PGENETICS-D-25-00928

The neuropeptide neuromedin U receptor NMUR-1 buffers insulin receptor signaling in bacteria-dependent C. elegans survival

PLOS Genetics

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Fengwei Yu

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Aimée Dudley

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Anne Goriely

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Reviewers’ comments:

Reviewer’s Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This is a very nice paper by Deniz Sifoglu et al in Joy Alcedo’s lab examining how the neuromedin U receptor regulates lifespan in the nematode C. elegans. They build on their previous work showing that nmur-1 regulates lifespan in a bacterial LPS-specific manner. Here they show that many of these effects are though modulation of one specific cause of death related to pharyngeal swelling. Interestingly, nmur-1 does not have a simple effect on lifespan, instead its effect on lifespan is dependent on the bacterial LPS. Even more interestingly, the type of influence of NMUR-1 on lifespan depends on the level of signaling by the insulin receptor DAF-2. At normal levels of DAF-2, NMUR-1 increases lifespan, but at low levels of DAF-2 (which increase lifespan), NMUR-1 shorten lifespan. Thus, as the authors conclude, it appears that NMUR-1 limits the dynamic range of the lifespan effects of DAF-2 by influencing both its lower and upper bounds. I think this is a conceptually important paper and , therefore, support its publication.

I have a series of minor suggestions that I think may make the paper easier to read to non-geneticists. The use of the word activity (eg "nmur-1 adjusts and buffers insulin receptor activity") can be quite un-intuitive to non-geneticists. I suppose by genetic activity they mean, how much a gene influences a phenotype (?), here lifespan and P death. But this influence on activity, confusingly, is not necessarily direct, it can be upstream or downstream of the gene function, and it is confusing if they refer about the activity of the DAF-2 protein or the daf-2 gene, or both. I understand that activity packs all these meanings in one word, but I suggest they unpack these terms because the readers may not know what they mean. Similarly, it would help if when they refer to the normal gene activity they wrote nmur-1(+) instead of nmur-1, to more explicitly differentiate it from the mutant allele.

The paper ends kind of with a teaser for future studies, showing that nmur-1 regulates the expression of daf-28/insulin. It would be nice if they also looked at whether the LPS-affecting E. coli mutants phenocopy those effects. But I don’t think it is essential.

In many figures the authors plot survival curves with "P-deaths only." I suppose that what they are doing is to censor the non-P deaths? In any case, they should explain in the methods what is it that they are doing to make those plots, and ideally in the main text.

Reviewer #2: In this article by Sifoglu et al., the authors report that differences in C. elegans death on two different E. coli strains can be attributed to differences in bacterial LPS structures and nmur-1 activity. Mutants deficient in nmur-1 exhibit greater P-deaths on OP50 but not on CS180 bacteria. However, the authors make the surprising observation that nmur-1 and daf-2 double mutants are long-lived with fewer P-deaths than daf-2 single mutants. They determine that these phenotypes are dependent on daf-16, the downstream transcription factor of daf-2 signaling. This suggests that depending on the level of insulin signaling, nmur-1 can have opposing roles on P-deaths on OP50. Lastly, the authors demonstrate that rescuing nmur-1 activity in sensory neurons in nmnur-1 mutants rescues P-deaths on OP50. Overall, the findings of the paper and convincing, logical and rigorous and will appeal to a broad genetics audience. The data reveals a fascinating interaction between a host’s genetic background, insulin signaling, and different bacterial food sources. Addressing the following concerns will strengthen these findings and the interpretation of the data:

Major concerns:

1) In figure 6A, the authors claim that nmur-1 mutants require daf-16 for P-body death. Both double mutants daf-16;nmur-1 and single mutants exhibit the same level of P-deaths, suggesting that daf-16 and nmur-1 are acting in the same pathway. However, this does not provide an order to claim that nmur-1 acts upstream of daf-16 as depicted in Figure 7.

2) Interestingly, reducing daf-2 (thereby activating daf-16) protects against death and nmur-1 loss-of-function mutants are further protected. This suggests that nmur-1 is acting in a parallel protective pathway with daf-16 in daf-2 mutants. In Figure 6B, the authors demonstrate that the triple mutant (daf-2, daf-16, and nmur-1) exhibit the same lifespan and P-deaths as single mutants. This suggests that daf-16 is the decisive node, because in daf-16 mutants, the level of daf-2 or nmur-1 does not change the outcome. Nmur-1 could be acting upstream of daf-16 or in parallel, but one cannot conclude which from the provided data. The authors should use a daf-16 reporter or a sod-3p::GFP reporter to determine if nmur-1 regulates daf-16 directly and if daf-16 activity changes in the daf-2;nmur-1 double mutant.

3) The authors show osm-6p::nmur-1; nmur-1 mutant animals have fewer P-body deaths. Is there increased daf-16 signaling in these animals?

4) The authors show that daf-28 is increased in Fig 6D. However, there is no data demonstrating that daf-28 mutants suppress P-deaths induced by loss of nmur-1 on OP50 or that increasing daf-28 levels alone is sufficient to induce P body death on OP50. Elevated daf-28 could be correlation and the evidence does not exist to place it in the Figure 7 unless there is a ? mark after daf-28.

5) Figure 7 is very confusing. The use of “high activity” of daf-2 does not make sense to describe basal levels. To generate a high level of insulin/IGF signaling the authors should use gain of function mutants for pdk-1 or akt-1. I recommend the authors draw out the schematic for normal/wild-type conditions and the daf-2 mutant/reduced insulin signaling condition separately.

6) The authors claim that the data in Figure “suggests that wild-type nmur-1 in sensory neurons inhibits P-deaths when daf-2 activity is high but promotes P deaths under low daf-2 activity.” However, the gray and green dotted lines indicating the daf-2 mutant and daf-2 mutant with wild-type nmur-1 in sensory neurons show similar levels of P-deaths (ie. not more p-deaths in green dotted line compared to gray dotted line). Also, stating “high” level of daf-2 suggests higher than normal/wild-type.

Minor concerns:

1. Some areas of the text have confusing wording. For example, “The same construct rescued both the P-death and non-P death phenotypes…” makes it sound to the reader as though there are other phenotypes associated with P-death or non-P death that were assayed. I recommend, “The same construct rescued P-death and non-P death...”

2. The phrase “inhibits healthy pharynges” is nonsensical.

3. It is not clear from the plot that 3F is the difference between the wild-type and daf-2 mutant background which could lead to misunderstanding if the legend is not read carefully. I suggest adding a title to the plot “Difference in Median (daf-2mutant-wild-type)” or something similar.

Reviewer #3: This manuscript provides strong genetic evidence for the function of nmur-1 in regulating P-death, both in the presence and absence of daf-2. Overall, the study is well written, and I have no major concerns regarding the experimental design or data interpretation.

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Dear Dr Alcedo,

We are pleased to inform you that your manuscript entitled "The neuropeptide neuromedin U receptor NMUR-1 buffers insulin receptor signaling in bacteria-dependent C. elegans survival" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Fengwei Yu

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PLOS Genetics

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PLOS Genetics

Anne Goriely

Editor-in-Chief

PLOS Genetics

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Comments from the reviewers (if applicable):

Reviewer’s Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: I want to thank the authors for addressing the issues I raised (as well as those by the other reviewers). As with my previous recommendation, I think the paper should be accepted.

I have to say that I still find a lot of jargon in the paper very confusing. For example "wild type" is used as a genotype [eg daf-2(+)], that much is clear and is a very standard use. But in a lot of contexts it is also used to imply how the level of expression of a protein (or gene) in wild type animals under specific experimental conditions affects a phenotype [eg "Because wild-type DAF-28 increases P-deaths"]. This is confusing for several reasons, but especially because if the level of protein changes as part of the life of a wild type animal due to some sort of developmental or environmental or tissue-specific input, all of those levels are by definition "wild type". So many wild type levels are possible and normal. I would suggest that rather than trying to define what the right wild type level is, that the authors just refer to the level of protein or gene function that is occurring in the context they describe, relative to a context they define as a reference: for example none, low, medium/normal, high, very high.

Reviewer #2: The authors have addressed all substantive concerns in a thorough manner. The revisions have improved the clarity, rigor, and overall quality of the work, and I support its publication in PLOS Genetics.

Reviewer #3: Congratulations on the beautifully work! All concerns addressed!

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics  data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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