PONE-D-20-03925

Stanniocalcin-1 (STC-1), a downstream effector molecule in latanoprost signaling, acts independent of the FP receptor for intraocular pressure reduction

PLOS ONE

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Reviewer concerns are clearly detailed and should be carefully addressed.

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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: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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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

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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

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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: In this manuscript, authors studied whether STC-1, a downstream effector molecule in latanoprost signaling lowers IOP wild-type mice compared to to latonoprost. Moreover, authors examined whether latanoprost and STC-1 require the FP receptor for IOP reduction. Major findings are ; STC-1 is

equivalent in IOP reduction comppared to latonoprost and non-additive in the early IOP response in wild-type mice. Additionally, unlike latanoprost, STC-1 does not use the FP receptor for IOP reduction.

This is solid study and important in the field glaucoma. Authors need to just clarify the following minor comments.

1) Figure 4 is misplaced. It appears that figures are not in sequence. Figure 4 is kept first.. this needs to be corrected.

2)it is unclear whether IOPs are measured in masked manner. Since IOP differences are small, it is important to have masked measurements.

3) Based on discussion, STC-1 may be working on different pathways than latonoprost. However, this does not explain why STC-1 ko prevents latonoprost lowering IOPs (in previous manuscript). It seems more likely that STC1 may be working completely different pathway. Authors need to just clarify where crosstalk between STC-1 and latonoprost signaling is occurring.

4) It is not clear whether STC-1 has any effects on TM outflow. Considering previous studies that point out cytoprotective effects of STC-1 against mitochondrial stress, oxidative etc..it may have a protective role in TM dysfunction in glaucoma. Authors should clarify this as well.

5) Would STC1 lower IOP in similar fashion in ocular hypertensive or glaucoma mice? Please discuss this as well.

Reviewer #2: Manuscript ID: PONE-D-20-03925

Title: Stanniocalcin-1 (STC-1), a downstream effector molecule in latanoprost signaling, acts independent of the FP receptor for intraocular pressure reduction

Authors: Gavin W. Roddy, Tommy A Rinkoski, Kjersten J Monson, Uttio Roy Chowdhury, Michael P Fautsch.

Summary and Impression: Roddy et al present a paper surrounding Sanniocalcin-1 (STC-1), a molecule shown to be a downstream effector in latanoprost signaling. The authors suggested that use of the FP receptor for IOP lowering may be circumvented by using this molecule in lieu of PTGFR ligands such as latanoprost. To address this the authors, use a mouse model to show that STC-1 lowers IOP upon tonometry in comparable measures to latanoprost free acid at similar doses. They also show that the STC1 is able to lower IOP in a PTGFR knockout model in the same mouse background. This has implications for glaucoma treatments in cases where PGF2a analogues cannot be used due to genetic factors.

Overall the data is very interesting and obviously the authors have put a lot of work into this, especially in creation of the mouse model, and the data is believable. However, the presentation of the data, and some of the analysis should be redone. The manuscript itself could use some reorganization/restructuring. First off, the figures are out of order and have no explicit indication that Figure 1 is indeed Figure 1, which made reviewing this a little confusing. Additionally, the way the methods are laid out are confusing as is and I would request the authors rewrite parts of it for the sake of clarity. I also have concerns about some of the statistics. My major and minor issues are outlined below.

Major Issues:

1) I believe a Student paired T-test is inappropriate for the comparisons that are being done. As it stands now the authors do a series of paired tests between their groups of which N=6. The way the statistics are set up now lend itself to multiple testing errors (i.e. multiple pairwise comparisons). I suggest that the authors redo their stats with a Two-way ANOVA with an appropriate post-hoc test (A Bonferroni posthoc would be best for small data sets) rather than the series of paired tests. The authors are trying to demonstrate differences between treatments-controls and between treatments, thus a two-way ANOVA would be appropriate. This should take care of the below:

A) In Figure 1B, the authors should do statistical tests between the treatment groups as it appears there is a lower IOP in the double treatment group, which may indicate an effect on the contralateral eye. Otherwise it should be shown that there Is no statistical difference between the two.

B) In Figure 4B similarly the authors should show statistical differences (or not) between the wildtype and FP knockout groups.

2) Portions of the manuscript have discussion and results blended together. The results section of a scientific manuscript should be for simply reporting the data, whereas interpretation or rationale should be provided in the discussion. For example, under the heading “Determining the effect of FP receptor inhibition on IOP….” Just report the data you obtained. The rationale for the design and the interpretation should be moved to discussion. This is also true in the figure legends, please remove rationale and interpretation from the figures. Additionally, in figure legends, please list statistical test, and expression of error calculations (i.e standard error or std deviation?)

3) Could the authors comment on why uterine tissue was used for the FP receptor quantification. The tests performed concern eye tissue (whole eye for example) which would be the proper control post-mortem.

4) The discussion is not very robust. This should be used to place your data in the context of the literature which I feel the authors have not done here. One aspect of this study that I believe the authors aren’t haven't fleshed out well enough is that they have identified a molecule which may circumvent the FP receptor. They point out in passing that there are people with SNPs in the PTGFR gene that have reduced responses. Dr. Michael Walter’s group (I will freely admit that some of this is work I had a hand in) has published work on so-called ‘recalcitrant glaucoma’ in Axenfeld-Rieger syndrome. Strungaru et al (PMID: 17197537) showed that ARS patients with POAG do not respond to IOP lowering medications (including prostaglandin medications). Doucette et al. (PMID: 29847662) showed that FOXC1 controls expression of the FP receptor. Ultimately, my suggestion is to rewrite this part of the discussion to drive home this part of the data. There is real world applicability here. I’m not asking the authors to cite the above, but thought it would provide some additional context to the author’s dataset.

Minor Issues:

1) Introduction: “G-coupled protein receptor”. Should be G-protein coupled receptor

2) Introduction: “Up to 20% of patients are either minimally response of non-responsive” Could the authors expand on this point a little as I think it would add some implications to the data the authors present. i.e. What type of patients don’t respond or is this consistent across subtypes of glaucoma? Primary open angle? Anterior segment dysgeneses? Closed angle?

3) Introduction: “pigmentation of the iris”. Most likely the authors are referring to hyper-pigmentation or darkening of the iris here. Make this clear

4) Methods: “Animal Experiments” very minor, but mention that you’re using mice here. “Animals” is all that’s used to described the procedures here.

5) Perhaps this is vernacular I’m unfamiliar with, but the uses of the term “treatment nadir” is not something I’ve come across. Could the authors clarify what they mean by this in the text? I see its clarified in the legend of Figure 1, but this needs to either be changed or stated earlier.

6) “Testing LFA and STC-1 in FP receptor knockout mice” – “However, addition of STC-1 to FP receptor (-/-) lowered IOP….”. This seems to be an unfinished sentence.

7) When referring to genes, convention is to italicize

8) Please clarify between cessation and washout periods. As one sounds like stopping treatment whereas the other sounds like a physical washing of the eyes.

9) What is the vehicle for these drugs?

10) I urge the authors to consider using * and/or ** for statistical significance, rather than listing the p-values below 0.00001. This makes the figures much less cumbersome

11) Figure 3C there are no error bars or statistical analysis. While the effect is clearly obvious this needs to be done

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Reviewer #1: Yes: Gulab Zode

Reviewer #2: Yes: Lance P Doucette

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Stanniocalcin-1 (STC-1), a downstream effector molecule in latanoprost signaling, acts independent of the FP receptor for intraocular pressure reduction

PONE-D-20-03925R1

Dear Dr. Roddy,

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

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With kind regards,

Ted S Acott, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

concerns addressed

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: 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?

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: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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 #2: Yes

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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: No

Reviewer #2: Yes

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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 #2: I believe the authors have addressed the reviewer's comments sufficiently. I thank the editors for the opportunity to review this paper, and wish the authors the best of luck in their future endeavors.

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Reviewer #1: Yes: GulabZode

Reviewer #2: Yes: Lance P Doucette