Abstract
Autophagy has been linked to longevity in many species, but the underlying mechanisms are unclear. Using a GFP-tagged and a new tandem-tagged Atg8/LGG-1 reporter, we quantified autophagic vesicles and performed autophagic flux assays in multiple tissues of wild-type Caenorhabditis elegans and long-lived daf-2/insulin/IGF-1 and glp-1/Notch mutants throughout adulthood. Our data are consistent with an age-related decline in autophagic activity in the intestine, body-wall muscle, pharynx, and neurons of wild-type animals. In contrast, daf-2 and glp-1 mutants displayed unique age- and tissue-specific changes in autophagic activity, indicating that the two longevity paradigms have distinct effects on autophagy during aging. Although autophagy appeared active in the intestine of both long-lived mutants, inhibition of intestinal autophagy significantly abrogated lifespan extension only in glp-1 mutants. Collectively, our data suggest that autophagic activity normally decreases with age in C. elegans, whereas daf-2 and glp-1 long-lived mutants regulate autophagy in distinct spatiotemporal-specific manners to extend lifespan.
Highlights
Macroautophagy is a multistep cellular recycling process in which cytosolic components are encapsulated in membrane vesicles and degraded in the lysosome
We confirmed that the GFP::LGG-1-positive punctae likely represented APs and not GFP::LGG-1 aggregates, since WT C. elegans expressing a mutant form of GFP-tagged LGG-1/Atg8 protein (G116A) that is expected to be defective in lipidation and autophagosomemembrane targeting (Manil-Segalen et al, 2014) showed no punctae formation in the intestine or muscle, and only a small increase in the pharynx over this 10-day time period (Figure 1—figure supplement 1, see figure legend for comment on neurons, which have yet to be evaluated in detail)
We found that (i) aging of wild-type (WT) animals is accompanied by increased numbers of autophagic vesicles in all tissues examined that likely reflects a reduction in autophagy activity; (ii) long-lived daf-2 insulin/IGF-1 mutants and germline-less glp-1 mutants differentially regulate autophagy spatially and temporally compared to WT animals, and (iii) glp-1 mutants, but not daf-2 mutants, require autophagy genes in the intestine for
Summary
Macroautophagy (hereafter referred to as autophagy) is a multistep cellular recycling process in which cytosolic components are encapsulated in membrane vesicles and degraded in the lysosome. APs fuse with lysosomes to form autolysosomes (AL), where degradation of cargo takes place (Mizushima, 2007). As interest in this pathway and its pathophysiological roles has increased, it has become clear that measurement of autophagic vesicle levels at steady state, without monitoring the overall pathway flux, can lead to controversial results. Autophagy is commonly monitored by enumerating APs under steady-state conditions, referred to as the AP pool size (Loos et al, 2014), using a GFPtagged Atg marker. A tandem-tagged mCherry-GFP-Atg reporter, which monitors both IMs/APs (yellow [green/ red] punctae) and ALs (red punctae due to GFP fluorescence quenching in the acidic autolysosome environment) can help distinguish between these possibilities (Kimura et al, 2007). When used in combination with chemical inhibitors of autophagy, such as
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