-->PONE-D-26-14007-->-->N-acetyl-L-leucine normalizes Transcription Factor EB activity by stereospecific bidirectional modulation in a Hela cell model of Niemann-Pick disease type C-->-->PLOS One

Dear Dr. Galione,

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.

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PLOS One

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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: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->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

Reviewer #3: Yes

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-->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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This is a very interesting work showing a potential for N-acetyl-L-leucine for NPC disease as well as other neurodegenerative diseases. The manuscript is clear and well written.

I have one minor comment. At the end of Results section, authors describe the effects of NALL on the ratio of phosphor S122 TFEB/total TFEB in NPC1-/- cells (Figure 6B). Authors mentioned “However, the major effect was a larger significant decrease in this ratio in NPC1-/- cells, indicating that a primary effect of the drug is the inhibition of TFEB activation in NPC1-/- cells”. “Decrease in this ratio” should be changed to “increase in this ratio” to reflect the inhibition of TFEB activation.

Reviewer #2: In this manuscript, Davis et al. investigate the modulation of TFEB by N-acetyl-leucine in wild-type cells and models of Niemann-Pick disease type C. While the study addresses a relevant topic for the disease, additional experimental evidence and clarification of key points are required to strengthen the manuscript.

In Figures 2, 3, 5, and 6, the authors present quantifications of immunofluorescence data, but representative images of the cells are not shown. These images are essential for evaluating the quality and interpretation of the data. The authors are requested to include representative immunofluorescence images in the corresponding figures.

1. The graphs should be presented in a consistent format to facilitate interpretation of the results. Currently, Figures 5 and 6 display data in different formats, which makes comparison difficult. Please standardize the graphical representation across figures.

2. In Figure 6, treatment with N-acetyl-leucine does not induce translocation of endogenous TFEB, in contrast to previous experiments where translocation of overexpressed TFEB was observed.

How can this discrepancy be explained?

What is the biological relevance of this finding, given that the endogenous protein is not affected?

Additionally, the authors are encouraged to analyze the effects of the enantiomers of N-acetyl-leucine on endogenous TFEB.

3. In figure 6b the data appear to indicate that untreated NPC1 KO cells have lower levels of phosphorylated TFEB, while treatment restores these levels to those observed in wild-type cells. This observation should be clarified and discussed in more detail.

4. To support the claim that the compound affects TFEB activity in NPC1 KO cells, the authors should assess the expression of TFEB target genes (e.g., LAMP1, cathepsins). In addition, lysosomal modulation should be evaluated, for example LAMP1 staining o lysotracker evaluation with flow cytometry analysis. These data are necessary and should be included.

Reviewer #3: Levacetylleucine (N-Acetyl-L-leucine) is an approved therapeutic for the lysosomal storage disease Niemann-Pick disease type C (NPC) and restores both lysosome and mitochondrial function in NPC models, though the precise mechanism of action remains unclear.

Here the authors build on their previous important finding that N-acetylation increases leucine uptake via monocarboxylate transporters, to show that levacetylleucine normalises aberrant activity of TFEB, the master regulator of lysosome biogenesis and autophagy. They find that treatment with levacetylleucine rapidly activates overexpressed, but not endogenous, TFEB in control cells. Since TFEB activation has been proposed as a potential therapeutic approach for NPC, taken alone this could implicate TFEB activation in the mechanism of action of levacetylleucine. However, increased TFEB activity has been reported in NPC which the authors here confirm in their models. Importantly they go on to show that rather than further increasing the already hyperactive TFEB, levacetylleucine treatment reduces TFEB activity, normalising it almost to control levels, consistent with restoration of lysosomal homeostasis in levacetylleucine-treated models of NPC. Interestingly effects on TFEB are specific to levacetylleucine, with the D-enantiomer or NADLL racemate having no effect. Overall this is a significant finding that will be of great interest to the community.

The manuscript is very well-written and presents robust data with rigorous experimental design, including validation of findings by multiple methods, giving a high level of confidence in the findings.

Minor comments/suggestions:

• Including example images and blots throughout would be nice.

• The authors suggest that L-leucine is able to regulate TFEB via mTORC1 but no effect on mTORC1 is shown and a lack of effect of levacetylleucine on mTOR phosphorylation was previously reported (PMID: 33738443). It would be interesting to see if changes in TFEB activity correlate with changes in mTORC1 phosphorylation.

• The apparent contradiction between the reported mTORC1 hyperactivation in NPC (PMID: 28336668) and the increased TFEB activity shown here should be discussed in more detail – how do the authors reconcile this? mTORC1-independent mechanisms of TFEB regulation should also be considered/discussed.

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Reviewer #1: Yes:  Byeongwoon Song

Reviewer #2: No

Reviewer #3: No

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NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

-->PONE-D-26-14007R1-->-->N-acetyl-L-leucine normalizes Transcription Factor EB activity by stereospecific bidirectional modulation in a Hela cell model of Niemann-Pick disease type C-->-->PLOS One

Dear Dr. Galione,

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 Jul 30 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.

Please include the following items when submitting your revised manuscript:-->

• A 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.

As the corresponding author, your ORCID iD is verified in the submission system and will appear in the published article. PLOS supports the use of ORCID, and we encourage all coauthors to register for an ORCID iD and use it as well. Please encourage your coauthors to verify their ORCID iD within the submission system before final acceptance, as unverified ORCID iDs will not appear in the published article. Only  the individual author can complete the verification step; PLOS staff cannot  verify ORCID iDs on behalf of authors.

We look forward to receiving your revised manuscript.

Kind regards,

David Chau

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

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.

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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

Reviewer #3: (No Response)

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

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

Reviewer #2: Yes

Reviewer #3: (No Response)

**********

-->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: (No Response)

Reviewer #3: (No Response)

**********

-->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

Reviewer #3: (No Response)

**********

-->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: (No Response)

Reviewer #3: Comments have mostly been addressed (with the exception of effects on mTORC1 in NPC) and the manuscript is improved. As a very minor additional suggestion I think it should be made clear in the title/legend for the NALL mechanism scheme (Figure 6) that this refers to wild-type cells (and does not apply to NPC) as it shows NALL stimulating TFEB activation, whereas the authors have shown the opposite in models of NPC1 deficiency.

**********

-->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.

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Reviewer #2: No

Reviewer #3: 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.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

-->

N-acetyl-L-leucine normalizes Transcription Factor EB activity by stereospecific bidirectional modulation in a Hela cell model of Niemann-Pick disease type C

PONE-D-26-14007R2

Dear Dr. Galione,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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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,

David Chau

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: (No Response)

Reviewer #3: (No Response)

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #3: 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 #1: Yes

Reviewer #3: 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 #1: Yes

Reviewer #3: 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 #1: (No Response)

Reviewer #3: (No Response)

**********

-->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 #1: No

Reviewer #3: No

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