Dear Dr. Auld,

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Academic Editor

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?

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The manuscript by Paluri and Auld investigates the role of miR-184 as a regulator of pleated septate junctions (pSJs) during the development of the Drosophila blood-brain barrier (BBB). The authors characterize miR-184 expression using a transgenic fluorescent reporter and show that it is restricted to tissues that do not form pSJs. In contrast, ectopic expression of miR-184 in subperineurial glia disrupts the localization of pSJ proteins in both the central nervous system and peripheral nerves. Interestingly, this mislocalisation occurs without a detectable decrease in the mRNA levels of core pSJ components. Instead, they observe an increase in nrv2 transcripts, a phenotype that can be phenocopied by RNAi-mediated downregulation of neurexin-IV.

Overall, this is a well-executed and thoughtfully written study. The manuscript is clear and the discussion addresses most of the relevant limitations of the experimental approaches. While the conclusions may be of limited impact—given that miR-184 is not endogenously expressed in the Drosophila BBB—this work provides a detailed description of miR-184 expression and its potential role in preventing ectopic expression of pSJ components. I recommend acceptance of the manuscript with minor revisions.

Minor comments.

1. I suggest including a figure that shows the miR-184 binding sites in Nrx-IV used for generating the NLS-YFP sensor construct. It would also be of interest to indicate predicted or potential miR-184 binding sites in other core pSJ components.

2. While the manuscript focuses on larval tissues, it would strengthen the study to analyze miR-184 expression in the adult BBB to rule out expression later in development.

3. The authors report that misexpression of miR-184 using SPG-GAL4 (moody-GAL4) is lethal. However, this driver is also known to be active in other tissues such as the midgut, salivary glands, and fat body. To ensure that the observed lethality results specifically from BBB defects, the experiment should be repeated using mdr65-GAL4 (R54C07), which has more restricted expression in subperineurial glia.

4. It would be interesting to assess whether co-expression of UAS-Nrx-IV lacking the 3′UTR (thus evading miR-184 regulation) together with miR-184 can rescue the loss of other pSJ components such as Kune and Mcr. This would strengthen the conclusion that Nrx-IV is a primary functional target of miR-184 in this context.

Reviewer #2: 

The paper by Sravya Paluri and Vanessa J. Aull is an impeccable descriptive study of the expression of microRNA miR-184. In it, the authors employ appropriate tools to investigate its expression in larval tissues, identifying the specific tissues and cells where the microRNA is expressed. They then focus on the brain, particularly on the glia forming the blood-brain barrier (BBB), the cells of the SPG. In these cells, they describe the effects of downregulating the microRNA on the morphology and permeability of the barrier, as well as on the abundance of various proteins associated with pleatet septate junctions (pSJs), which are key cellular components in forming the barrier in SPG cells. They found that miRNA downregulation disrupts barrier formation, increasing its permeability, alters the expression of several pSJ proteins, and affects larval locomotion. Notably, it does not alter the mRNA levels of the proteins that constitute the SJs.

However, they observed that the NRV2 protein, a component of the pSJ but not a direct target of the microRNA, had increased levels, suggesting a regulatory relationship among pSJ proteins.

Although mainly descriptive, the results are of interest to the community working on microRNAs and septate junctions. While it is not surprising that proteins forming part of a larger complex such as the SJ might increase their expression as a compensatory mechanism, this phenomenon has been previously described in synapses, particularly in the postsynaptic density, among other contexts.

Minor comments:

- It could be interpreted that the only target of miR-148 is septate junction proteins. Whether this is the case or not, it would be important to clarify this point in the introduction.

- Why is the Sinu label in the brain's SPG so faint, while it is clear in the SPG of peripheral nerves? Does this suggest that the SPG of peripheral nerves has a different type of SJ?

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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

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microRNA-184 distribution and consequences on glial septate junctions and the blood-brain barrier

PONE-D-25-37097R1

Dear Dr. Auld,

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.

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Kind regards,

Carlos Oliva, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: The revised manuscript have addressed all the comments and I do not have further questions. THe work is sound and well performed.

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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

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