMCR-007). The authors acknowledge Microscopy Australia (ROR: 042mm0k03) resources at the Future Industries Institute, University of South Australia, enabled by NCRIS.

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. In the online submission form, you indicated that The data underlying the results presented in the study are available from the corresponding author under reasonable request.

All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either 1. In a public repository, 2. Within the manuscript itself, or 3. Uploaded as supplementary information.

This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval. 

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[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: Yes

**********

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

The PLOS Data policy

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 authors report aging effects on the aging effects on autophagy flux across three tissues (peripheral blood monocytes, heart, and motor cortex neurons) by examining a tandem-fluorescent LC3B mouse at 6, 12 and 18 months of age. In addition, mice fed a high-fat diet were also evaluated at each group. Both sexes were studied.

The study addresses a significant gap in the field using a novel mouse model and a naturally aging model.

MAJOR COMMENTS:

1. The presentation of the mouse phenotype is incomplete. This aspect is particularly important given the naturally aging model. Specifically, information regarding appearance and weight gain is in Suppl. Fig. 1, but additional data seem necessary (e.g., survival curves or tumor free survival) to better understand the model (some of this data appears to have been collected given the n of animals indicated in Suppl. Fig. 1). Other behavioral and motor performance characteristics would be desirable. Similarly, information on non-transgenic mouse of the same background seems important to understand possible differences in aging effects.

2. The high fat diet mice did not develop fasting hyperglycemia (suppl. Fig. 1) despite apparently greater weight gain at all ages

3. The aging phenotype of many mouse models is very limited by 18 months of age (survival rates are usually >75%) and the lack of an older group with lower survival rates is not justified in the manuscript.

4. The characterization of autophagy flux is limited to the GFP:RFP ratio in cells of interest, but other measurements (e.g., puncta per cell, LC3 abundance are not presented).

5. The presumed DAPI+ of lipofuscin granules is intriguing. It would be expected that pigment granules containing lipofuscin would be cytosolic (mostly lysosomes) and lacking DNA (e.g., PLoS ONE 2024; 19(7): e0306275. https://doi.org/10.1371/journal.pone.0306275). This aspect of the work is insufficiently presented to provide confidence in this methodological approach to address autofluorescence in the brain, particularly in older animals.

6. Many essential methodological aspects of confocal fluorescence imaging (e.g., image acquisition, selection of dynamic range, quantification of puncta) are not provided. These aspects are particularly important for the model characterization and the tissue-based measures. For example, sarcomeres appear visible on some of the GFP images of the heart.

7. Corroborative information would be useful in comparing the measured changes in autophagy flux with other measures of autophagy. For instance, there appears to be very low levels of GFP detected suggesting the near absence of autophagosomes. Whether there are global changes in LC3 protein is not clear.

8. The analyses of GFP:RFP ratio presented in figs. 2-4 only reflect mean values per cell/field, clustered across animals, and there is no information presented on the variability across cells within animals, which may be of interest given expected increases in heterogeneity with older age.

9. Concluding statements regarding aging effects on autophagy flux in the heart and brain (results sections and first paragraph of the Discussion) do not seem to be supported by the figures. There are no significant post-hoc differences highlighted in Fig 3B or 4B/D, even if a main age effect is reported. None of the diet effects were significant in the 2-way ANOVA and a significant interaction with age was only reported in the male heart.

MINOR COMMENTS:

1. The quantification of GFP:RFP ratios is insufficiently presented in the results and figure legends to facilitate interpretation (Fig. 2 uses “median fluorescence intensity”, Figs. 3 and 4 “puncta per field”.

2. Post hoc analysis in suppl. Fig. 1B are not presented despite a significant age effect.

3. Statements in the text regarding tumor frequency seem to be based on only 8 animals per age/diet/sex group (suppl. Fig. 1D, 1E). A tumor-free survival curve is likely obtainable for the aging colony.

4. The section on motor cortex neurons suggests that p62/SQSTM1 puncta were identified, but there is no such data in any of the figures (including supplemental).

Reviewer #2: In their manuscript, Carosi et al. analyze autophagy in PBMCs, heart and motor cortex neurons during aging and high-fat diet. For that, they use a transgenic mouse expressing a tandem- fluorescent LC3 reporter (RFP-GFP-LC3), which allows the quantification of autophagic activity. They show that autophagy changes differently across the different tissues analyzed, with differences in response to aging and HFD, and between males and females. The manuscript addresses an interesting topic, and tries to elucidate how autophagy changes during aging and HFD in different tissues. However, there are some concerns the authors should address to add strength to their conclusions.

1. The main issue with the data shown in this manuscript is that the authors have only used the transgenic mouse model expressing RFP-GFP-LC3 reporter. Although very useful, it has some limitations (as the authors state in their conclusions). The activity and acidity of the lysosome are two key aspects in the interpretation of the results obtained using this technique. For example, changes in lysosomal degradation without changes in the pH would lead to the misinterpretation of the results. In this regard, I suggest the authors to evaluate lysosomal activity and pH. Although challenging in tissues in vivo, they can assess it easily in PBMCs using lysosensor and other probes that can be used for flow cytometry.

2. In Figure 2, the authors show autophagic flux in PBMCs isolated from male and female mice at different ages and diet. Although they show the data in PBMCs in general, it would be interesting to show autophagic flux in the different main cell populations ((T cells, B cells, monocytes, etc).

3. The addition of some data regarding the expression of the main autophagy regulators and proteins would add important information and would help to strengthen the conclusions of the study.

**********

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.

Dear Dr. Sargeant,

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 Jun 06 2025 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.

• 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 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,

Bin Wu, M.D. & Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

The authors have succesfully addressed my questions.

However, it would be important to provide quantifications of the WB for autophagic proteins and cathepsin D.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

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

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

**********

Reviewer #2: The authors have succesfully addressed my questions.

However, it would be important to provide quantifications of the WB for autophagic proteins and cathepsin D.

**********

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: 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

Autophagy across tissues of aging mice

PONE-D-24-49236R2

Dear Dr. Sargeant,

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. If you have any questions relating to publication charges, 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,

Bin Wu, M.D. & Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: