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. 2007 Sep 1;21(17):2161-71.
doi: 10.1101/gad.1573107.

Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation

Chanhee Kang  1 , Young-jai You, Leon Avery
Affiliations

Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation

Chanhee Kang et al. Genes Dev. .

Abstract

Autophagy is a major pathway used to degrade long-lived proteins and organelles. Autophagy is thought to promote both cell and organism survival by providing fundamental building blocks to maintain energy homeostasis during starvation. Under different conditions, however, autophagy may instead act to promote cell death through an autophagic cell death pathway distinct from apoptosis. Although several recent papers suggest that autophagy plays a role in cell death, it is not known whether autophagy can cause the death of an organism. Furthermore, why autophagy acts in some instances to promote survival but in others to promote death is poorly understood. Here we show that physiological levels of autophagy act to promote survival in Caenorhabditis elegans during starvation, whereas insufficient or excessive levels of autophagy contribute to death. We found that inhibition of autophagy decreases survival of wild-type worms during starvation, and that muscarinic signaling regulates starvation-induced autophagy, at least in part, through the death-associated protein kinase signaling pathway. Furthermore, we found that in gpb-2 mutants, in which muscarinic signaling cannot be down-regulated, starvation induces excessive autophagy in pharyngeal muscles, which in turn, causes damage that may contribute to death. Taken together, our results demonstrate that autophagy can have either prosurvival or prodeath functions in an organism, depending on its level of activation.

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Figures

Figure 1.
Figure 1.
Autophagy is required for optimal survival of C. elegans during starvation. Starvation survival analyses were performed as described in Materials and Methods. Each of the starvation survival curves presented in this and subsequent figures is the result of one representative trial. Additional trials are listed in Table 1. (A) Percent of worms surviving to adulthood on NGM plates with HB101 bacteria after incubation in M9 buffer in the absence of food for the indicated time. Error bars are standard errors estimated assuming a Poisson distribution. (B) Quantitation of autophagy in the pharyngeal muscles of control RNAi animals and bec-1(RNAi) animals in A. Error bars indicate standard error for proportions. (*) P < 0.001. (C) Representative images of control RNAi animals after 9 d of starvation. Differential interference contrast image of control RNAi animals (left) and green fluorescence image (right). The arrow shows representative GFP∷LGG-1-positive punctate structures that label preautophagosomal and autophagosomal structures. In the inset, the area marked by the box is magnified and contrast enhanced. (D) Representative images of bec-1(RNAi) animals after 9 d of starvation. Differential interference contrast image of bec-1(RNAi) animals (left) and green fluorescence image (right). (E) Pumping rates in control RNAi animals and bec-1(RNAi) animals after starvation. Each dot represents an individual worm. Horizontal lines represent the average (line) and SEM (error bars). (*) P < 0.0001 (Student’s t-test). (F) Retrospective pumping rates of surviving and nonsurviving bec-1(RNAi) animals after starvation. Each dot represents an individual worm. Horizontal lines represent the average (line) and SEM (error bars). (*) P < 0.0001 (Student’s t-test).
Figure 2.
Figure 2.
Overactivated muscarinic signaling induces excessive levels of autophagy and causes death in gpb-2 mutants after starvation. (A,D,F,H) Quantitation of autophagy in the pharyngeal muscles of indicated genotype after incubation in M9 buffer in the absence of food with or without either atropine (10 mM) or U0126 (25 μM) for the indicated time. Error bars indicate standard error for proportions. (*) P < 0.0001 for all except 2 d in F and H, for which P < 0.005. See Supplementary Figure S1 for representative images. (B,E,G,I) Percent of animals surviving to adulthood in A, D, F, and H. Error bars are standard errors estimated assuming a Poisson distribution. (C) The relative levels of PE-conjugated LGG-1–GFP and LGG-1–GFP were determined by Western blotting. Starved L1 worms were prepared and analyzed by immunoblotting using an anti-GFP antibody. The arrows indicate LGG-1–GFP and PE-conjugated LGG-1–GFP. The Western blot is representative of what was seen in two independent experiments.
Figure 3.
Figure 3.
DAPK-1 acts downstream from or in parallel to muscarinic signaling in the regulation of autophagy, probably with RGS-2. (A,C) Quantitation of autophagy in the pharyngeal muscles of the indicated genotype after incubation in M9 buffer in the absence of food for the indicated time. Error bars indicate standard error for proportions. (*) P < 0.005; (**) P < 0.0001. (B,D) Percent of worms surviving to adulthood in A and C. Error bars are standard errors estimated assuming a Poisson distribution. (E) The relative levels of phospho-MPK-1 and MPK-1 were determined by Western blotting. Starved L1 worms were prepared 32.5 h after egg preparation and were analyzed by immunoblotting using an anti-phospho-MPK-1 antibody and an anti-MPK-1 antibody. The Western blot is representative of what was seen in two independent experiments.
Figure 4.
Figure 4.
Unrestrained autophagy causes defects in the pharyngeal muscles of gpb-2 mutants and contributes to death after starvation. (A) Percent of worms surviving to adulthood on NGM plates with HB101 bacteria after incubation in M9 buffer in the absence of food for the indicated time. Error bars are standard errors estimated assuming a Poisson distribution. (B) Quantitation of autophagy in A. bec-1(RNAi) reduced excessive levels of autophagy in the pharyngeal muscles of gpb-2 during starvation. Error bars indicate standard error for proportions. (*) P < 0.0001. See Supplementary Figure S1 for representative images. (C,D) gpb-2 control RNAi animals (C) and gpb-2; bec-1(RNAi) animals (D) after 2 d of starvation followed by growth on GFP-expressing E. coli. (Left) Differential interference contrast image. (Right) Green fluorescence image. Arrows in C indicate unground bacteria in the intestine. Bars, 20 μm.
Figure 5.
Figure 5.
Muscarinic signaling positively regulates autophagy during starvation. (A) Quantitation of autophagy in the pharyngeal muscles of wild-type animals treated with atropine (10 mM) after incubation in M9 buffer in the absence of food for the indicated time. (C) Quantitation of autophagy in the pharyngeal muscles of ras(gf) animals. Error bars indicate standard error for proportions. (*) P < 0.01. (B,D) Percent of worms surviving to adulthood in A and C. Error bars are standard errors estimated assuming a Poisson distribution. See Supplementary Figure S1 for representative images. (E) Model of dual roles of autophagy in the survival of C. elegans.
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