Aspirin Activates Autophagy in C. elegans and Reduces Aging in an Autophagy Gene-Dependent Manner
(A) Representative confocal images (left panel) of GFP::LGG-1-expressing embryos treated with 1 mM aspirin compared to vehicle and quantification of GFP::LGG-1 puncta per embryo (right panel). Scale bar, 10 μm. Data represent means ± SEM of at least 15 images obtained across 2 independent experiments (∗∗∗p < 0.001, unpaired t test compared to vehicle-treated nematodes).
(B) Aspirin administration promotes proficient autophagic flux, as monitored by the reduction in levels of SQST-1/p62 autophagic substrate in the pharyngeal region of SQST-1::GFP transgenic animals at day 1 of adulthood. Representative images (left panel) and quantification (right panel) are shown. Scale bar, 20 μm. Data represent means ± SEM of n = 52 worms per group, pooled from three independent experiments (∗∗p < 0.01, unpaired t test).
(C) Aspirin stimulates autophagic flux. Representative confocal images (left panel) and quantification (right panel) of GFP::LGG-1 puncta in the hypodermal seam cells of L3–L4 larvae treated or not from the L4 stage of the first generation with 1 mM aspirin in the absence or in the presence of bafilomycin A1 (100 μg/mL). Scale bar, 10 μm. Data represent means ± SEM of n = 196–329 seam cells, pooled from two independent experiments (∗∗∗p < 0.001, one-way ANOVA compared to vehicle – BafA1 treatment; ###p < 0.001, one-way ANOVA compared to vehicle + BafA1 treatment).
(D) Administration of aspirin induces the autophagy-dependent increase of pnhx-2mCherry::LGG-1 puncta in the intestine of 2-day-old adults, which is lost upon siRNA-mediated depletion of BEC-1 and ATG-7. Epifluorescence images are depicted in the left panel (magnification indicates mCherry::LGG-1 puncta as detected in aspirin-treated worms) and quantified in the right panel. Scale bar, 100 μm. Data represent means ± SEM of n = 15–28 worms per group, pooled from two independent experiments (∗∗∗p < 0.001, one-way ANOVA compared to vehicle Co RNAi).
(E) CBP-1 depletion is epistatic to aspirin-induced autophagy. RNAi-driven elimination of cbp-1 in transgenic animals expressing the GFP::LGG-1 reporter leads to an increase in the number of GFP::LGG-1 puncta, which are not further increased by aspirin administration. Representative confocal images (left panel) and corresponding quantification (right panel) are shown. Scale bar, 10 μm. Data represent means ± SEM (##p < 0.005 and ∗∗p < 0.01, unpaired t test compared to Co RNAi-vehicle condition).
(F) Salicylate induces autophagy in C. elegans. Representative confocal images (left panel) and corresponding quantification (right panel) of GFP::LGG-1 puncta in transgenic embryos treated with vehicle or salicylate (1 mM). Vehicle bar is shared with experiments depicted in (A) as assays were conducted in parallel. Data represent means ± SEM of at least 15 images obtained in 2 independent experiments (∗∗∗p < 0.001, unpaired t test compared to vehicle-treated nematodes).
(G) Knockdown of dct-1, a putative ortholog to the mammalian NIX/BNIP3L and BNIP3, reduces the number of GFP::LGG-1-positive foci in the epidermis of aspirin-treated young adult wild-type animals. Representative confocal images (left panel) and corresponding quantification (right panel) are depicted. Scale bar, 10 μm. Values represent means ± SEM (∗∗p < 0.01, unpaired t test compared with Co RNAi vehicle; #p < 0.05, unpaired t test compared with Co RNAi aspirin). Co RNAi vehicle and aspirin bars are shared with data depicted in (E), as assays were conducted in parallel.
(H) Mitophagy is induced in nematodes treated with aspirin. Transgenic animals expressing the mt-Rosella biosensor in the body wall muscle cells were treated with 1 mM aspirin or vehicle control. Mitophagy induction is signified by the reduction of the ratio between pH-sensitive GFP to pH-insensitive DsRed. Data represent means ± SEM of n = 22–33 worms per group, pooled from two independent experiments (∗∗∗p < 0.001, unpaired t test).