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Optimized detection of caspase-6 activation in a murine inflammation model to inform neurodegenerative disease therapies

PLOS One

Dear Dr. Hardy,

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Xiangning Zhang, M.D., Ph.D.

Academic Editor

PLOS One

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Additional Editor Comments:

Re: PONE-D-25-61406

Optimized detection of caspase-6 activation in a murine inflammation model to inform neurodegenerative disease therapies, Prof Jeanne A Hardy.

Dear Dr. Hardy,

Thank you for an opportunity of letting us read your above-referenced manuscript. At the moment, it has been examined by two independent external reviewers, and enough reviews have reached us. One referee recommended accept, and another one recommend reject. In view of this, we recommend a major revision, asking you to revise the paper according to the comments by the second reviewer, in term of specificty of caspase-6 detection, to increase the amount of the studied materials. If you accept the suggestion, plz return your revised version of paper for further reviewing, subject to further recommendation for decision.

Best regards,

Xiangning Zhang, M.D., Ph.D.

Academic Editor.

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

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: No

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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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: This study describes a detailed study of the development of a mouse model for testing the effects of Caspase-6 elevation in the thymus, colon, and brain. The study has excellent controls and developed new more accurate ways to quantify Caspase-6 activation in mouse tissues. They showed that immunoblots were inadequate to detect Caspase-6 activation or that of its downstream targets. They also showed that home bred mice were much more reliable as test subjects than mice shipped from other sites. This study provides a good model system for testing the effects of caspase inhibitors on reducing or preventing immuno diseases or neurodegenerative diseases in mice.

Reviewer #2: In this manuscript, Sagarbarria et al propose a mouse model where caspase-6 activation can be induced and detected as a potential methodology for future testing of inhibitors. While this is an interesting concept and has significance for understanding the role of caspase-6 in neurodegenerative diseases, the methodology and results are not consistent enough that this method could be widely used.

Major concerns

1. Inconsistent quality of the blots

a. A lot them, especially the caspase-6 ones are very grainy. (technical tip: it is often easier to clearly capture caspase cleavage products with film rather than imaging systems like the chemidoc)

b. Many blot panels do not contain positive controls. E.g 1A and 1B. Blank blot panels are very hard to interpret.

c. Throughout, it would be better to have the samples that are being directly compared (e.g. the organs in figure 1) shown on a single membrane so comparisons can be accurately made. It seems from the supplemental data that in many cases they were but were split up in the figure. This should be avoided.

d. All protein size markers should be labeled on each blot, not just one and the appropriate areas of the blots should be shown. E.g cleaved lamin A is 25 kD but in Figure 1, that area of the blot is not shown.

e. All blots should be quantitated to show relative the protein amounts across experiments/samples and statistics performed. Correlation between lamin and cleaved caspase-6 should be shown where appropriate

2. Lack of specificity of caspase-6 measurements

a. There is no control to show that the lamin A cleavage detected is caspase-6 dependent. Do accomplish this properly the caspase-6 knockout would be needed. This is important since the Lamin A readout seems much more reliable than detection of caspase-6 cleavage

b. VEID is not specific for caspase-6 and can be cleaved more efficiently by caspase-3 (see PMID: 17975551). Therefore, specificity of this readout also needs to confirmed in a caspase-6 knockout

c. The data supporting that murine caspase-6 is activated by LPS, NLRP1 and caspase-1 is based on a single paper (ref 11). In that paper most of the results were from human neurons there was only one figure shown using mouse tissues (cortex). The NLRP1 dependent effects on VEIDase and tubulin cleavage were minor and they did not look as caspase-6 cleavage at all. Therefore,, more confirmation is needed to show that LPS is activating caspase-6 and not having other effects.

d. Based on the literature it is unclear is LPS activates NLRP1 or a contaminant. Human NLRP1 was initially published as being activated by LPS but the activating compound turned out to be muramyldipeptide a contaminant of the LPS prep. Mice predominantly express the isoform NLRP1b which is primarily activated by anthrax. It is not clear if the LPS used in this study is the ultrapure variety

e. Similarly, the rationale for focusing on the thymus and not the cortex as was done in ref 11 is unclear.

f. Additional caspase-6 substrates (tubulin, tau) should be measured to increase robustness and specificity

g. The correlation between caspase-6 cleavage and lamin cleavage does not appear to be consistent. Eg Figure 3B where there is an even level of lamin cleavage in the thymus following LPS treatment but the caspase-6 cleavage is highly variable in both the treated and untreated samples. Same for Figure 7A where there is no visible caspase-6 cleavage but lamin A cleavage

3. Sample size and rigor

a. The sample sizes are often too small. Eg Figure 1C-D the saline group has one sample, Figure 1E there are only 2 samples per group. Figure 4 C, as few as 2 samples per group

b. There is no justification for the different ages of used in different experiments and they are not always age matched when direct comparisons are being made. Eg in Figure 1A the cerebellum sample was from 6 month old mice while the rest were from 1 month old mice

c.

4. In figure 4B, there appears to be more caspase 6 cleavage and lamin A cleavage in the thymus with methylcellulose than corn oil so it is not clear why the authors concluded that methylcellulose was a superior vehicle

Minor

Needs a ref for LPS inducing NLRP1 in line129

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Reviewer #1: No

Reviewer #2: No

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Optimized detection of caspase-6 activation in a murine inflammation model to inform neurodegenerative disease therapies

PONE-D-25-61406R1

Dear Dr. Hardy,

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,

Xiangning Zhang, M.D., Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Attn.: Dr. Jeanne A Hardy,

University of Massachusetts

710 N Pleasant St

Amherst, MA 01003

UNITED STATES OF AMERICA

Re: MS No. PONE-D-25-61406R1 Optimized detection of caspase-6 activation in a murine inflammation model to inform neurodegenerative disease therapies, by Jeanne A Hardy et al.

Dear Dr. Hardy,

Thank you very much for sending us the above-referenced manuscript, with an opportunity of letting us consider it for publication with PlosOne. Comments from two external reviewers reached us; one recommends accept and one, reject. On going through your response of rebuttal opinion, we have found that questions raised by reviewers have been adequately responded, and we recommend an acceptance. It is appreciated that necessary revision is made according to reviewers' comments in the proof.

Best rgds,

Xiangning Zhang, M.D., Ph.D.

Academic Editor.

Reviewers' comments: