Regulation of the longevity response to temperature by thermosensory neurons in Caenorhabditis elegans

Abstract

Background: Many ectotherms, including C. elegans, have shorter life spans at high temperature than at low temperature. High temperature is generally thought to increase the "rate of living" simply by increasing chemical reaction rates. In this study, we questioned this view and asked whether the temperature dependence of life span is subject to active regulation.

Results: We show that thermosensory neurons play a regulatory role in the temperature dependence of life span. Surprisingly, inhibiting the function of thermosensory neurons by mutation or laser ablation causes animals to have even shorter life spans at warm temperature. Thermosensory mutations shorten life span by decreasing expression of daf-9, a gene required for the synthesis of ligands that inhibit the DAF-12, a nuclear hormone receptor. The short life span of thermosensory mutants at warm temperature is completely suppressed by a daf-12(-) mutation.

Conclusions: Our data suggest that thermosensory neurons affect life span at warm temperature by changing the activity of a steroid-signaling pathway that affects longevity. We propose that this thermosensory system allows C. elegans to reduce the effect that warm temperature would otherwise have on processes that affect aging, something that warm-blooded animals do by controlling temperature itself.

Figure 1. Thermosensory mutations decrease lifespan at warm temperature

A to D, Laser ablation of the thermosensory AFD neurons (A) or the ttx-1(p767) mutation [ttx-1(−)], which disrupts AFD neurons (B), shortened lifespan at 25°C [In this and other figures, (−) refers to a reduction or loss of function mutant allele and the specific allele is given in the corresponding figure legend.]. AFD ablation (C) or the ttx-1(p767) mutation (D) did not affect lifespan at 15°C. See table S2 for additional trials and statistical analysis for this and all other lifespan data. E to G, tax-2(p671) (E), tax-4(p678) (F) and tax-2(p671); tax-4(p678) (G) mutants lived shorter than wild type (WT) at 25°C. H, tax-2(p671); ttx-1(p767) double mutants did not live shorter than the single mutants. I, ttx-3(ks5) mutants lived shorter than wild type at 25°C. J, the lifespan of ttx-3(ks5) mutants was not shorter than that of wild type at 15°C.

Figure 1. Thermosensory mutations decrease lifespan at warm temperature

A to D, Laser ablation of the thermosensory AFD neurons (A) or the ttx-1(p767) mutation [ttx-1(−)], which disrupts AFD neurons (B), shortened lifespan at 25°C [In this and other figures, (−) refers to a reduction or loss of function mutant allele and the specific allele is given in the corresponding figure legend.]. AFD ablation (C) or the ttx-1(p767) mutation (D) did not affect lifespan at 15°C. See table S2 for additional trials and statistical analysis for this and all other lifespan data. E to G, tax-2(p671) (E), tax-4(p678) (F) and tax-2(p671); tax-4(p678) (G) mutants lived shorter than wild type (WT) at 25°C. H, tax-2(p671); ttx-1(p767) double mutants did not live shorter than the single mutants. I, ttx-3(ks5) mutants lived shorter than wild type at 25°C. J, the lifespan of ttx-3(ks5) mutants was not shorter than that of wild type at 15°C.

Figure 2. Thermosensory and chemosensory mutations affect lifespan independently of one another

A and B, Chemosensory-defective osm-3(p802) and osm-5(p813) mutants, which exhibit normal thermotaxis, were long-lived at both 25°C (A) and 15°C (B). C and D, At 25°C, thermosensory ttx-1(p767) mutations shortened lifespan but osm-3(p802) and osm-5(p813) mutations lengthened lifespan. When introduced by genetic crosses, the ttx-1(p767) mutation shortened the lifespan of osm-3(p802) (C) and osm-5(p813) (D) mutants. These data suggest that the thermosensory and chemosensory systems act independently of one another to influence lifespan.

Figure 3. Thermosensory mutations shortened the lifespans of daf-16(−) mutants, as well as long-lived calorically-restricted or respiration-defective mutants at 25°C

All panels show 25°C lifespans. A and B, The short lifespan conferred by daf-16(mu86) mutation was further decreased by tax-2(p671) (A) (2 out of 3 trials. See table S2D.) or ttx-1(p767) (2 out of 2 trials, table S2D) (B) mutations. C and D, The tax-2(p671) mutation decreased the long lifespans of feeding-defective eat-2(ad1116) mutants (C) and respiration-defective isp-1(qm150) mutants (D), as with wild type.

Figure 4. Thermosensory neurons influence lifespan at 25°C through the cytochrome P450 DAF-9

A and B, The short lifespan caused by daf-9(rh50) was not further shortened by ttx-1(p767) (A) or tax-2(p671) (B) mutations. C to G, Expression of daf-9 is reduced in thermosensory mutants. Functional Pdaf-9::daf-9::gfp [20] expression in control [daf-9::gfp] (C) or tax-2(p671) [tax-2(−); daf-9::gfp] (D) L4 animals. E, Quantification of fluorescence intensity of DAF-9::GFP from >20 worms for each data set in two independent trials. All animals in panel C, D and E carry daf-9(e1406) and dpy-7(sc27) mutations. Relative daf-9 mRNA level is decreased in ttx-1(p767) (F) or in tax-2(p671); tax-4(p678) (G) mutants compared to wild type. Error bars represent s.e.m (***P<0.0001, *P<0.05, two-tailed Student’s t-test). See table S3 for statistical analysis. H and I, Expression of daf-9::gfp in the neurosecretory XXX cells (Psdf-9::daf-9) suppressed the short lifespan of ttx-1(p767) (H) or tax-2(p671) (I) animals. See Figure S5 for lifespans of transgenic worms expressing daf-9 under daf-9 (Pdaf-9), neuronal (Pche-2) and hypodermal (Pdpy-7 and Pcol-12) promoters. All animals were grown at 25°C.

Figure 5. The regulation of lifespan by thermosensory neurons at 25°C is mediated by the nuclear hormone receptor DAF-12

A, The null daf-12(rh61rh411) mutation completely suppressed the short lifespan of daf-9(rh50) mutants. B and C, daf-12(rh61rh411) prevented the ttx-1(p767) (B) and tax-2(p671) (C) mutants from having shorter lifespan than normal.

Figure 6. ttx-1 mutations affect daf-9 mRNA levels at 25°C but not hsp-16.1 or hsp-70 mRNA levels in animals cultured continuously at 15°C or 25°C

Relative mRNA levels of daf-9 (A), hsp-16.1 (B) and hsp-70 (C) in N2 (wild type) and ttx-1(p767) animals grown at 15°C or 25°C. Synchronized L4 (A) or young-adult (day 1) (B and C) animals were used for RNA extraction and the subsequent experiments. We extracted RNA from L4 animals to examine daf-9 transcripts because that was the stage at which we assayed DAF-9::GFP fluorescence in live tax-2(p671) mutants (Figure 4, C to E). Error bars represent s.e.m from four independent biological replicates (*P<0.05, two-tailed Student’s t-test). See table S3, D to F for statistical analysis.

Figure 7. The lifespan-shortening effects of hsf-1 mutations and RNAi are greater at high temperature than at lower temperatures

A-C, The lifespan-shortening effect of hsf-1(sy441, RNAi) [hsf-1(sy441) mutants treated with hsf-1 RNAi] was the biggest (−65 to −66%) at high temperature (22.5°C) (A), intermediate (−47%) at an intermediate temperature (20°C) (B), and the smallest (−5%) at low temperature (15°C) (C). Note that we used 22.5°C as a high temperature here instead of 25°C because hsf-1(sy441) mutation results in lethal phenotype at 25°C [60]. Worms were grown on bacteria expressing hsf-1 double-stranded RNA to knock down hsf-1 further, since hsf-1(sy441) is not a null mutation. See Table S2F for lifespan data of hsf-1(sy441) and hsf-1(RNAi) animals.