Calcineurin inhibition enhances Caenorhabditis elegans lifespan by defecation defects-mediated calorie restriction and nuclear hormone signaling

  1. Priyanka Das
  2. Alejandro Aballay
  3. Jogender Singh

  • Curated by eLife

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    eLife assessment

    This useful study has the potential to reveal insights into how calcineurin influences C. elegans lifespan through its role in controlling the defecation motor program. Currently, the evidence in support of the conclusions is still incomplete, largely due to concerns about partial gene inactivation by RNAi. The inclusion of experiments using a tax-6 null allele would mitigate these concerns.

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Abstract

Calcineurin is a highly conserved calcium/calmodulin-dependent serine/threonine protein phosphatase with diverse functions. Inhibition of calcineurin is known to enhance Caenorhabditis elegans lifespan via multiple signaling pathways. Aiming to study the role of calcineurin in regulating innate immunity, we discover that calcineurin is required for the rhythmic defecation motor program (DMP) in C. elegans . Calcineurin inhibition leads to defects in the DMP, resulting in intestinal bloating, rapid colonization of the gut by bacteria, and increased susceptibility to bacterial infection. We demonstrate that intestinal bloating by calcineurin inhibition mimics calorie restriction that results in enhanced lifespan. The TFEB ortholog, HLH-30, is required for calcineurin inhibition-mediated lifespan enhancement by triggering lipolysis. Finally, we show that the nuclear hormone receptor, NHR-8, is upregulated by calcineurin inhibition and is required for increased lifespan. Our studies uncover a role for calcineurin in the C. elegans DMP and provide a new mechanism for calcineurin inhibition-mediated longevity extension.

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  1. Version published to 10.7554/elife.89572.1 on eLife
  2. Version published to 10.7554/elife.89572 on eLife
  3. eLife

    eLife assessment

    This useful study has the potential to reveal insights into how calcineurin influences C. elegans lifespan through its role in controlling the defecation motor program. Currently, the evidence in support of the conclusions is still incomplete, largely due to concerns about partial gene inactivation by RNAi. The inclusion of experiments using a tax-6 null allele would mitigate these concerns.

  4. eLife

    Reviewer #1 (Public Review):

    In this paper, the authors show that disruption of calcineurin, which is encoded by tax-6 in C. elegans, results in increased susceptibility to P. aeruginosa, but extends lifespan. In exploring the mechanisms involved, the authors show that disruption of tax-6 decreases the rate of defecation leading to intestinal accumulation of bacteria and distension of the intestinal lumen. The authors further show that the lifespan extension is dependent on hlh-30, which may be involved in breaking down lipids following deficits in defecation, and nhr-8, whose levels are increased by deficits in defecation. The authors propose a model in which disruption of the defecation motor program is responsible for the effect of calcineurin on pathogen susceptibility and lifespan, but do not exclude the possibility that calcineurin affects these phenotypes independently of defecation.

  5. eLife

    Reviewer #2 (Public Review):

    The manuscript titled "Calcineurin Inhibition Enhances Caenorhabditis elegans Lifespan by Defecation Defects-Mediated Calorie Restriction and Nuclear Hormone Signaling" by Priyanka Das, Alejandro Aballay, and Jogender Singh reveals that inhibiting calcineurin, a conserved protein phosphatase, in C. elegans affects the defecation motor program (DMP), leading to intestinal bloating and increased susceptibility to bacterial infection. This intestinal bloating mimics calorie restriction, ultimately resulting in an enhanced lifespan. The research identifies the involvement of HLH-30 and NHR-8 proteins in this lifespan enhancement, providing new insights into the role of calcineurin in C. elegans DMP and mechanisms for longevity.

    The authors present novel findings on the role of calcineurin in regulating the defecation motor program in C. elegans and how its inhibition can lead to lifespan enhancement. The evidence provided is solid with multiple experiments supporting the main claims.

    Strengths:
    The manuscript's strength lies in the authors' use of genetic and biochemical techniques to investigate the role of calcineurin in regulating the DMP, innate immunity, and lifespan in C. elegans. Moreover, the authors' findings provide a new mechanism for calcineurin inhibition-mediated longevity extension, which could have significant implications for understanding the molecular basis of aging and developing interventions to promote healthy aging.

    1. The study uncovers a new role for calcineurin in the regulation of C. elegans DMP and a potential novel pathway for enhancing lifespan via calorie restriction involving calcineurin, HLH-30, and NHR-8 in C. elegans.
    2. Multiple signaling pathways involved in lifespan enhancement were investigated with fairly strong experimental evidence supporting their claims.

    Weaknesses:
    The manuscript's weaknesses include the lack of mechanistic details regarding how calcineurin inhibition leads to defects in the DMP and induces calorie restriction-like effects on lifespan.

    The exact site of calcineurin action, i.e., whether in the intestine or enteric muscles (Lee et al., 2005), and the possible molecular mechanisms linking calcineurin inhibition, DMP defects, and lifespan were not adequately explored. Although characterization of the full mechanism is probably beyond the scope of this paper, given the relative simplicity and advantages of using C. elegans as a model organism for this study, some degree of rigor is expected with additional straightforward control experiments as listed below:

    The authors state that tax-6 knockdown animals had drastically reduced expulsion events (Figure 2G), leading to irregular DMP (Lines 144-145), but did not describe the nature of DMP irregularity. For example, did the reduced expulsion events still occur with regular intervals but longer cycle lengths? Or was the rhythmicity completely abolished? The former would suggest the intestine clock is still intact, and the latter would indicate that calcineurin is required for the clock to function. Therefore, ethograms of DMP in both wild-type and tax-6 mutant animals are warranted to be included in the manuscript. Along the same line, besides the cycle length, the three separable motor steps (aBoc, pBoc, EMC) are easily measurable, with each step indicating where the program goes wrong, hence the site of action, which is precisely the beauty of studying C. elegans DMP. Unfortunately, the authors did not use this opportunity to characterize the exact behavior phenotypes of the tax-6 mutant to guide future investigations. Furthermore, it is interesting that about 64% of tax-6 (p675) animals had normal DMP. The authors attributed this to p675 being a weak allele. It would be informative to further examine tax-6 RNAi as in other experiments or to make a tax-6 null mutant with CRISPR. In addition, in one of the cited papers (Lee et al., 2005), the exact calcineurin loss-of-function strain tax-6(p675) was shown to have normal defecation, including normal EMC, while the gain-of-function mutant of calcineurin tax-6(jh107) had abnormal EMC steps. It wasn't clear from Lee et al., 2005, if the reported "normal defecation" was only referring to the expulsion step or also included the cycle length. Nevertheless, this potential contradiction and calcineurin gain-of-function mutant is highly relevant to the current study and should be further explored as a follow-up to previously reported results. For some of the key experiments, such as tax-6's effects on susceptibility to PA14, DMP, intestinal bloating, and lifespan, additional controls, as the norm of C. elegans studies, including second allele and rescue experiments, would strengthen the authors' claims and conclusions.

    The second weakness of this manuscript is the data presentation for all survival rate curves. The authors stated that three independent experiments or biological replicates were performed for each group but only showed one "representative" curve for each plot. Without seeing all individual datasets or the averaged data with error bars, there is no way to evaluate the variability and consistency of the survival rate reported in this study.

    Overall, the authors' claims and conclusions are justified by their data, but further experiments are needed to confirm their findings and establish the detailed mechanisms underlying the observed effects of calcineurin inhibition on the DMP, calorie restriction, and lifespan in C. elegans.

  6. Version published to 10.1101/2023.06.21.545873v1 on bioRxiv