A neuronal GPCR is critical for the induction of the heat shock response in the nematode C. elegans

Abstract

In the nematode Caenorhabditis elegans, the heat shock response (HSR) is regulated at the organismal level by a network of thermosensory neurons that senses elevated temperatures and activates the HSR in remote tissues. Which neuronal receptors are required for this signaling mechanism and in which neurons they function are largely unanswered questions. Here we used worms that were engineered to exhibit RNA interference hypersensitivity in neurons to screen for neuronal receptors that are required for the activation of the HSR and identified a putative G-protein coupled receptor (GPCR) as a novel key component of this mechanism. This gene, which we termed GPCR thermal receptor 1 (gtr-1), is expressed in chemosensory neurons and has no role in heat sensing but is critically required for the induction of genes that encode heat shock proteins in non-neural tissues upon exposure to heat. Surprisingly, the knock-down of gtr-1 by RNA interference protected worms expressing the Alzheimer's-disease-linked aggregative peptide Aβ3-42 from proteotoxicity but had no effect on lifespan. This study provides several novel insights: (1) it shows that chemosensory neurons play important roles in the nematode's HSR-regulating mechanism, (2) it shows that lifespan and heat stress resistance are separable, and (3) it strengthens the emerging notion that the ability to respond to heat comes at the expense of protein homeostasis (proteostasis).

Figure 1.

Knock-down of gtr-1 sensitizes worms to heat stress. A–B, Worms expressing sid-1 under the regulation of the pan-neuronal unc-119 promoter (strain TU3335) were treated with RNAi toward either gcy-8 or gtr-1 or left untreated (EV), exposed to 35°C, and their survival rates were recorded in 3 h intervals (A). Similarly, the survival rates of RNAi-treated TU3335 worms (as in A) exposed to 35°C for 10 h were recorded (B). gtr-1 RNAi-treated animals exhibited reduced survival rates compared with their untreated counterparts. The survival rates of gtr-1 and gcy-8 RNAi-treated animals were comparable. C–F, gtr-1 RNAi-treated wild-type worms (strain N₂) (C,D) and CF512 animals (E,F) exhibited significantly reduced survival rates in 35°C compared with their untreated counterparts. Three independent single-time-point survival assays with each strain (12 and 15 h of exposure to 35°C for N₂ and CF512 worms, respectively) showed that daf-16 and gtr-1 RNAi-treated animals exhibited similarly reduced lifespan after exposure to heat compared with untreated animals (D,F). G, Similarly to daf-16, the knock-down of gtr-1 by RNAi abolished the elevated stress resistance of daf-2(e1370) mutant worms that were exposed to heat (35°C) for 20 h. In B, D, F, and G, bars represent mean survival ± SEM, p < 0.01. H, CF512 worms that were treated with RNAi toward the 3′UTR region of gtr-1 and exposed to 35°C exhibited significantly reduced survival rates compared with the control group (EV).

Figure 2.

gtr-1 is expressed in chemosensory neurons. A, Fluorescent visualization of worms that express tdTomato under the regulation of gtr-1 promoter (strain EHC101) revealed that gtr-1 is expressed in neurons of the head ganglia, the ventral cord, and the tail. Scale bar, 50 μm. B–C, gtr-1 is expressed in neither the AFD thermosensory neurons (labeled by GFP driven by the AFD-specific, gcy-8 promoter; B) nor in AIY interneurons (labeled with GFP driven by the ttx-3 promoter; C). D, Visualization of worms that express GFP under the regulation of the chemosensory specific lin-11 promoter (green channel) and tdTomato driven by the gtr-1 promoter (red channel) shows colocalization. In B and D, scale bars, 10 μm.

Figure 3.

Temporal analysis of gtr-1 expression. A, Eggs of EHC101 worms were visualized in 30 min intervals by a fluorescent microscope. tdTomato signal was first observed in a single location at the 1.5-fold embryonic stage (arrow). At the 3-fold stage, clear signals were detected in two areas corresponding to the head and tail regions of the developing embryo (arrows), whereas at late morphogenesis, tdTomato signals were seen in the head and tail and along the developing embryo's body. B, tdTomato signals were observed in the head, tail, and ventral cord of L1 larvae and in ADL and AIZ neurons of L2 and L3 larvae.

Figure 4.

Knock-down of gtr-1 by RNAi has no effect on heat sensing. A, daf-2(e1370) mutant worms were placed on temperature gradient plates. The plates were photographed right after placing the worms and 12 min thereafter. Both untreated (EV; top) and gtr-1 RNAi-treated (bottom) animals migrated away from regions of high (∼38°C) and low (∼6°C) temperatures to populate the central region of the plates where the temperatures were similar to their cultivation temperature (18–20°C, rectangles). B, Four independent experiments as in A confirmed that untreated and gtr-1 RNAi-treated worms exhibited indistinguishable thermotactic behavior.

Figure 5.

Knock-down of gtr-1 prevents the induction of the heat shock response downstream of both DAF-16 and HSF-1. A, Worms expressing GFP under the control of the hsp-16.2 promoter (strain CL2070) that were treated with gtr-1 RNAi and exposed to heat shock exhibited reduced GFP signal compared with their untreated (EV) counterparts. B–C, Measurement of GFP signal intensities (B) and Western blot analysis using GFP antibody (C) indicated that knock-down of gtr-1 resulted in remarkable reduction in the induction of hsp-16.2 by heat. This effect was significant (p < 0.01) but less prominent than the effect of hsf-1 RNAi. D–E, Knock-down of gtr-1 by RNAi reduces the signal intensity of worms expressing GFP under the regulation of hsp-70 (C12C8.1) promoter as visualized by fluorescent microscope. This effect was most prominent in the pharynx (D) and less in the spermatheca (“S”). Signal quantification (>20 worms per group; E) confirmed the significance of this observation (p < 0.01). F, Knock-down of gtr-1 by RNAi reduced the induction of hsp-70 (C12C8.1) by heat shock as measured by qPCR in CF512 worms (p = 0.012). G–H, Induction level of hsp-12.6 was significantly (p < 0.01) reduced by the knock-down of gtr-1 by RNAi as visualized (G) and quantified (H) in EHC102 worms that express tdTomato under the regulation of the hsp-12.6 promoter. This effect was most prominent in the vulva (insets). I, qPCR analysis confirmed the necessity of gtr-1 for the induction of hsp-12.6 in heat-shocked daf-2(e1370) worms (error bars represent ± SEM).

Figure 5.

Knock-down of gtr-1 prevents the induction of the heat shock response downstream of both DAF-16 and HSF-1. A, Worms expressing GFP under the control of the hsp-16.2 promoter (strain CL2070) that were treated with gtr-1 RNAi and exposed to heat shock exhibited reduced GFP signal compared with their untreated (EV) counterparts. B–C, Measurement of GFP signal intensities (B) and Western blot analysis using GFP antibody (C) indicated that knock-down of gtr-1 resulted in remarkable reduction in the induction of hsp-16.2 by heat. This effect was significant (p < 0.01) but less prominent than the effect of hsf-1 RNAi. D–E, Knock-down of gtr-1 by RNAi reduces the signal intensity of worms expressing GFP under the regulation of hsp-70 (C12C8.1) promoter as visualized by fluorescent microscope. This effect was most prominent in the pharynx (D) and less in the spermatheca (“S”). Signal quantification (>20 worms per group; E) confirmed the significance of this observation (p < 0.01). F, Knock-down of gtr-1 by RNAi reduced the induction of hsp-70 (C12C8.1) by heat shock as measured by qPCR in CF512 worms (p = 0.012). G–H, Induction level of hsp-12.6 was significantly (p < 0.01) reduced by the knock-down of gtr-1 by RNAi as visualized (G) and quantified (H) in EHC102 worms that express tdTomato under the regulation of the hsp-12.6 promoter. This effect was most prominent in the vulva (insets). I, qPCR analysis confirmed the necessity of gtr-1 for the induction of hsp-12.6 in heat-shocked daf-2(e1370) worms (error bars represent ± SEM).

Figure 6.

gtr-1 is dispensable for the determination of lifespan, for innate immunity, and for the developmental functions of the IIS. A–B, gtr-1 RNAi-treated and untreated (EV) CF512 worms had indistinguishable lifespans (A). Similarly, the knock-down of gtr-1 by RNAi had no significant effect on the lifespans of daf-2(e1370) mutant worms (B). C–D, Knock-down of gtr-1 affected neither the egg-laying pattern of daf-2(e1370) mutant worms (C) nor the percentage of dauer larvae in a worm population that was hatched and grown at 25°C (D). E, In contrast to hsf-1 RNAi, gtr-1 RNAi treatment during larval development had no effect on the survival rates of daf-2(e1370) mutant worms that were fed with the pathogenic bacteria P. aeruginosa during adulthood (p = 0.63). F, Knock-down of octr-1 by RNAi had no effect on the heat stress resistance of daf-2(e1370) mutant worms.

Figure 7.

Knock-down of gtr-1 partially protects from Aβ proteotoxicity. A, CL2006 worms (expressing Aβ_3–42 in their body wall muscles) were either left untreated (EV) or were treated with daf-2, hsf-1, or gtr-1 RNAi and the rates of paralysis within the worm populations were recorded daily. The rate of paralysis within the gtr-1 RNAi-treated population was lower than that of the control group (EV), but higher than that of daf-2 RNAi-treated worms. B, The counter-proteotoxic effect of gtr-1 RNAi treatment was confirmed by three independent paralysis assays. Bars represent the relative slopes of the paralysis graphs as in A (p = 0.045).