Co-chaperone p23 regulates C. elegans Lifespan in Response to Temperature

Abstract

Temperature potently modulates various physiologic processes including organismal motility, growth rate, reproduction, and ageing. In ectotherms, longevity varies inversely with temperature, with animals living shorter at higher temperatures. Thermal effects on lifespan and other processes are ascribed to passive changes in metabolic rate, but recent evidence also suggests a regulated process. Here, we demonstrate that in response to temperature, daf-41/ZC395.10, the C. elegans homolog of p23 co-chaperone/prostaglandin E synthase-3, governs entry into the long-lived dauer diapause and regulates adult lifespan. daf-41 deletion triggers constitutive entry into the dauer diapause at elevated temperature dependent on neurosensory machinery (daf-10/IFT122), insulin/IGF-1 signaling (daf-16/FOXO), and steroidal signaling (daf-12/FXR). Surprisingly, daf-41 mutation alters the longevity response to temperature, living longer than wild-type at 25°C but shorter than wild-type at 15°C. Longevity phenotypes at 25°C work through daf-16/FOXO and heat shock factor hsf-1, while short lived phenotypes converge on daf-16/FOXO and depend on the daf-12/FXR steroid receptor. Correlatively daf-41 affected expression of DAF-16 and HSF-1 target genes at high temperature, and nuclear extracts from daf-41 animals showed increased occupancy of the heat shock response element. Our studies suggest that daf-41/p23 modulates key transcriptional changes in longevity pathways in response to temperature.

Fig 1. daf-41/ZC395.10 regulates dauer formation and stress resistance.

(A) An alignment of protein sequences between C. elegans DAF-41, D. melanogaster CG16817 and Homo sapiens p23/PTGES3. The similarity between DAF-41 and p23/PTGES3 is 44.6%. (B) Schematic illustration of the daf-41, and deletion alleles of ok3015 and ok3052. Black arrows indicate the direction of transcription. Red area indicates HSP20-like co-chaperone domain. (C) daf-41 mutants constitutively formed dauer larvae (Daf-c) weakly at 25°C and strongly at 27°C. (D) Dauer alae of daf-41(ok3052) animals grown at 27°C are indicated by the white arrows. (E) daf-41(ok3052) worms were resistant for oxidative stress (20mM of H₂O_2, 2.5 hrs) and heat stress (35°C, 8 hrs). gst-4(ok2358) worms were also slightly stress tolerant. (F) daf-41p::gfp (i.e. dpy-5(e907); sEx10796 [rCes daf-41::gfp + pCeh361]) worms were labeled with DiI and photos taken at the young adult stage. Patterns of gene expression of daf-41p::gfp (green), DiI (red), and merged figures are shown, with arrows indicating individual neurons.

Fig 2. Genetic interactions of daf-41 with dauer signaling pathways.

(A) daf-41(ok3052) Daf-c phenotypes were partially suppressed in af-16(mgDf50) and completely suppressed in daf-12(rh61rh411) backgrounds. (B) daf-41(ok3052) Daf-c phenotypes were suppressed in various chemotaxis mutant backgrounds. (C-D) daf-21(p673) had no additive effect on Daf-c phenotypes at 25°C, and modestly reduced dauer formation at 27°C in the daf-41(ok3052) background. (E) daf-11(m47) had no additive effect on dauer formation in the daf-41(ok3052) background at 22.5°C. (F) hsf-1(sy441) strongly enhanced dauer formation of daf-41(ok3052) at 22.5°C. (G) Cultures of daf-41(ok3052), hsf-1(sy441) and daf-41;hsf-1 are shown grown at 22.5°C. White arrows indicate dauer larvae. All error bars indicate S.D. *, p<0.05; **, p<0.01 versus daf-41(ok3052); ††, p<0.01 versus daf-21(p673)A or hsf-1(sy441) by t-test. (H) daf-41 regulates dauer formation via daf-10, daf-12 and daf-16 similar to daf-21. However, hsf-1 suppresses dauer formation in daf-21 but not daf-41. Note that the model reflects genetic interactions, not necessarily direct biochemical interactions.

Fig 3. daf-41 mutants extend lifespan at elevated temperatures and shorten life span at lower temperatures.

(A) daf-41(ok3052) mutant animals have a lifespan similar to wild type N2 worms at 20°C (B) daf-41(ok3052) animals showed extended lifespan at 25°C (C) daf-41(ok3052) animals showed reduced lifespan at 15°C. (D) Lifespan curves of N2 and daf-41(ok3052) at different temperatures were plotted onto the same graph as indicated. (E) Mean lifespan of 3 individual experiments were plotted. Error bars, S.D. *, p<0.05; n.s., no significant difference versus N2 by t-test. (F) daf-41(+) transgenes rescued Daf-c phenotypes of daf-41(ok3052) at 27°C. dhEx906 and dhEx907 are independent daf-41(+) transgenic lines. dhEx909 and dhEx910 are pges-2(+) transgenes under control of daf-41 5’ and 3’ regulatory elements. Error bars, S.D.; **, p<0.001 versus non-transgenic worms by t-test. (G) Mean lifespan from 3 individual experiments at 15°C, 20°C and 25°C were plotted. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05 versus daf-41(ok3052); n.s., no significant difference by t-test. (H) Gene expression of daf-41 was slightly reduced at 15°C, but no significant differences were measured between 20°C and 25°C. Error bars, S.D. *, p<0.05; n.s., no significance by t-test.

Fig 4. daf-41 longevity is dependent on daf-16/FOXO.

(A) daf-16(mgDf50) equally reduced the lifespan of daf-41(ok3052) and N2 worms at 20°C. (B) daf-16(mgDf50) abolished daf-41 longevity at 25°C. (C) daf-16(mgDf50) did not further reduce the short life span of daf-41(ok3052) worms at 15°C. (D) Mean lifespan from 3 individual experiments were plotted for the indicated genotypes. Error bars; S.D.; **,p<0.01 versus N2; ††, p<0.01 versus daf-41(ok3052) by t-test. (E) DAF-16 target genes, sod-3, dod-3 and lipl-4, were significantly upregulated in response to warm temperature in daf-41(ok3052) relative to N2. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus daf-41(ok3052) of 20°C by t-test.

Fig 5. daf-41(ok3052) longevity is hsf-1 dependent.

(A) hsf-1(sy441) shortened both N2 and daf-41(ok3052) life span at 20°C. (B) hsf-1(sy441) abolished daf-41(ok3052) longevity at 25°C. (C) hsf-1(sy441) further reduced daf-41(ok3052) short life span at 15°C. (D) Mean lifespan from of 3 individual experiments were plotted for indicated genotypes and conditions. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus daf-41(ok3052) by t-test. (E) daf-41(ok3052) enhanced the upregulation of HSF-1 target genes, hsp-16.2, hsp-4, and hsp-70, in response to warm temperature. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus daf-41(ok3052) of 20°C by t-test. (F) HSF-1 binding activity to HSE was 1.5 fold increased in daf-41(ok3052) at 25°C. Error bars, S.E.M; **, p<0.01 versus N2. (G) At 15°C, daf-21(p673) mutation enhanced the longevity of N2 and daf-41(ok3052). At 20°C, daf-21 mutant animals lived slightly longer than N2. At 25°C, daf-21(p673) animals lived slightly longer than WT but the mutation reduced longevity in the daf-41(ok3052) background.

Fig 6. The short life span of daf-41(ok3052) at 15°C is daf-12 dependent.

(A-B) daf-12(rh61rh411) reduced longevity of daf-41(ok3052) at 20°C and 25°C. (C) daf-12(rh61rh411) partly rescued the short life span of daf-41(ok3052) at 15°C. (D) Mean lifespan from 3 individual experiments are plotted with indicated genotypes and conditions. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus daf-41(ok3052) by t-test. (E) Transcriptional targets of DAF-12, cdr-6 and fard-1, were reduced with temperature in N2, but this tendency was reversed in the daf-41(ok3052) background. n = 4 biological replicates. Error bars, S.E.M; *, p<0.05 versus N2 of 20°C; †, p<0.05 versus daf-41(ok3052) of 20°C by t-test.

Fig 7. daf-41 partially interacts with the chemosensory and thermosensory apparatus to regulate longevity.

(A) Mean lifespan of 3 individual experiments were plotted. The triple mutant of gcy-23(nj37) gcy-8(oy44) gcy-18(nj38) (gcy triple) caused a parallel reduction of lifespan in N2 and daf-41(ok3052), respectively at 25°C. The gcy triple mutant did not further shorten the life span of daf-41(ok3052) at 15°C. (B) daf-10 mutation increased lifespan in parallel to daf-41 at 15°C and 20°C. daf-10 mutation did not further extend the life span of daf-41(ok3052) worms at 25°C. Error bars, S.D.; *, p<0.05; **, p<0.01 versus N2; †, p<0.05; ††, p<0.01 versus daf-41(ok3052) by t-test. (C) A schematic model describing the regulatory mechanism of longevity by daf-41 at different temperatures. At 25°C, daf-41 negatively regulates the transcriptional activities of DAF-16 and HSF-1 and their down-regulation results in normal life span. Thermotaxis and steroidal signaling may regulate longevity in parallel to daf-41. At 15°C, daf-41 (+) contributes to longevity possibly via daf-16/FOXO. daf-41(+) may also prevent life shortening activities of daf-12(+), while hsf-1 may promote longevity in parallel. These are working models that we interpret with caution, and may reflect direct or indirect interactions.