Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans

  1. Lingxiu Xu
  2. Chengxuan Han
  3. Lei Chun
  4. XZ Shawn Xu
  5. Jianfeng Liu

  • Curated by eLife

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    eLife Assessment

    This is an important study that addresses the temporal aspects of cell non-autonomous regulation of lifespan. It demonstrates that the same neurons and neurotransmitter have distinct impacts on longevity at different ages. The data convincingly supports the authors' claims.

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Abstract

The nervous system modulates aging by secreting signaling molecules to cell-nonautonomously regulate the physiological state of distal tissues such as the gut. However, the underlying mechanisms are not well understood. Here, using C. elegans as a model, we identified two distinct neuroendocrine signaling circuits through which motor neurons signal the gut in early life to shorten lifespan but in mid-late life to extend lifespan. Both circuits employ the same neurotransmitter acetylcholine (ACh), while recruiting two different gut ACh receptors ACR-6 and GAR-3 to regulate the transcription factor DAF-16 and HSF-1 in early and mid-late life, respectively. Strikingly, the gut expression of ACR-6 is restricted to early life, whereas that of GAR-3 is confined to mid-late life, providing a potential mechanism for the temporal control of the two circuits. These results identify a novel mechanism that empowers the nervous system to bidirectionally regulate longevity by differentially signaling the gut at different life stages.

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  1. eLife

    eLife Assessment

    This is an important study that addresses the temporal aspects of cell non-autonomous regulation of lifespan. It demonstrates that the same neurons and neurotransmitter have distinct impacts on longevity at different ages. The data convincingly supports the authors' claims.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    This manuscript addresses the temporal patterns in how cholinergic signaling to the gut affects the lifespan of the worm C. elegans, which should make the manuscript of wide interest to those who study aging, as well as those who study the brain-gut axis in health and disease. The authors show that early acetylcholine (ACh) signaling to the intestine via the ACR-6 receptor shortens worm lifespan, which depends on the DAF-16/FOXO transcription factor. However, later ACh signaling to the intestine via the GAR-3 receptor extends lifespan, which in turn depends on the heat shock factor HSF-1. The authors also show a potential mechanism through which these two temporal patterns of ACh signaling might be coordinated to influence longevity in the worm, and possibly in other animals.

    Strengths:

    The authors observed that the functional ablation of acr-2-expressing cholinergic neurons in C. elegans (Pacr-2::TeTx) produced a lifespan curve that intersects the lifespan curve of a wild-type population. The first quartile of Pacr-2::TeTx worms shows a longer lifespan than the first quartile of wild-type worms, whereas the last quartile of Pacr-2::TeTx worms exhibits a shorter lifespan than wild type. These observations raised the hypothesis that cholinergic neurons have two opposing effects on longevity: an early longevity-inhibiting effect and a later longevity-promoting effect. Much of the data support the authors' conclusions.

    The authors have also addressed the points raised in the previous review.

  3. eLife

    Reviewer #3 (Public review):

    I very much enjoyed reading Lingxiu Xu et al.'s paper "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans," where they investigate the mechanisms by which motor neurons regulate lifespan in C. elegans worms. In this paper, they first discover that interfering with synaptic release in cholinergic motor neurons affects lifespan. Using mutants and gene knockdowns they show that these effects are due to the neurotransmitter acetylcholine. They show that the effects these motor neurons on lifespan are opposite, depending on timed genetic interventions promoting synaptic release. If these interventions occur during development, lifespan is shortened, but if they occur starting on day 7 of adulthood, then lifespan is lengthened. They then show that the transcription factor daf-16 is required for the former effect, while the transcription factor hsf-1 is required for the latter one. In addition, these early and late effects, they find, required the acetylcholine receptors acr-6 and gar-3, respectively, and intestinal expression of these genes rescues the respective phenotypes. Interestingly, tagging the endogenous acr-6 and gar-3 genes with mCherry, they find that the ACR-6 and GAR-3 proteins are expressed in the intestine, ACR-6 during development and GAR-3 during adulthood. Based on these findings they propose a model where acetylcholine from motor neurons regulates lifespan by modulating different receptors expressed at different times. These receptors, in turn, affect lifespan in opposing ways via different transcription factors.

    Comments on revisions:

    I am grateful to the authors for their effort to address my comments and suggestions, and for the thoughtful discussion of their efforts to strengthen the claims supporting their model.

  4. eLife

    Reviewer #4 (Public review):

    This is a very interesting study, where the authors discovered two neuroendocrine signaling circuits with opposite effects on organismal longevity elicited by motor neurons at different ages.

    Interestingly, both systems employ the same neurotransmitter (that is, acetylcholine) and signal the intestine. However, one has effects on early life to shorten lifespan whereas the other system is activated in mid-life to extend lifespan. At the mechanistic level, this bidirectional regulation is possible through the recruitment of two different ACh receptors in the gut: ACR-6 and GAR-3. The authors found that ACR-6 expression in the intestine is restricted to early life, whereas GAR-3 expression in the gut is confined to mid-late life. Interestingly, ACR-6 modulates the transcription factor DAF-16, but GAR-3 regulates HSF-1.

    The study combines different approaches, including inducible systems (AID) which are critical for the conclusions of the paper. The conclusions are well supported by the experiments and results. The data provide a potential mechanism for the temporal control of lifespan and shed light on the complex role of the nervous system in organismal aging. These results can have important implications to understand how organismal aging is regulated in a temporal manner by cell non-autonomous mechanisms.

    The paper has significantly improved after addressing all the Reviewers' comments and I did not observe significant weaknesses in the study.

  5. eLife

    Author Response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public Review):

    While the authors have proved their hypothesis by temporally increasing the activity of cholinergic neurons at different life stages through the auxin-inducible degron system, their work raises two major concerns. First, they might want to discuss the conflicting data from Zullo et al (Nature 2019, vol 574, pp 359-364). For example, the authors show that increasing the activity of acr-2-expressing neurons after the 7th day of adulthood increases lifespan. However, Zullo et al (2019) show that the reciprocal experiment, inhibiting cholinergic neuron activity on the 1st day or the 8th day of adulthood, also increases lifespan. Is this because the two studies are using different promoters, that of the acr-2 ACh receptor (this work) versus that of the unc-17 vesicular ACh transporter (Zullo et al., 2019)? The two genes are expressed in different subsets of cells that do not completely overlap. CeNGEN shows that acr-2 is expressed in motor and non-motor neurons, but some of these neurons are also different from those that express unc-17. Is it possible that different cholinergic neurons also have opposite lifespan effects during adulthood? Or is it because both lack of signaling and hypersignaling can lead to a long-life phenotype? Leinwand et al (eLife 2015, vol 4, e10181) previously suggested that disturbing the balance in neurotransmission alone can extend lifespan. A simple discussion of these possibilities in the Discussion section is likely sufficient. Or can the auxin treatment and removal be confounding factors? Loose and Ghazi (Biol Open 2021, vol 10, bio058703) show that auxin IAA alone can affect lifespan and that this effect can depend on the time the animal is exposed to the auxin.

    We thank the reviewer for the thoughtful comments and valuable suggestions. In response, we have expanded the Discussion section to address the points raised, as detailed below.

    We fully agree with the reviewer that the different results between our study (activating acr-2-expressing neurons) and Zullo et al. (inhibiting unc-17- expressing neurons) are most likely due to the distinct cholinergic neurons targeted. Our new preliminary data further support this neuron-specific model, as inhibition of acetylcholine synthesis at mid-late life stages produces opposing lifespan effects in different cholinergic neurons. At the same time, we cannot rule out the alternative possibility raised by the reviewer (eLife, 2015) that both activation and inhibition of neuronal activity may extend lifespan by similarly disrupting the balance of neurotransmission. This hypothesis requires further experimental validation in the context of cholinergic motor neurons. Regarding the potential technical concern related to auxin exposure (Biol Open, 2021), our control experiments using 0.5 mM auxin did not show non-specific lifespan effects.

    Accordingly, in the revised manuscript, we have discussed the first two possibilities in the Discussion by stating (page 17-18): “Nevertheless, it is still unclear whether other neuronal populations share similar temporal regulatory mechanisms. A previous study reported that inhibiting cholinergic neurons activity (using unc-17 promoter) extends lifespan regardless of timing[2], which is different from the temporal lifespan regulation we observed in cholinergic motor neurons (using acr-2 promoter). This discrepancy is likely due to differences in subsets of neurons, as the unc-17 promoter labels a broad repertoire of cholinergic neurons, while the acr-2 promoter mainly marks cholinergic motor neurons[53]. Thus, the distinct lifespan-modulating effects of cholinergic motor neurons may be overshadowed by opposing contributions from other cholinergic subtypes when a mixed population is manipulated. Alternatively, both activation and inhibition of cholinergic activity may perturb neurotransmission balance, leading to similar effects on lifespan[54]. It will be interesting to test these hypotheses in future studies.”

    Second, the daf-16-dependence of the early longevity-inhibiting effect of ACh signaling needs clarification and further experimentation. The authors present a model in Figure 6D, where DAF-16 inhibits longevity. This contradicts published literature. Libina et al (Cell 2003, vol 115, pp 489-502) have shown that intestinal DAF-16 increases lifespan. From the authors' data, it is possible that ACh signaling inhibits DAF-16, not promotes it as they have drawn in Figure 6D.

    We thank the reviewer for this important point. We agree that intestinal DAF-16 promotes longevity. Our original model Figure 6D aimed to show that the larval pathway shortens lifespan by inhibiting DAF-16, not that DAF-16 itself shortens lifespan. The arrowhead style used in the original Fiugure 6D might have given an impression that DAF-16 shortens lifespan. Our apologies. We have now fixed this error in Figure 6D. In addition, as suggested, we have performed additional daf-16 experiments (see below).

    In Figure 3F, the authors used Pacr-2::TeTx, which inhibits cholinergic neuron activity, to show an increase in the expression of DAF-16 targets. Why did the authors not use the worms that express the transgene Pacr-2::syntaxin(T254I), which increases cholinergic neuron activity? What happens to the expression of DAF-16 targets in these animals? Do their expression go down? What happens if intestinal daf-16 is knocked down in animals with increased cholinergic neuron activity, instead of reduced cholinergic neuron activity?”

    Thanks for these insightful questions. In Figure 3F-H, we used TeTx instead of syntaxin(T254I) to investigate the function of DAF-16 in the early stage pathway based on the two main reasons. First, Pacr-2::TeTx transgene extends lifespan in early life by inhibiting cholinergic activity, which provides a genetic background complementary to that of syntaxin(T254I) for characterizing the role of DAF-16. Second, TeTx pathway is expected to activate DAF-16 and upregulate its target genes. This approach is more sensitive than measuring gene downregulation in Pacr-2::syntaxin(T254I) transgenic worms.

    We fully agree with the reviewer that performing the corresponding experiments in the syntaxin(T254I) background would strengthen the overall evidence. As suggested, we have now examined the expression of DAF-16 target genes in Pacr-2::syntaxin(T254I) transgenic worms, and performed intestine-specific RNAi of daf-16 in the same background. We found that these worms exhibit downregulation of DAF-16 target genes. Furthermore, intestinal daf-16 knockdown did not further shorten the already reduced lifespan of these transgenic worms. Together, these results from both the TeTx and syntaxin(T254I) lines confirms that cholinergic motor neurons require DAF-16 in the intestine to regulate lifespan. These new data has now been described in Figure S5A-5D (page 11-12): “As expected, the expression level of sod-3 and mtl-1, two commonly characterized DAF-16 target genes, was upregulated in transgenic worms deficient in releasing ACh from cholinergic motor neurons (Figure 3F), and downregulated in transgenic worms with enhanced ACh release from cholinergic motor neurons (Figure S5A), consistent with the notion that DAF-16 acts downstream of cholinergic motor neurons.”, and “RNAi of daf-16 in the intestine abolished the ability of cholinergic motor neurons to regulate lifespan at early life stage (Figure 3G, 3H and Figure S5C-S5E).”

    Recommendations for The Authors:

    Reviewer #1 (Recommendations for The Authors):

    (1) “The Methods section needs to be clarified/expanded.”

    (a) “For example, are the authors using indole-3-acetic acid or a synthetic auxin? How long does it take for syntaxin to be made after the removal of the auxin?”

    We have now included auxin information and recovery time in the Method for auxin treatment by stating (page 24): “natural auxin indole-3-acetic acid (G&K Scientific)”, and “Expression of syntaxin(T254I) can be suppressed by auxin treatment and restored in 24 hours following auxin removal.”

    (b) “How much FUDR was used in some of the lifespan assays?”

    2 μg/mL FUDR was used in some of the lifespan assays. We have now included the concentration in the Method for lifespan assay by stating (page 23 line 526): “2 μg/mL 5-Fluoro-2’-deoxyuridine (FUDR) was included in assays involving TeTx transgene worms, unc-31 and unc-17 mutant worms, which show a defect in egg laying.”

    (c) “In line 494 of the Methods section, worms were anesthetized with 50 mM sodium azide. That concentration seems a bit high.”

    It is an error indeed. We used 5 mM NaN3. This has now been fixed in the text and in line 548.

    (d) “What are the concentrations of the transgenes used in the extrachromosomal arrays?”

    We have now included the concentrations in the Method for strains and genetics by stating (line 507-509 on page 22): “Microinjections were performed using standard protocols. Each plasmid DNA listed above in the transgenic line was injected at a concentration of 50 ng/μL. Each marker for RNAi was co-injected at a concentration of 25 ng/μL.”

    (2) “Gene expression can vary in different parts of the worm intestine. Do the measurements in Figure 6C represent the entire intestine or only certain parts of the intestine?”

    We have now included the intestine area used for quantification in the Method for microscopy by stating (page 24): “and the entire intestine area was selected by ImageJ”, and in the legends of Figure 6C by stating (page 36): “The entire intestinal area was selected for measurement.”

    (3) “In Figure S1C, does tph-1 have a slight effect? Might serotonin partly counteract the effects of ACh?”

    We thank the reviewer for raising this interesting point regarding the potential role of serotonin. We have re-examined our data in Figure S2C (the original Figure S1C) and agree that loss of tph-1 partly counteracted the lifespan-shortening effect of Pacr-2::syntaxin(T254I) transgene in early life stage, thought the whole-life suppression effect is slight. To assess whether the acr-2 promoter-driven manipulation might directly affect serotonergic neurons, we checked the CeNGen. We found that the transcript expression of acr-2 can be detected in serotonergic neurons (ADF, HSN, and NSM), but the levels are extremely low. In this regard, it is unlikely that the Pacr-2::syntaxin(T254I) transgene exerts its primary effect by substantially altering serotonin release. While a potential indirect interaction between cholinergic and serotonergic signaling in lifespan regulation remains, it falls beyond the primary focus of the current study. We would like to follow up this in future studies. We have now pointed this out in the text by stating (page 9):“As a control, we also tested mutants deficient in other types of small neurotransmitters, including glutamate (eat-4), GABA (unc-25), serotonin (tph-1), dopamine (cat-2), tyramine (tdc-1), and octopamine (tbh-1), but detected no effect, with the exception of tph-1, which showed a modest, partial suppression of the phenotype (Figure S2A-S2F). This observation suggests that the lifespan effects of cholinergic signaling can be modulated by serotonin.”

    (4) “Where else is GAR-2 expressed? Might there be redundancies between neuronal and intestinal GAR-2?”

    We appreciate this insightful question. Based on available single-cell gene expression atlases of C. elegans at both embryonic and adult stages[1,2], gar-2 expression has been detected not only in neurons and the intestine, but also in additional tissues such as the muscle. Regarding the observed lack of effects upon neuronal or intestinal gar-2 RNAi on the ability of cholinergic motor neurons to extend lifespan in mid-late life, and also suggested by another reviewer, we performed muscle-specific RNAi experiments. Together with our previously presented data, the results show that intestinal (but not neuronal or muscle) RNAi of gar-3 abolished the ability of cholinergic motor neurons to extend lifespan at mid-late life stages, while muscle-specific (but not neuronal or intestinal) RNAi of gar-2 suppresses this effect. This finding indicates that GAR-3 and GAR-2 mediate cholinergic signaling in distinct peripheral tissues, with GAR-3 primarily in the intestine and GAR-2 primarily in muscle, to produce their effects on longevity. Given our focus on neuron-gut signaling, the role of GAR-2 in the muscle will be further investigated in future studies. The new data have now been described in Figure S8 by stating (page 13-14): “RNAi of gar-2 in the intestine (Figure 4D and 4E), but not in neurons or the muscle (Figure 4D-4F, and Figure S8A, S8D-S8E), abolished the ability of cholinergic motor neurons to extend lifespan at mid-late life stage. Thus, GAR-3 may function in the intestine to regulate lifespan. Surprisingly, RNAi of gar-2 in the muscle (Figure S8A-S8C), but not in neurons or the intestine (Figure S7F-S7H) had an effect on the ability of cholinergic motor neurons to extend lifespan in mid-late life, indicating that GAR-2 acts in the muscle to regulate lifespan.”

    (1) Packer, J. S. et al. A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution. Science 365, doi:10.1126/science.aax1971 (2019).

    (2) Roux, A. E. et al. Individual cell types in C. elegans age differently and activate distinct cell-protective responses. Cell Rep 42, 112902, doi:10.1016/j.celrep.2023.112902 (2023).

    (3) Chun, L. et al. Metabotropic GABA signalling modulates longevity in C. elegans. Nat Commun 6, 8828, doi:10.1038/ncomms9828 (2015).

    (4) Izquierdo, P. G. et al. Cholinergic signaling at the body wall neuromuscular junction distally inhibits feeding behavior in Caenorhabditis elegans. J Biol Chem 298, 101466, doi:10.1016/j.jbc.2021.101466 (2022).

    (5) “In line 344, please correct "fwork" to "work".”

    This has now been fixed.

    (6) “In line 360, please correct "acts" to "act".”

    This has now been fixed.

    (7) “Please check citations within the main text. Some of the citations do not fit the cited material. For example, in line 112, reference 28 is not about GABAergic neurons.”

    We thank the reviewer for pointing out these important details. We have now carefully checked and corrected the citations throughout the manuscript as suggested.

    Reviewer #2 (Recommendations for The Authors):

    (1) “How are the authors assessing the efficacy of the TeTx manipulations in their strains? Likely TeTx has a concentration-dependent effect. Are there any phenotypes associated with the loss of cholinergic signaling? Also, does TeTx expression in cholinergic neurons alter the neuronal activity of other associated neurons, or alter muscle integrity?”

    Thanks for the question. Our observations show that overexpression of TeTx results in defects including small size, slow growth, egg-laying deficiencies, and severe locomotion impairment, which are all associated with the loss of cholinergic signaling. While we did not directly examine the activity of interconnected neurons in our strains, we tested the muscle integrity by recording muscle reaction to 1 mM levamisole and found that overexpression of TeTx does not affect muscle integrity. To circumvent these pleiotropic complications, we instead employed Syntaxin(T254I) transgenic worms, which exhibits only slight locomotion defects, to further characterize the temporal effect of cholinergic motor neurons on lifespan. This data has now been described in Figure S1A by stating (page 6): “Overexpression of TeTx induces characteristic phenotypes of cholinergic deficiency, such as developmental delay and severe locomotion impairment[32], yet does not compromise muscle function (Figure S1A).”

    (2) “The authors are expressing TeTx throughout the lifespan of the animal, including during development. How does this contribute to the organismal phenotype?”

    As described above, chronic TeTx expression from egg stage results in developmental delay, which is similar to the development phenotype of unc-17 mutant worms defective in acetylcholine transmission. However, unc-17 mutation has no effect on lifespan[3], which is different from TeTx overexpression, indicating that the developmental delay caused by TeTx overexpression may not affect the lifespan phenotype.

    (3) Chun, L. et al. Metabotropic GABA signalling modulates longevity in C. elegans. Nat Commun 6, 8828, doi:10.1038/ncomms9828 (2015).

    (3) “A previous study has shown that increasing cholinergic activity by altering ACR-2 expression can cause neurodegeneration (DOI: https://doi.org/10.1523/JNEUROSCI.1515-10.2010). Does overexpressing syntaxin, or AID-mediated degradation of syntaxin cause motor neuron degeneration, which could also contribute to the lifespan phenotype?”

    We thank the reviewer for raising this important point regarding potential motor neuron degeneration. In response, we performed confocal microscopy to assess the motor neurons. We found that worms expressing the transgene Pacr-2::syntaxin::mCherry do not exhibit a defect in the number or morphology of labeled neuronal cell bodies compared to control worms expressing Pacr-2::mCherry. This observation indicates that chronic, increased cholinergic activity through syntaxin overexpression, under our experimental conditions, does not induce motor neuron degeneration. This data has now been described in Figure S1B by stating (page 7): “This transgene simply shortened lifespan without causing a pleotropic effect (Figure 1B), and critically, without inducing motor neuron degeneration (Figure S1B).”

    (4) “Figures 1I-1L: The authors do not show how long it takes for the expression of syntaxin to be restored following the removal of auxin from plates. This would be important to assess the age-dependent effects of neuronal signaling.”

    We thank the reviewer for pointing this out. In general, complete restoration of syntaxin expression occurred within 24 hours after auxin withdrawal. We have now pointed this out in the text by stating (the last sentence on page 24):“Expression of syntaxin(T254I) can be suppressed by auxin treatment and restored in 24 hours following auxin removal.”

    (5) “In Figures S1A-E: Although the mutant backgrounds decrease the lifespan of animals expressing the Pacr2::syntaxin(T254I) transgene, the lifespan of these transgenic animals appears to be extended compared to what was shown in Figure 1B. Is this the case? (can these experiments be repeated alongside wild-type N2s to assess if their lifespan is indeed extended compared to the N2?). Also, if so, could it be that the lifespan effects are modified to different extents by other small neurotransmitters?”

    We thank the reviewer for pointing this out. All the experiments presented in current Figure S2 (original Figure S1) were performed with wild-type N2 controls, which are now included in the updated Figure S2. This data shows that, in the Pacr-2::syntaxin(T254I) transgenic background, loss of unc-25 (GABA) or tph-1 (serotonin) leads to a further extension of lifespan, while loss of other genes had no effect. Importantly, while unc-25 mutation also extends lifespan in wild-type worms, tph-1 mutation does not. This observation indicates that the lifespan effects of cholinergic signaling can be modulated by serotonin. We have now pointed this out in the text by stating (page 9):“As a control, we also tested mutants deficient in other types of small neurotransmitters, including glutamate (eat-4),, GABA (unc-25), serotonin (tph-1), dopamine ,(cat-2), tyramine (tdc-1), and octopamine (tbh-1), but detected no effect, with the exception of tph-1, which showed a modest, partial suppression of the phenotype (Figure S2A-S2F). This observation suggests that the lifespan effects of cholinergic signaling can be modulated by serotonin.”

    (6) “RNAi of several of the receptors appear to modulate wild-type lifespan. Although I understand that this is not the main focus of the manuscript, the fact that this occurs should be mentioned in the results and discussed later on.”

    We thank the reviewer for pointing this out. As suggested by the reviewer, we have now pointed this out in the text by stating (page 9):“Notably, RNAi of several ACh receptors such as acr-11 appears to shorten wild-type lifespan, whereas RNAi of several other ACh receptors such as acr-9 extends wild-type lifespan, suggesting lifespan-modulating potential of ACh receptors (Figure S3).”

    (7) “Cholinergic signaling and ACR-6 have been previously shown to regulate pharyngeal pumping/feeding behavior. (https://doi.org/10.1016/j.jbc.2021.10146”). Could the requirements for ACR-6/cholinergic signaling in longevity be related to caloric restriction/nutritional intake which in turn could be expected to alter DAF-16 and HSF-1 activity? These previous studies should be referenced and discussed.”

    Thanks for the suggestion. As suggested by the reviewer, we have examined the pumping rate of acr-6 mutant worms. Our results showed that acr-6 mutation slightly reduced the pumping rate. As the decrease is relatively minor, we do not expect a major DR effect, though we cannot completely rule out such a possibility. Furthermore, as acr-6 acts in the pharynx to regulate pumping but in the intestine to regulate the role of cholinergic signaling in lifespan, we do not expect this would have a major contribution to our pathway. This new data has now been described in Figure S4I. As suggested by the reviewer, we have now pointed this out in the text by stating (page 10): Previous data has shown that cholinergic signaling and ACR-6 may control pharyngeal pumping[42]. As expected, we found that acr-6 mutation slightly reduced pumping rates (Figure S4G).”

    (8) “The expectation for the studies in Figure 3/DAF-16, is that animals expressing Ex[Pacr-2::syntaxin(T254I)], should have downregulated DAF-16 in the intestine. This needs to be shown through some method (increased daf-16 activation upon loss of cholinergic signaling does not necessarily imply that the converse is also true).”

    We thank the reviewer for the insightful suggestion. The reviewer has suggested us performing additional measurements to confirm that DAF-16 is the downstream transcription factor in the intestine. Specifically, the reviewer suggested testing if syntaxin(T254I) transgene signaling could inhibit DAF-16 activity. We have now followed the reviewer’s suggestion by performing two different assays. First, as also suggested by the first reviewer, we detected the expression of DAF-16 target genes in Pacr-2::syntaxin(T254I) transgenic worms, which exhibited downregulation of these genes, consistent with the notion that increasing cholinergic motor neuron activity inhibits DAF-16. This data has now been described in Figure S5A. Second, we performed an assay to detect DAF-16 subcellular localization pattern in the intestine. We found that acr-6 RNAi notably promotes nuclear translocation of DAF-16, suggesting that ACR-16 inhibits DAF-16, which is consistent with our model. This new data has now been described in Figure S5E. As suggested by the reviewers, we have now pointed this out in the text by stating (page 11): “As expected, the expression level of sod-3 and mtl-1, two commonly characterized DAF-16 target genes, was upregulated in transgenic worms deficient in releasing ACh from cholinergic motor neurons (Figure 3F), and downregulated in transgenic worms with enhanced ACh release from cholinergic motor neurons (Figure S5A), consistent with the notion that DAF-16 acts downstream of cholinergic motor neurons. To obtain further evidence, we assessed the subcellular localization pattern of DAF-16::GFP fusion and found that acr-6 RNAi notably promoted nuclear translocation of DAF-16, confirming that ACh signaling inhibits DAF-16 activity (Figure S5B).”

    (9) “Similarly, it would be good to have additional lines of evidence that signaling through GAR-3 impinges on HSF1, and that the lifespan effects are not due to non-specific effects of hsf-1 knockdown, which could lead to several un-related deficiencies and compromise lifespan (Figure 5b).”

    We thank the reviewer for the valuable suggestions. The reviewer correctly noted that the observed lifespan effect from hsf-1 RNAi could involve non-specific deficiencies. In response, we performed an assay to detect HSF-1 subcellular localization in the intestine upon gar-3 overexpression by using the strain EQ87 (iqIs28[pAH71(hsf-1p::hsf-1::gfp) + pRF4(rol-6)]). We found that the induced nuclear translocation of HSF-1 was weak. This result suggests that GAR-3 may modulate HSF-1 activity through a mechanism distinct from, or more subtle than, robust nuclear accumulation, or that its effect is highly dependent on the expression level and timing.

    (10) “Figure 6: An N2 control should be provided to assess the specificity of the mCherry signal from the intestine (given autofluorescence in the animals' gut).”

    Thanks for the suggestion. As suggested by the reviewer, we have now included the control in Figure S10.

    Reviewer #3 (Recommendations for The Authors):

    (1) “While the model is consistent with the data, there are alternatives that were not addressed. Additionally, there are some deficiencies in the interpretation of results that should be addressed, in my opinion. Possibly most importantly given the claims, the authors should address an alternative model: that it is the level of acetylcholine signaling that matters. Is it possible that the level auxin-inducible degradation of syntaxin(T254I) in acr-2 expressing cells is age dependent, such that one level increases lifespan and the other shortens it, and that the timing doesn't matter at all? A chronic dose response to auxin concentration would address if the level of syntaxin is a non-monotonic determinant of lifespan.”

    We sincerely thank the reviewer for raising this important alternative model. The reviewer suggested that the apparent temporal effect we observed might instead be explained by an age-dependent change in the efficiency of AID system in degrading syntaxin(T254I) in acr-2 expressing cells. That is, different levels of acetylcholine signaling, rather than timing, produce opposite lifespan outcomes. We agree that this is a formal possibility that our current data cannot fully rule out. On the other hand, other data in the manuscript suggests otherwise. For example, the expression of ACR-6 and GAR-3 in the intestine exhibited a temporal switch in early and mid-late life, providing support for a time-dependent mechanism. In addition, the differential requirement of the downstream transcription factors DAF-16 and HSF-1 in the early and mid-late life, respectively, provides further evidence supporting a temporal mechanism. Thus, while we agree that the possibility raised by the reviewer cannot be formally ruled out, the temporal mechanism we proposed may play an important role.

    The reviewer suggested performing a chronic dose-response experiment with varying auxin concentrations. Actually when we first employed the AID system to temporally manipulate motor neuron output at different life stages, we tested potential effects of auxin concentration. Using the soma-expressed TIR1 system, we found that, restoring syntaxin(T254I) activity from day 10 of adulthood extends lifespan, regardless of whether the prior suppression was maintained with 0.1 mM or 0.5 mM auxin. This suggests that the pro-longevity effect is likely not triggered by differences in the efficacy of prior suppression within this concentration range. We acknowledge that the tested dose range may not cover potential threshold concentrations. Furthermore, we cannot exclude the possibility of a non-linear relationship between auxin concentration and degradation efficiency. We agree that a comprehensive chronic dose-response analysis remains a valuable future direction, and we plan to employ more precise tools in the future to investigate the interplay between signal level and temporal context in lifespan regulation. The auxin concentration data have now been described in Figure S1C-1D by stating (page 7): “Comparable outcomes were obtained with both 0.1 mM and 0.5 mM auxin treatments (Figure S1C-1D).” As suggested by the reviewer, we have discussed the alternative model in the Discussion by stating (page 19): “An alternative mechanism based on differential levels of cholinergic signaling could also contribute to the observed lifespan effects.”

    (2) “Several times, including in several section headings, it is claimed that daf-16 (eg line 205-206) and acr-6 (eg line 185-186) function "early in life". This was not tested, so the claim is not warranted. For instance, these genes could act later in life to respond to signals made or sent early in life, or they could act both early and late, or only early (as they claim).”

    We thank the reviewer for this precise and important clarification. The reviewer is correct that our genetic interventions do not by themselves define the temporal window.

    Our experimental rationale was based on the observation that the lifespan-shortening effect of Pacr-2::syntaxin(T254I) expression is similar whether it is induced throughout life or specifically during larval stages (early life), indicating the detrimental effect results from enhanced motor neuron output in early life. Therefore, we used the lifelong expression paradigm as a tool to genetically dissect the downstream pathway triggered by early-life neuronal activation. We acknowledge the reviewer's point that this design does not formally prove that daf-16 or acr-6 acts only in early life; they could be required continuously or again later. However, we would like to note that our expression data show that the gut expression of ACR-6 is restricted to early life, which is consistent with a primary early-life function in this context.

    To reflect this more accurate interpretation, we have revised all relevant statements, including section headings. We now consistently state that daf-16 is required for the lifespan-shortening effect of cholinergic motor neuron, rather than claiming it functions "in early life". We have also toned down the discussion regarding their temporal function by stating (page 12): “Because this lifespan-shortening effect results from enhanced motor neuron output in early life and overwrites its beneficial effect at later stages, we propose this signaling circuit mediates the lifespan-shortening effect in early life.”

    (3) “In line 118, they note that such intervention led to a complex effect on the lifespan curve "by initially promoting worm's survival followed by inhibiting it at later stages." I think that while findings from later experiments support a time-dependent lifespan effect stemming from syntaxin function in the cholinergic motor neurons, this experiment's TeTx expression in those neurons is not time-dependent. Lifespan is an endpoint measure, so there is no sense in which a non-timed perturbation has an early or late effect on an individual. Rather, the effect on survival they observed is at the population level, their intervention increases the average lifespan while decreasing the worm-to-worm variation in lifespan.”

    We thank the reviewer for the critical and precise comment regarding our interpretation of the survival curves of TeTx transgenic worms. As suggested by the reviewers, we have revised the text by stating (page 6): “Surprisingly, such intervention led to a complex effect on the population survival curve by reducing both early mortality and the proportion of long-lived individuals (Figure 1A). Specifically, the 25% lifespan of these worms was prolonged, while their 75% and maximal lifespan were slightly shortened, leading to a mean lifespan slightly increased or unchanged compared to that of wild-type worms. This suggests that inhibiting cholinergic motor neurons may exert temporally distinct effects on survival, leading to decreased individual variation in lifespan.”

    (4) “The layout of the plots separating the responses of wild type and mutants to different panels makes it often difficult to interpret the results. For instance, do acr-6, gar-3, and other receptor mutants or knockdowns affect lifespan on their own? If they do, it matters to the interpretation whether they live longer or shorter than the wild type: which of the mutants phenocopy the lack of a lifespan-extending signal that activates them? Which phenocopy lacks a lifespan-shortening signal that activates them? Could they phenocopy the effect of an inhibitory signal? And critically, are the effects of these mutants on lifespan consistent with their model?”

    “The paper would be stronger if they determined when ACR-6 and GAR-3 functions are necessary and sufficient. Is it possible that the receptor doesn't matter, just that there be one of the two expressed in the intestine, and that other mechanisms determine the lifespan response to modulation of syntaxin(T254I)? What does time-dependent knockdown of these receptors do to daf-16 and hsf-1 localization and to the transcription of the targets of these transcription factors?”

    We thank the reviewer for these insightful comments. We have addressed the points as follows:

    As suggested, we have reorganized the lifespan data in Figure S4 to directly compare wild type and mutant/RNAi conditions within the same panels. This new presentation clarifies the autonomous effects of these genes. The data shows that loss of acr-6 or gar-2 (via RNAi or mutation) has minimal effect on lifespan. Notably, acr-8 RNAi shortens lifespan, whereas the acr-8 mutation does not, supporting our hypothesis of tissue-specific or compensatory roles for this receptor, as detailed in our following response to point (5). The reviewer's key question regarding when these receptors are necessary and sufficient is central to our model. We agree with the reviewer that complementary loss-of-function experiments with temporal precision, such as time-specific knockdown of the two receptors, would provide even stronger evidence. To this end, we attempted to generate endogenous degron-tagged alleles of acr-6 and gar-3 to apply the AID system for precise, stage-specific degradation. Unfortunately, despite multiple design attempts and screening efforts, we were unable to obtain homozeygous strains with the desired genomic edits using the same gRNA we used to knock in mCherry or other gRNAs. This is rather frustrating. Consequently, we are currently unable to perform the ideal temporally controlled loss-of-function experiments suggested by the reviewer.

    (5) “Why does RNAi but not mutation of acr-8 and gar-2 suppress the lifespan shortening effect of Pacr-2::syntaxin(T254I)?”

    Thanks for this important question regarding the differential effects of feeding RNAi versus mutation of acr-8 and gar-2. The discrepancy likely arises from the potential off-target effects of RNAi. RNAi is not strictly specific as it may target other related genes, generating a non-specific effect, whereas precise mutations in acr-8 and gar-2 alone may not produce the same effect.

    (6) “sid-1(-); Ex[Pacr-2::tetx lives longer than sid-1(-); in daf-16(+) worms in Figure 3G; so it is very hard to interpret the lack of effect of Pacr-2::tetx in daf-16(-) worms, since this transgene behaves differently in sid-1 mutants than in wild type worms. This would be clear if the two plots were combined (appropriately, since it is the same experiment). It looks like daf-16 RNAi has a shortening effect in the sid-1 mutant, but not in in sid-1 mutants expressing Pacr-2::text.”

    Thanks for this helpful suggestion. As suggested by the reviewer, we have now merged Figure 3G and 3H into one figure to present as Figure S5F. This combined presentation clarifies the comparison and shows that intestinal daf-16 RNAi shortens lifespan in both sid-1 mutants and sid-1 mutants expressing Pacr-2::TeTx.

    Reviewer #4 (Recommendations for The Authors):

    (1) “Lines 50-52: I would replace "leading to increased incidents in age-related diseases and probability of death" with "leading to the onset of age-related diseases and increased probability of death". Instead of "such an aging process" I would use "the aging process".”

    This has now been fixed.

    (2) “Figure 2E-F: By rescuing the expression of ACR-6 in neurons or intestinal cells alone, the authors show that the release of ACh from cholinergic neurons has effects on the intestine to shorten lifespan. Is ACR-6 expressed in other tissues (e.g. muscle?) It might be interesting to assess whether ACh also regulates lifespan through activating the ACR-6 receptor in other tissues or specifically targets the intestine. This question is partially answered with the tissue-specific RNAi experiments for DAF-16, but it is possible that ACR-6 also modulates other pathways beyond the tested transcription factors.”

    Analyzing the role of other tissues could also be applied to understand how GAR-3 influences lifespan. Along these lines, it would be interesting to expand the tissue-specific knockdown experiments for GAR-3 to other tissues. More importantly, these experiments can address whether activation of ACR-6 and GAR-3 can also have different effects on lifespan by regulating distinct tissues in addition to the intestine, and not only due to temporal expression patterns. For instance, whereas DAF-16 regulates lifespan primarily through its effects in the intestine, HSF1 could have effects on additional tissues. Although it would interesting to perform these experiments, I understand that the authors main focus is the nervous system-gut axis.

    We thank the reviewer for the insightful suggestions regarding the potential tissue-specific functions of ACR-6 and GAR-3. As noted in our response to point #6, endogenous expression imaging indicates that ACR-6 and GAR-3 are primarily expressed in neurons and the intestine with weak expression of GAR-3 in the muscle, so we tested the muscle. We found that muscle-specific RNAi of gar-2 abolished the ability of cholinergic motor neurons to extend lifespan at mid-late life stages, whereas muscle-specific RNAi of gar-3 does not. This result further supports that GAR-3 primarily exerts this effect in the intestine.

    (3) “Can the authors specify in the corresponding figure legend at what age they tested sod-3 and mtl-1 expression in Pacr-2::TeTx worms (Figure 3F)? This is important to support the conclusions of the paper. Along these lines, can the authors also specify at what age they quantified the expression of HSF-1 targets (Figure 5F).”

    Thanks for the suggestion. As recommended, we have now provided the worm age in Figure 3F (day 1 adult) and Figure 5F legends (day 10 adult).

    (4) “To further strengthen the authors' conclusions, it might be interesting to examine the intracellular localization of DAF-16 in the intestine of Pacr-2::TeTx and syntaxin(T254I) worms compared to controls.”

    We thank the reviewer for this valuable suggestion, which was also raised by another reviewer. In response, we examined the subcellular localization of DAF-16 in the intestine. Direct imaging in the Pacr-2::TeTx or Pacr-2::syntaxin(T254I) backgrounds was technically challenging because their fluorescent protein tags (YFP or mCherry) would interfere with the detection of DAF-16::GFP. Therefore, we adopted an alternative approach by modulating the activity of acr-6, the intestinal acetylcholine receptor that transmits cholinergic signals from motor neurons to DAF-16. We found that acr-6 RNAi promotes the nuclear translocation of DAF-16. These new data are presented in Figure S5E by stating (page 11): “To obtain further evidence, we assessed the subcellular localization pattern of DAF-16::GFP fusion and found that acr-6 RNAi notably promotes nuclear translocation of DAF-16, confirming that ACh signaling modulate DAF-16 activity (Figure S5B).”

    (5) “The results with gar-2 RNAi are fascinating. I am very curious (and I assume potential readers too) about what tissues mediate the mid-late life effects of GAR-2 in longevity. Perhaps the authors could add experiments in a couple of other tissues known to regulate organismal lifespan (e.g. muscle). However, I totally understand why the authors focused on GAR-3, especially because both GAR-3 and ACR-6 have effects on the intestine and this is sufficient for the main conclusions of the paper.”

    We sincerely thank the reviewer for the insightful suggestion and for highlighting the potential role of GAR-2. In response, we performed muscle-specific RNAi experiments. Together with our previously presented data, the results show that intestinal (but not neuronal or muscle) RNAi of gar-3 abolished the ability of cholinergic motor neurons to extend lifespan at mid-late life stages, while muscle-specific (but not neuronal or intestinal) RNAi of gar-2 suppresses this effect. This finding indicates that GAR-3 and GAR-2 mediate cholinergic signaling in distinct peripheral tissues, with GAR-3 primarily in the intestine and GAR-2 primarily in the muscle, to produce their effects on longevity. Given our focus on neuron-gut signaling, the role of GAR-2 will be investigated in future studies. The new data have now been described in Figure S8 by stating (page 13-14): “RNAi of gar-3 in the intestine (Figure 4D and 4E), but not in neurons or the muscle (Figure 4D-4F, and Figure S8A, S8D-S8E), abolished the ability of cholinergic motor neurons to extend lifespan at mid-late life stage. Thus, GAR-3 may function in the intestine to regulate lifespan. Surprisingly, RNAi of gar-2 in the muscle (Figure S8A-S8C), but not in neurons or the intestine (Figure S7F-S7H) had effect on the ability of cholinergic motor neurons to extend lifespan in mid-late life, indicating that GAR-2 acts in the muscle to regulate lifespan.”

    (6) “Figure 6: It seems that the genes are also expressed in the muscle. Can the authors include images of other tissues in supplementary figures?”

    Thanks for the suggestion. As suggested by the reviewer, we have now included images of whole worms expressing mCherry, which was knocked in the endogenous locus off gar-3 or acr-6 by CRISPR in Figure S10. However, we did not detect strong expression of gar-3 or acr-6 in the muscle under the conditions examined, which may be limited by the low endogenous protein expression level of the two genes in the muscle, though the CeNGEN website shows they are expressed in the muscle. Determining the precise spatiotemporal expression profiles of these receptors will likely require more sensitive methods. We plan to address this important question in future studies by using such refined approaches.

  6. Version published to 10.7554/elife.97829.2 on eLife
  7. Version published to 10.7554/elife.97829 on eLife
  8. Version published to 10.7554/elife.97829.1 on eLife
  9. eLife

    eLife assessment

    This study reports that the timing of 'brain-to-gut' signaling influences the lifespan of the C. elegans model. The main finding, that modulating the same neurotransmitter, Acetylcholine, at different ages elicits lifespan shortening - or extending - effects utilizing different receptors, is important and of broad interest to the longevity field as recognized by all the reviewers. The data is largely consistent with the authors' model, but the strength of the evidence is incomplete. The study requires several rigorous experiments detailed by the reviewers to substantiate the main conclusions.

  10. eLife

    Reviewer #1 (Public Review):

    Summary:

    This manuscript addresses the temporal patterns in how cholinergic signaling to the gut affects the lifespan of the worm C. elegans, which should make the manuscript of wide interest to those who study aging, as well as those who study the brain-gut axis in health and disease. The authors show that early acetylcholine (ACh) signaling to the intestine via the ACR-6 receptor shortens worm lifespan, which depends on the DAF-16/FOXO transcription factor. However, later ACh signaling to the intestine via the GAR-3 receptor extends lifespan, which in turn depends on the heat shock factor HSF-1. The authors also show a potential mechanism through which these two temporal patterns of ACh signaling might be coordinated to influence longevity in the worm, and possibly in other animals.

    Strengths:

    The authors observed that the functional ablation of acr-2-expressing cholinergic neurons in C. elegans (Pacr-2::TeTx) produced a lifespan curve that intersects the lifespan curve of a wild-type population. The first quartile of Pacr-2::TeTx worms shows a longer lifespan than the first quartile of wild-type worms, whereas the last quartile of Pacr-2::TeTx worms exhibits a shorter lifespan than wild-type. These observations raised the hypothesis that cholinergic neurons have two opposing effects on longevity: an early longevity-inhibiting effect and a later longevity-promoting effect. Much of the data supports the authors' conclusions.

    Weaknesses:

    While the authors have proved their hypothesis by temporally increasing the activity of cholinergic neurons at different life stages through the auxin-inducible degron system, their work raises two major concerns. First, they might want to discuss the conflicting data from Zullo et al (Nature 2019, vol 574, pp 359-364). For example, the authors show that increasing the activity of acr-2-expressing neurons after the 7th day of adulthood increases lifespan. However, Zullo et al (2019) show that the reciprocal experiment, inhibiting cholinergic neuron activity on the 1st day or the 8th day of adulthood, also increases lifespan. Is this because the two studies are using different promoters, that of the acr-2 ACh receptor (this work) versus that of the unc-17 vesicular ACh transporter (Zullo et al., 2019)? The two genes are expressed in different subsets of cells that do not completely overlap. CeNGEN shows that acr-2 is expressed in motor and non-motor neurons, but some of these neurons are also different from those that express unc-17. Is it possible that different cholinergic neurons also have opposite lifespan effects during adulthood? Or is it because both lack of signaling and hypersignaling can lead to a long-life phenotype? Leinwand et al (eLife 2015, vol 4, e10181) previously suggested that disturbing the balance in neurotransmission alone can extend lifespan. A simple discussion of these possibilities in the Discussion section is likely sufficient. Or can the auxin treatment and removal be confounding factors? Loose and Ghazi (Biol Open 2021, vol 10, bio058703) show that auxin IAA alone can affect lifespan and that this effect can depend on the time the animal is exposed to the auxin.

    Second, the daf-16-dependence of the early longevity-inhibiting effect of ACh signaling needs clarification and further experimentation. The authors present a model in Figure 6D, where DAF-16 inhibits longevity. This contradicts published literature. Libina et al (Cell 2003, vol 115, pp 489-502) have shown that intestinal DAF-16 increases lifespan. From the authors' data, it is possible that ACh signaling inhibits DAF-16, not promotes it as they have drawn in Figure 6D. In Figure 3F, the authors used Pacr-2::TeTx, which inhibits cholinergic neuron activity, to show an increase in the expression of DAF-16 targets. Why did the authors not use the worms that express the transgene Pacr-2::syntaxin(T254I), which increases cholinergic neuron activity? What happens to the expression of DAF-16 targets in these animals? Do their expression go down? What happens if intestinal daf-16 is knocked down in animals with increased cholinergic neuron activity, instead of reduced cholinergic neuron activity?

  11. eLife

    Reviewer #2 (Public Review):

    Summary:

    In the manuscript "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans", Xu and colleagues examine the role of cholinergic signaling by C. elegans motor neurons in modulating lifespan. The authors show that manipulating motor neuronal activity using genetic techniques can be beneficial or detrimental to lifespan, depending on when motor neuron activity is modulated.

    Strengths:

    A large body of data showing the effects of knockdown of cholinergic receptors and neurotransmitters on lifespan is presented. This would be of value to the community.

    Weaknesses:

    However, the studies are incomplete. More rigorous approaches would be needed to support the key conclusions, and substantiate the main findings and pathway components.

  12. eLife

    Reviewer #3 (Public Review):

    I very much enjoyed reading Lingxiu Xu et al.'s paper "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans," where they investigate the mechanisms by which motor neurons regulate lifespan in C. elegans worms. In this paper, they first discover that interfering with synaptic release in cholinergic motor neurons affects lifespan. Using mutants and gene knockdowns they show that these effects are due to the neurotransmitter acetylcholine. They show that the effects of these motor neurons on lifespan are opposite, depending on timed genetic interventions promoting synaptic release. If these interventions occur during development, the lifespan is shortened, but if they occur starting on day 7 of adulthood, then lifespan is lengthened. They then show that the transcription factor daf-16 is required for the former effect, while the transcription factor hsf-1 is required for the latter one. In addition, these early and late effects, they find, required the acetylcholine receptors acr-6 and gar-3, respectively, and intestinal expression of these genes rescues the respective phenotypes. Interestingly, tagging the endogenous acr-6 and gar-3 genes with mCherry, they find that the ACR-6 and GAR-3 proteins are expressed in the intestine, ACR-6 during development, and GAR-3 during adulthood. Based on these findings they propose a model where acetylcholine from motor neurons regulates lifespan by modulating different receptors expressed at different times. These receptors, in turn, affect lifespan in opposing ways via different transcription factors.

  13. eLife

    Reviewer #4 (Public Review):

    This is a very interesting study, where the authors discovered two neuroendocrine signaling circuits with opposite effects on organismal longevity elicited by motor neurons at different ages.

    Interestingly, both systems employ the same neurotransmitter (that is, acetylcholine) and signal the intestine. However, one has effects on early life to shorten lifespan whereas the other system is activated in mid-life to extend lifespan. At the mechanistic level, this bidirectional regulation is possible through the recruitment of two different ACh receptors in the gut: ACR-6 and GAR-3. The authors found that ACR-6 expression in the intestine is restricted to early life, whereas GAR-3 expression in the gut is confined to mid-late life. Interestingly, ACR-6 modulates the transcription factor DAF-16, but GAR-3 regulates HSF-1.

    The study combines different approaches, including inducible systems (AID) which are critical for the conclusions of the paper. The conclusions are well supported by the experiments and results. The data provide a potential mechanism for the temporal control of lifespan and shed light on the complex role of the nervous system in organismal aging. These results can have important implications for understanding how organismal aging is regulated in a temporal manner by cell non-autonomous mechanisms. I didn't observe significant weaknesses in the study, but I have several comments that I hope the authors will address.

  14. Version published to 10.1101/2024.04.12.589179 on bioRxiv