PREreview del Dietary restriction promotes neuronal resilience via ADIOL

Reviewer report

Dietary restriction promotes neuronal resilience via ADIOL

Ana Guijarro-Hernández¹, Shinja Yoo¹, George A. Lemieux¹, Sena Komatsu¹, Abdullah Q. Latiff¹, Rishika R. Patil¹, and Kaveh Ashrafi¹

¹ Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA

Summary

This study identifies a physiological role of the steroid hormone ADIOL in mediating the beneficial effects of dietary restriction. Fasting and caloric restriction upregulate enzymes involved in ADIOL biosynthesis, and elevated ADIOL acts in the nervous system to lower kynurenic acid levels via NHR-91 signaling specifically in the RIM neuron. Enhancing ADIOL signaling improves several healthspan markers during aging, whereas loss of ADIOL signaling reduces healthspan even under normal or caloric-restriction–mimetic conditions. These effects occur without altering lifespan, indicating that ADIOL selectively enhances healthspan. Overall, the work positions ADIOL as a key mediator of the neuroprotective and pro-healthspan effects of dietary restriction and a potential therapeutic route for age-related neurodegeneration.

The authors previously demonstrated that ADIOL promotes learning by reducing kynurenic acid levels (DOI: 10.1101/gad.350745.123). Here, they extend these findings to dietary restriction-mediated benefits. Given that ADIOL is conserved between worms and humans, and that dietary restriction exerts beneficial effects across species, the findings may have broader relevance to human health. While the manuscript presents interesting observations, such as ADIOL pathway-dependent increase in pumping rate upon fasting, additional experiments and clarification are required to fully support the authors’ claims.

Major comments

1. On page 6 (3rd and 4th paragraph), the authors interpret the suppressed fasting-induced pumping increase in cyp-44A1 (CYP11A1), cyp-13A4 (CYP17A1), and F12E12.11 (17β-HSD) mutants as evidence that fasting enhances ADIOL production through upregulation of these genes. However, in Fig. 2b, cyp-13A4 expression is decreased upon fasting, F12E12.11 shows no significant change, and cyp-44A1 is only marginally upregulated. These data do not convincingly support their proposed model that fasting enhances ADIOL production through upregulation of these genes. To address this discrepancy, it might be helpful for the authors consider examining the expression or functional requirement of other ADIOL biosynthetic genes that are clearly upregulated by fasting.

2. On page 8 (4th and 5th paragraphs), the authors conclude that ADIOL promotes healthspan based on enhanced pumping and thrashing during aging. However, ADIOL treatment also increases these activities in Day 1 adults, which are already at their peak physiological state in Fig 3b-e. The magnitude of improvement appears comparable between Day 1 and Day 5 animals. This raises the possibility that ADIOL broadly enhances neuromuscular performance independent of aging or healthspan, and it might not support their claim that ADIOL promotes healthspan. It would be helpful for the authors to clarify whether the effects are aging-specific or reflect general neuromuscular stimulation in the text.

3. The title states that dietary restriction promotes neuronal resilience via ADIOL, yet the neuronal evidence that supports the central claim in the title is limited to the data that nhr-91 expression in RIM neurons rescues the reduced pumping rate in Fig 1c and 4c. The study does not test ADIOL’s effects on learning or neuronal resilience per se. Although mxl-3 and daf-2 mutants were tested for learning, these genes influence numerous pathways beyond dietary restriction, making the interpretation less specific. Moreover, pumping, thrashing, and locomotion can reflect muscular rather than neuronal effects. A more specific title reflecting the presented data may be considered (e.g., "The ADIOL-kynurenic acid pathway mediates beneficial effects of dietary restriction.")

Minor comments

1. In Graphical Abstract, the arrows from CHOL to ADIOL are confusing, as PREG and DHEA might be seen as both receiving inputs and providing outputs. To improve interpretability, the authors can modify the curved arrows to direct ones between the molecules.

2. In Abstract, it might be necessary to stress that the effects of kynurenic acid are known in humans to clarify what is known in C. elegans vs humans. The sentence "kynurenic acid, a neuroactive metabolite linked to cognitive decline and neurodegeneration." could be "kynurenic acid, a neuroactive metabolite linked to cognitive decline and neurodegeneration in humans."

3. In Abstract (last sentence: "This positions ADIOL as a promising mimetic of dietary restriction effects on healthspan that could be used as a therapeutic strategy for age-related neurodegenerative conditions."), steroid hormones can have both positive and negative effects. Thus, it would be more informative if the authors discuss if there are any negative consequences of elevating ADIOL in worms in the Results or Discussion section.

4. F12E12.11 has a gene name of nta-1. Please consider using the updated name of the gene to improve reader understanding.

5. In Fig 1c, cex-1p::nhr-91 was used to express nhr-91 in RIM specifically. However, given the reported nonspecific posterior intestinal expression associated with unc-54 3′UTR used in their constructs (Thomas Boulin, John F. Etchberger, and Oliver Hobert, 2006, WormBook), it would strengthen the author's conclusion that RIM neurons mediate the effects of ADIOL if they directly demonstrated that cex-1p::nhr-91 expression is restricted to RIM and not present in other cells.

6. In Fig 1c and Fig 4c, Different nhr-91 constructs were used for expressing nhr-91 in RIM (cex-1p::nhr-91) and RIC (tbh-1p::nhr-91cDNA::SL2::GFP) (Supplementary Table 1). This difference is not discussed, and the functionality of the nhr-91cDNA::sl2::GFP transgene is not demonstrated. It remains possible that a nonfunctional transgene in RIC fails to rescue the phenotype. It would be more supportive if the authors demonstrate the transgene functionality with an additional experiment. If the nhr-91cDNA::sl2::GFP transgene is functional, the rescue experiment would further support that nhr-91 does not act in RIC.

7. In page 5 (1st paragraph), the authors refer to mxl-3 mutants as a caloric restriction model because starvation represses mxl-3 expression. However, starvation and caloric restriction are distinct interventions, and physiological responses are different depending on caloric restriction between starvation (e.g. starvation can induce developmental arrest as a stress response. To improve reader understanding, the authors should consider clarifying this distinction in this paragraph.

8. For clarity, it would be helpful if the authors could define several acronyms that are used throughout the text, including HSD, F17, and KAT.

9. In page 5, A brief explanation of the behavioral learning assay used would improve clarity, as multiple learning paradigms exist.

10. In Fig 2b, consider adding a legend explaining what the numerical values represent.

11. In Fig 2c, the use of 2–dCt rather than fold change, which is used in Fig 2d,e. It might be helpful to explain why the authors used different analysis methods for clarity.

12. In page 7 (2nd paragraph) and Extended Fig 2a, the Day 1 pumping rates are different for the three genotypes, and this result is not consistent with the data in Fig 1a-c. The authors might need to explain this discrepancy for clarification.

13. On page 8 (1st paragraph), the authors did not explain what FUDR is or why assays were conducted without FUDR to better assess age-related effects. Please consider explaining the rationale of using FUDR in the experiments to improve reader understanding.

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.