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

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?

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.

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.

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.