A TRPV channel modulates C. elegans neurosecretion, larval starvation survival, and adult lifespan

Abstract

For most organisms, food is only intermittently available; therefore, molecular mechanisms that couple sensation of nutrient availability to growth and development are critical for survival. These mechanisms, however, remain poorly defined. In the absence of nutrients, newly hatched first larval (L1) stage Caenorhabditis elegans halt development and survive in this state for several weeks. We isolated mutations in unc-31, encoding a calcium-activated regulator of neural dense-core vesicle release, which conferred enhanced starvation survival. This extended survival was reminiscent of that seen in daf-2 insulin-signaling deficient mutants and was ultimately dependent on daf-16, which encodes a FOXO transcription factor whose activity is inhibited by insulin signaling. While insulin signaling modulates metabolism, adult lifespan, and dauer formation, insulin-independent mechanisms that also regulate these processes did not promote starvation survival, indicating that regulation of starvation survival is a distinct program. Cell-specific rescue experiments identified a small subset of primary sensory neurons where unc-31 reconstitution modulated starvation survival, suggesting that these neurons mediate perception of food availability. We found that OCR-2, a transient receptor potential vanilloid (TRPV) channel that localizes to the cilia of this subset of neurons, regulates peptide-hormone secretion and L1 starvation survival. Moreover, inactivation of ocr-2 caused a significant extension in adult lifespan. These findings indicate that TRPV channels, which mediate sensation of diverse noxious, thermal, osmotic, and mechanical stimuli, couple nutrient availability to larval starvation survival and adult lifespan through modulation of neural dense-core vesicle secretion.

Figure 1. Loss of unc-31 enhances L1 starvation survival.

(A) Multiple independent experiments at room temperature (∼23°C) showing the range and reproducibility of L1 starvation survival distribution for wild-type (N2) animals. Survival curves represent counts on 1000–3000 animals per experiment. In aggregate, average mean survival for wild type was 12.2 days (standard error: ±0.4 days). (B) Multiple unc-31 loss-of-function mutations extend L1 starvation survival by ∼50%. This difference is statistically significant as determined by log-rank test (p,<0.00001); see Table S1 for details. (C) Predicted protein structure of UNC-31 with functional domains. The locations of the four new unc-31 mutations, ft1–ft4, are indicated. ft1 causes a W₅₉₇ TGG->TGA stop mutation. ft2 causes a 2 bp deletion after Q₁₃₀₄ resulting in a frame-shift that produced a stop codon four amino acids after the deletion. ft3 causes a L₆₁₀ TTA->TAA stop mutation. ft4 causes a R₂₉₆ CGA->TGA stop mutation. Amino acid positions are numbered based upon Wormbase Release WS189 predicted protein structure of UNC-31. C2 = Ca²⁺ binding domain. PH = pleckstrin homology domain. DUF1041 = domain of unknown function likely to be involved in vesicle secretion.

Figure 2. Extended survival of unc-31 mutants depends on daf-16.

Effects of mutations that cause (A) paralysis, (B) defects in synaptic vesicle release, (C) defects in synthesis of biogenic amines, (tph-1: serotonin, tdc-1: tyramine and octopamine, tbh-1: octopamine, and cat-2: dopamine) (D) defects in glutamate signaling, (E) defects in neuropeptide processing, and (F) loss of DAF-16/FOXO transcription factor, on L1 starvation survival. Statistical analyses of data shown in (A–F) are reported in Table S1. Assays reported in (A–B) were conducted at 20°C resulting in proportional extensions in mean and maximal survival of all strains.

Figure 3. Insulin-independent dauer and longevity mutants do not enhance L1 starvation survival.

(A) Effects of mutations in TGF-β signaling pathway on starvation survival. (B–C) Effects of insulin-independent adult longevity mutants on starvation survival. In the case of clk-1 and isp-1, the starvation assays were conducted at 20°C. Statistical analyses of data shown in (A–C) are reported in Table S1.

Figure 4. Expression of unc-31 in ADL and ASH sensory neurons is sufficient to partially abrogate extended starvation survival.

(A–H) unc-31 cDNA separated from a mCherry reporter by an intercistronic region was expressed in various tissues of unc-31(ft1) mutants and starvation survivals of transgenic animals and non-transgenic siblings were assayed. The mCherry reporter allowed for verification of expression patterns ascribed to each promoter. Examples of the expression pattern of each promoter are shown. For each transgenic line, “+” designates transgenic animals and “−” designates non-transgenic siblings. Expression of unc-31 using (A) a pan-neuronal egl-3 promoter, and (C) a ciliated sensory neuron osm-6 promoter, fully abrogate unc-31(ft1) extended starvation survival, while expression using (B) myo-2, a pharyngeal muscle promoter, and (D) glr-5, an interneuron promoter, do not alter extended starvation survival. (E–H) Reconstitution of wild-type unc-31 in unc-31(ft1) mutants with promoters that target various subsets of ciliated sensory neurons. Individual neurons targeted by each promoter are listed in Table S2. Graphs (except for the myo-2 negative control) depict averages of 2–4 independent transgenic lines and their non-transgenic siblings along with standard error of the mean for each time point.

Figure 5. Defective cilia function extends L1 starvation survival.

(A–B) Effects of ciliary defects on starvation survival. Only mutations that cause severe defects in cilia function enhance starvation survival. (C) Extended L1 starvation survival of cilia defective osm-6 mutants requires daf-16. (D) Effects of mutations in TRPV family members on starvation survival. ocr-2 mutants extend starvation survival while other TRPV members do not alter survival. (E) Extended survival of ocr-2 mutant is dependent on daf-16. (F) Loss-of-function mutation in G_α odr-3 reduces starvation survival. Statistical analyses of data shown in (A–F) are reported in Table S1.

Figure 6. Loss of ocr-2 reduces neural insulin secretion and increases lifespan.

(A–B) Neuronal secretion from ADL neurons was assessed by monitoring the uptake of a fluorescently-tagged insulin, DAF-28::mCherry expressed exclusively in ADL neurons from an shr-220 promoter, into coelomocytes. Representative images with arrows pointing to coelomocytes (A) and corresponding quantitations normalized to wild type (B) are shown. Bars indicate standard error of the mean. (C) Lifespans of two independent ocr-2 mutants on plates containing FUDR, a drug that inhibits progeny production. Loss of ocr-2 substantially extends adult lifespan (p-value<0.0001 as determined by log-rank test; detailed statistical analyses are reported in Table S3). While these two ocr-2 mutants were generated in two independent laboratories and were not outcrossed to our lab's wild-type strain, they had similarly extended mean and maximal lifespans, suggesting that background differences are unlikely to contribute significantly to their observed lifespan phenotypes.

Figure 7. Model of L1 starvation survival regulation by ocr-2 and unc-31.

Under favorable environmental conditions, OCR-2, TRPV channel in sensory neurons allows influx of cations such as calcium. In response, calcium-activated UNC-31 promotes release of cargoes contained in dense-core vesicles of sensory neurons. Whether calcium influx from OCR-2 channels directly activates UNC-31 or whether it does so through an intermediate signaling cascade is not yet known. Similarly, the epistatic relationship between unc-31 and ocr-2 remains to be established but ocr-2 is likely to act upstream of unc-31 based upon their molecular functions. Active release of dense-core vesicle cargoes ultimately inhibits the FOXO transcription factor, DAF-16. This is most likely through direct signaling by insulin-like peptides through the DAF-2 insulin receptor, although alternative mechanisms are possible. Inhibition of DAF-16 favors pathways that promote growth and development. Inhibition of DAF-16 is relieved during unfavorable environmental conditions such as limited nutrient availability, leading to activation of starvation survival mechanisms. Loss of ocr-2, unc-31, and daf-2 extend starvation survival either by pre-conditioning animals through expression of starvation resistance genes even during periods of nutrient availability or by preventing spurious inactivation of DAF-16 during starvation.