PREreview of Dietary restriction promotes neuronal resilience via ADIOL

 In this manuscript, the authors propose that the pro-healthspan effects of dietary restriction (DR) are mediated by the steroid hormone ADIOL. They conclude that in the nematode C. elegans, DR induces intestinal ADIOL production, which then acts neuronally to promote healthspan. Importantly, the authors make a distinction between healthspan and lifespan; ADIOL improves only health measures but does not alter the longevity of C. elegans. They additionally suggest that ADIOL acts mechanistically through a downstream kynurenic acid (KynA) signal to mediate these pro-healthspan effects and that some pro-healthspan effects of dietary restriction (DR) can be attributed to this ADIOL pathway.

The effects of ADIOL on healthspan are convincing, and there is strong evidence presented that this effect is indeed decoupled from lifespan: ADIOL enhances multiple measures of fitness without extending lifespan and additionally does not seem to be the molecule responsible for longevity effects in long-lived mutants. The paper would be improved if the authors could clarify and/or further substantiate three of their main claims, as explained below: 1. KynA is the downstream effector of ADIOL.  2. ADIOL is a neuroprotective/anti-aging signal. 3. DR induces upregulation of ADIOL signaling. Clarifying these issues would provide a more complete and more precise mechanistic understanding of the entire pathway behind DR’s leading to enhanced healthspan, which would be a more impactful statement of the paper.

Major points:

1. A central explicit statement in the paper is that KynA is the (neuronal) effector metabolite that ADIOL induces to mediate its healthspan effects (Abstract/line 6, Page 4/line 4, Page 10/line 31, and the text/figure titles of Fig 3 and 4). The study utilizes mutants from a previous publication of the authors (Lemieux,.. Ashrafi, 2023, PMID: 38092521) defective in key genes that act directly in the KynA pathway, kmo-1, nkat-1 and aat-1 (Figure 4d-e). It would greatly strengthen the authors’ claims about KynA’s being downstream of ADIOL if they were to include those mutants in more of their analyses / epistasis analyses. In particular, extending their inclusion of kmo-1 and nkat-1 mutants to the aging healthspan assays of learning and pharyngeal pumping in Fig. 3a-b and testing double mutants of nkat-1 and nhr-91 in healthspan assays would clarify the relative contribution of ADIOL on the KynA pathway in an aging context and distinguish the findings described in this manuscript from their KynA investigations reported in earlier papers. This issue is especially pertinent as the authors state in the discussion that “KMO-1 activity may influence KynA production independently of ADIOL”, indicating that it is possible for KynA to not be directly downstream of or influenced by ADIOL.

2. The authors use the term “neuronal resilience” (in the title) and “neuroprotective” (in the abstract) to describe ADIOL’s effects on C. elegans physiology. Such terms seem to imply that ADIOL protects neurons against a decline of the functional measures of learning, pharyngeal pumping and motility.

i) Though nhr-91 mutants show an earlier decline in healthspan as measured by the learning assay (Fig. 3a), all other aging assays and experiments (Fig. 3-4) demonstrate that ADIOL addition alone at day 1 already induces enhanced measures of pro-healthspan attributes. The authors should consider more precisely defining/characterizing the effects of ADIOL; it does not just prevent or protect against age-related decline but also actively raises the baseline measures of healthspan. One way to disentangle these issues would be to test earlier-aged worms, for example at the L4 stage, to determine if there is an age at which ADIOL does not simply increase the healthspan baseline.

ii) Relatedly, from a loss of function direction for ADIOL, how do nhr-131 mutants perform in the learning assay in Fig. 3a at earlier stages, such as L4? Are they healthspan defective in general or do they have an accelerated aging-related decline that happens earlier than day 1?

iii) As the authors were also able to measure ADIOL and KynA levels in previous publications (e.g. Ref 9), direct measurements of ADIOL and KynA levels across aging timepoints should be possible and also would help bolster a direct connection of ADIOL to aging.

iv) Furthermore, all these measures are a function of both neuronal and muscular contributions; ADIOL has a likely intestinal origin and thus likely non-cell autonomous effects, and there is insufficient evidence to rule out an effect of muscle contribution here (see also minor point 1 about ADIOL’s site of action).

v) If the authors were willing to rephrase, they could consider a straightforward term to use: “pro/enhanced healthspan” instead of “resilience” or “protective”.

3. Fig. 2 shows expression data of ADIOL synthesis genes to support the claim that DR activates ADIOL signaling. Though there is a general trend across the aggregated data that supports the stated conclusions, some of these observations do not follow the general trend and are not as clear cut. In particular, the model in Fig. 2f attributes the effects of 2h-fast, and daf-2 and mxl-3 mutations to particular enzymatic reactions, but the data do not follow as cleanly. For example, if the enzymatic reactions are linearly arranged as shown, the nhr-131 mutation would be expected to show strong effects that block upstream processing of ADIOL; accordingly, we would not see an increase of cyp-33C5 expression in the nhr-131 background in Fig. 2b-c in the 2h fast condition. The authors do recognize that the results are not clear cut with phrases such as “some cases (Page 6 line 16)” and “partial requirements likely reflect enzymatic redundancy” (Page 6 line 23). However, given the claim as written in the title of the section is: “ADIOL levels increase under fasting and CR conditions through the upregulation of CYP11A1, CYP17A1 and 17-βHSDs orthologs (Page 5 line 25)”, the authors either should consider strengthening their claim by direct measurements of ADIOL (and KynA) in DR conditions or rephrasing the wording they use in their section title etc..

Minor points:

1. The authors specify that the effects of ADIOL-enhanced healthspan are neuronally mediated. I understand that their previous work has shown neuronal contributions to the effects of KynA on healthspan (Lemieux,.. Ashrafi, 2023). However, it would help readers of this paper if such prior evidence were clearly and precisely spelt out in the appropriate areas; in particular, I refer to the expression patterns of ADIOL-related genes and gene promoters used for rescue experiments. Both the cex-1 and tbh-1-based promoters that are used to show RIM and RIC expression have non-neural expression, so it is important to be precise about what is demonstrated and what is proposed about ADIOL’s site of action and about the difference between necessity and sufficiency in wording. In particular, the two pieces of data used to show a RIM-specific site of action for ADIOL in aging provide relatively weak evidence: (1) in Fig.3d the magnitude of rescue of nhr-91 in RIM for pharyngeal pumping is not particularly striking, and (2) in Fig. 4c the baseline rate of thrashing is already higher in nhr-91 mutants at day 5 compared to wild-type and is in fact an opposite result to what is expected of an ADIOL-insensitive phenotype. More context about ADIOL-gene expression data would also be helpful, i.e. where are nhr-91 and nhr-131 expressed?

2.     Given the major point about ADIOL’s ability to enhance baseline measures of healthspan, in Fig, 1b, it seems relevant to assess what ADIOL addition does to well-fed WT worms. That would be informative, as the combination of exogenous ADIOL and 2h fast might already be hitting a ceiling effect.

3.     The mxl-3 mutant has a striking learning assay phenotype, but its enhanced lifespan phenotype seems really weak. In fact, it seems that the median survival of the mxl-3 mutant (Fig 5e) here is much lower compared to reported values. (17 days (Fig 5e) vs ~28 days (Ref 43)). It would be helpful for the authors to clarify any potential reasons for their thoughts about this discrepancy.

4.     In the molecular pathways depicted in the graphical abstract and figures, molecules like hormones (ADIOL) are not distinguished stylistically from proteins like NHR-31 and NHR-131. Furthermore, the same colors are used for both PREG and NHR-91 as well as CHOL and KynA, even though these are different molecules acting in different parts of the pathway. The authors might want to consider marking the differences in these pathway nodes by changing styles and colors to enhance readability.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.