mTOR regulates longevity through a bile-acid like hormonal mechanism and DHS-26/DHRS1

Reviewer #1 (Public review):

This manuscript describes a novel downstream mechanism of mTORC1 deficiency-mediated lifespan extension in C. elegans. The authors demonstrated that the biosynthesis and the nuclear hormone receptor daf-12 binding of a bile acid-like hormone, dafachronic acid (DA), are essential for TORC1 mutant raga-1 to extend lifespan. Through RNA-seq and RNAi lifespan screen, they also discovered that a dehydrogenase, dhs-26, which is expressed in the canal-associated neurons, is regulated by DA/daf-12 and downstream of the mTORC1-DA signaling for lifespan extension. The authors also explored the conservation of mTOR/DA/daf-12/dhs-26 signaling in the mouse model. This work demonstrates significant findings that will advance the aging field and will be of interest to many researchers in this field. The conclusions are mostly well supported by data with proper controls.

Some suggestions to strengthen the manuscript include:

(1) Other mTOR activity perturbation or mutants should be used to support some of the core lifespan experiments. It will strengthen the conclusions made from raga-1 mutant only, although there is evidence from TOR RNAi in Figure 1g to support the daf-12 data in Figure 1d.

(2) The authors showed in Figure 1h and 1i that DA supplementation rescued the shortened lifespan of raga-1;daf-9 but not raga-1;daf-12; and also rescued the shortened lifespan of raga-1; dnh-26 in Fig. 5e. Does DA supplementation itself extend lifespan? If its level is increased by mTORC1 inhibition and it is downstream of mTORC1 inhibition, it should theoretically extend lifespan. But from the reported publications, it seems that the DA supplementation lifespan modulation is highly dependent on genetic backgrounds. It will strengthen the conclusions if the authors provide the wild-type condition DA supplementation lifespan data and also related discussions about it.

Reviewer #2 (Public review):

Summary

This manuscript by Schilling et al. presents an important advancement in our understanding of how mTOR signaling regulates organismal aging. While the longevity-promoting effects of reduced mTOR activity have been extensively documented across species, the mechanisms by which mTOR communicates systemic metabolic information to regulate lifespan remain unclear. In this study, the authors provide strong evidence that longevity induced by reduced TORC1 signaling requires the bile acid-like steroid hormone dafachronic acid (DA) and its cognate nuclear receptor DAF-12. Furthermore, through a combination of transcriptomics and functional genomics, they identify the conserved short-chain dehydrogenase DHS-26/DHRS1 as a previously unrecognized downstream effector of this pathway. The work integrates genetics, lifespan analyses, sterol measurements, transcriptomics, proteomics, endogenous genome engineering, and comparative mammalian datasets. The resulting model, in which mTOR influences lifespan through regulation of endocrine steroid signaling, represents a conceptual advance that links nutrient sensing, metabolism, and organismal aging. Although several mechanistic questions remain unresolved, the study is comprehensive, technically rigorous, and likely to be of broad interest to investigators studying aging, metabolism, endocrine signaling, and cellular stress responses.

Strengths:

One of the major strengths of this manuscript is its conceptual novelty. Rather than reinforcing the well-established role of mTOR as a longevity regulator, the study proposes a specific endocrine mechanism that links reduced mTOR activity to increased lifespan through steroid hormone signaling. This advances the field beyond descriptive observations of mTOR-dependent longevity and introduces a model in which bile acid-like hormones function as systemic mediators of nutrient-sensing pathways. The idea that endocrine steroid signaling may serve as a downstream effector of mTOR provides a new perspective on how longevity signals are coordinated at the organismal level.

The genetic evidence supporting this model is particularly strong. In Figure 1, the authors use a series of epistasis experiments to demonstrate that mutations in daf-36, daf-9, and daf-12 suppress lifespan extension in raga-1 mutants. The DA supplementation experiments further strengthen the pathway ordering by rescuing longevity in hormone-deficient backgrounds while failing to restore lifespan in receptor-deficient animals. Importantly, the direct quantification of endogenous DA levels elevates the study by providing biochemical support for the proposed model.

The transcriptomic analyses presented in Figure 2 provide a valuable systems-level perspective on the interaction between mTOR and steroid signaling pathways. The observation that DAF-12 profoundly reshapes the RAGA-1 transcriptional program highlights the importance of steroid signaling in mediating the physiological consequences of reduced mTOR activity. The enrichment of metabolic, lysosomal, and peroxisomal pathways is consistent with established longevity-associated programs and generates a valuable resource for future mechanistic studies.

Figure 3 effectively integrates discovery-driven and hypothesis-driven biology. The authors use transcriptomic information to prioritize candidate genes and then perform a functional genomic screen to identify factors required for RAGA-1-mediated lifespan extension. This approach converges on DHS-26, which subsequently emerges as a central mechanistic component of the study. The progression from transcriptomics to functional validation is well executed.

In Figure 4, the generation of CRISPR-engineered dhs-26 deletion mutants and endogenous tagged reporter strains provides strong validation for DHS-26 function. The demonstration that dhs-26 deletion selectively abolishes RAGA-1-dependent longevity without substantially affecting wild-type lifespan strongly supports its role as a context-dependent mediator of mTOR signaling. Furthermore, the conservation analyses linking DHS-26 to mammalian DHRS1 provide biological context and enhance the broader significance of the findings.

In Figure 5, multiple independent experimental approaches converge on the conclusion that DHS-26 participates in DA-dependent lifespan regulation. The rescue of lifespan by DA supplementation, reductions in DA levels in raga-1;dhs-26 mutants, reporter-based analyses of DAF-12 activity, and proteomic profiling collectively support a mechanistic model. The proposed positive feedback relationship between DA/DAF-12 signaling and DHS-26 is intriguing and offers a plausible explanation for how endocrine signaling may amplify longevity-promoting responses. Finally, the incorporation of mammalian datasets showing regulation of DHRS1 by rapamycin and FXR signaling provides a promising avenue for future studies investigating conservation of this pathway.

Weaknesses:

Despite the many strengths of the study, important mechanistic questions remain unresolved. The most significant limitation is that the precise molecular connection between reduced mTOR activity and increased DA production remains unclear. While the genetic and biochemical data convincingly place DA/DAF-12 signaling downstream of mTOR, the study does not establish whether mTOR regulates DA biosynthesis, degradation, intracellular trafficking, sterol uptake, or hormone availability. The observed increase in endogenous DA levels is statistically significant but relatively modest, and the mechanistic basis for this increase remains speculative. Additional experiments examining sterol flux, enzyme activity, or intracellular sterol trafficking would substantially strengthen the proposed model.

The transcriptomic analyses in Figure 2 are informative but correlative. Because the RNA-sequencing was performed at a single adult time point, it remains difficult to distinguish primary transcriptional responses from secondary adaptive changes. Similarly, while pathway enrichment analyses identify plausible processes, they do not establish direct regulatory relationships. Additional temporal analyses or direct assessment of DAF-12 occupancy at candidate loci would strengthen mechanistic interpretations and help distinguish direct from indirect targets.

A major unresolved question concerns the biochemical function of DHS-26 itself. While the genetic evidence clearly establishes DHS-26 as an important regulator of RAGA-1-mediated longevity, its endogenous substrate and enzymatic activity remain unknown. The manuscript presents evidence linking DHS-26 to sterol metabolism, but direct biochemical characterization is lacking. Thus, the mechanistic model remains somewhat incomplete. Defining the substrates and products of DHS-26 activity would greatly strengthen the study and provide important insight into how this enzyme influences DA availability.

Another area requiring additional clarification is the proposed neuroendocrine role of DHS-26. The expression of DHS-26 in canal-associated neurons is interesting and raises the possibility that these cells participate in systemic longevity regulation. However, the current data do not establish whether DHS-26 functions autonomously within these neurons or whether expression in other cell types contributes to the observed phenotypes. Tissue-specific rescue or depletion experiments would strengthen the neuroendocrine model and help establish physiological sites of action.

Finally, the mammalian data presented in Figure 5 are supportive and suggestive of evolutionary conservation, but they remain correlative. While regulation of DHRS1 expression by rapamycin and FXR signaling is interesting, these observations do not yet demonstrate functional conservation of the longevity mechanism itself. Additional studies directly testing DHRS1 function in mammalian systems will be required before stronger conclusions regarding conservation can be drawn.

In summary, this manuscript provides a significant contribution to the aging field and introduces a model linking mTOR signaling, endocrine steroid hormones, and longevity. The study is comprehensive, technically sophisticated, and supported by multiple complementary approaches. Although some mechanistic questions remain open regarding the precise regulation of DA production, the biochemical function of DHS-26, and the extent of conservation, these limitations represent opportunities for future investigation. Overall, the work substantially advances our understanding of how nutrient-sensing pathways regulate aging and is likely to stimulate considerable interest within the fields of aging biology, metabolism, and endocrine signaling.

Reviewer #3 (Public review):

Summary:

This interesting manuscript provides evidence that the well-established consequences of (reduced) mTOR activity on longevity are, at least in part, mediated by regulation of dafachronic acid (DA) availability and its signalling via its nuclear receptor DAF-12 in C.elegans, with some supporting evidence derived from mouse studies that similar processes may be functional in mammalian systems, i.e., be evolutionarily conserved. Earlier studies by the group have established that DA/DAF-12 signaling promotes adult longevity in several contexts. DA is a bile acid look-alike, and DAF-12 is a homolog of mammalian bile acid-activated nuclear receptors FXR and VDR: recent experimental studies and human cohort studies have indicated a role of (specific) bile acids in mammalian longevity.

The hypothesis that mTOR and DA/DAF-12 signaling interact to modulate longevity in C.elegans is novel and of great potential interest. The hypothesis has rigorously been tested in a series of well-performed experiments employing mutant strains, functional genomic screens, and DA exposures, etc.. It is convincingly demonstrated that DA/DAF-12 does not directly impact mTOR (assayed on AMPK phosphorylation) and acts downstream of the pathway. The short-chain hydrogenase DHS-26 (mammalian homologue DHRS1) was identified as a downstream target and modulator of this mTOR-DA-DAF12 axis by modulating the lifespan of the mTOR regulator raga-1. As the components of this axis are expressed in different cell types of the worms, this finding indicates a neuroendocrine mode of action. Mode of action of DHS-26 appears to be based on modulation of cholesterol and lathosterol, i.e., substrate availability for DA production.

Strengths:

Overall, the manuscript is well-written and builds up the story in a clear fashion. The conclusions are based on solid data and of relevance for ageing research, also because the mechanism identified appears to be evolutionary conserved.

Weaknesses:

No overt weaknesses were identified by this reviewer.