Age-associated and tissue-specific decline in autophagic activity in the nematode C. elegans

ABSTRACT

Macroautophagy/autophagy is a cellular recycling process that is required for the extended life span observed in many longevity paradigms, including in the nematode C. elegans. However, little is known regarding the spatiotemporal changes in autophagic activity in such long-lived mutants as well as in wild-type animals during normal aging. In a recent study, we report that autophagic activity decreases with age in several major tissues of wild-type C. elegans, including the intestine, body-wall muscle, pharynx, and nerve-ring neurons. Moreover, long-lived daf-2/insulin-signaling mutants and glp-1/Notch receptor mutants display increased autophagic activity, yet with different time- and tissue-specific differences. Notably, the intestine appears to be a critical tissue in which autophagy contributes to longevity in glp-1, but not in daf-2 mutants. Our findings indicate that autophagic degradation is reduced with age, possibly with distinct kinetics in different tissues, and that long-lived mutants increase autophagy in a tissue-specific manner, resulting in increased life span.

KEYWORDS:

• aging
• Atg8/LC3/LGG-1
• autophagy
• C. elegans
• daf-2
• glp-1
• tissue-specificity

Autophagy, a multistep cellular recycling pathway, plays important roles in many physiological processes, including aging and age-related diseases. Several conserved longevity paradigms, including reduced activity of the nutrient sensor MTOR, require autophagy genes for their life-span extension. Based on these studies, it has been proposed but not proven that autophagic activity declines with age, whereas in long-lived animals, autophagy may be increased allowing for life-span extension.

In a recent study, we provide evidence supporting this hypothesis. Our investigations reveal that in the nematode C. elegans, autophagic activity is reduced with age, whereas long-lived daf-2/insulin/IGF-1 receptor mutants and germline-less glp-1/Notch mutants maintain autophagic activity later in life in a distinct tissue-specific manner.

To monitor autophagic activity in C. elegans, we used GFP-tagged and newly constructed mCherry::GFP tandem-tagged LGG-1 (C. elegans ortholog of Atg8) reporters. Autophagy begins with the formation of a phagophore that elongates and engulfs cargo, forming a double-membrane autophagosome (AP). The lysosome fuses with the AP forming an autolysosome (AL) where acidic enzymes facilitate degradation of cargo (Fig. 1A). Using these two reporters, we quantified the number of GFP-positive puncta as a readout for APs. However, the acidity of the AL quenches GFP fluorescence; thus, we quantified the number of mCherry-only puncta as a readout for ALs. To infer whether autophagic activity is altered, we modulated autophagic flux by injecting bafilomycin A₁ (BafA), an inhibitor of lysosomal acidification, and monitored changes in fluorescent puncta numbers. With these tools, we assessed autophagic activity compared with age in a tissue-specific manner in wild-type and long-lived animals.

Figure 1. Spatiotemporal analysis of autophagic activity in C. elegans. (A) Different LGG-1/Atg8 reporters were used in combination with bafilomycin A₁ (BafA) to quantify fluorescent puncta as a readout for autophagosomes (AP) and/or autolysosomes (AL) to assess autophagic activity in tissues of C. elegans (i.e., intestine, pharynx [the foregut], body-wall muscle, and neurons. (B) Assessment of autophagic activity in young (top) and old wild-type (bottom) animals shows an age-associated decline. This trend was seen in all tissues, albeit with different kinetics. (C) Assessment of autophagic activity in long-lived mutants (i.e., in DAF-2/insulin/IGF-1 receptor daf-2(e1370) and GLP-1/Notch receptor glp-1(e2144) mutants) indicate a general increase in autophagic activity in young (top) and old (bottom) animals, compared to wild-type, and showed age- and tissue-specific differences between the 2 long-lived mutants. Future experiments are needed to directly test autophagic activity in C. elegans. See text for details. PG, phagophore.

Our studies indicate that in vivo autophagic activity decreases with age in the intestine, body-wall muscle, pharynx (the feeding organ of the animal), and nerve-ring neurons of wild-type C. elegans. While each of these major tissues show an overall decrease in autophagic activity, each have their own characteristic decline, with neurons possibly maintaining autophagic activity better in older animals (Fig. 1B). In turn, long-lived daf-2/InR mutants generally have increased numbers of APs and ALs compared to wild-type animals. Using the BafA autophagic flux assay, we observed that daf-2/InR mutants show a decline in autophagic activity with age in all tissues examined although to a lesser extent compared to wild type (Fig. 1C). Similarly, autophagic activity remains active throughout adulthood in all tissues examined of long-lived glp-1/Notch animals (Fig. 1C). Further investigation into autophagic rates is needed to accurately identify differences in autophagic flux between long-lived mutants and wild-type animals with age. Interestingly, we observed that while both daf-2/InR and glp-1/Notch mutants have active autophagy with age, they differ in the manner and the tissue in which this activity changes with age, suggesting that these mutants regulate longevity by different mechanisms.

To start addressing how tissue-specific autophagy may contribute to life-span extension, we focused on the intestine of daf-2/InR and glp-1/Notch mutants, where autophagy is active in both mutants. The intestine of daf-2/InR and glp-1/Notch animals has also been previously identified as a critical tissue for life span, because the downstream effector DAF-16, a FOXO transcription factor, functions in this tissue to ensure longevity. We used intestinal-specific RNAi to inhibit autophagy genes and observed that intestinal autophagy appears to be required in glp-1/Notch mutants, but not in daf-2/InR mutants. This finding further demonstrates the differential regulation of autophagy and longevity in these long-lived animals, and suggests that autophagic activity in the intestine may not be sufficient for longevity and instead, another tissue, such as muscle, or perhaps degradation of a specific cargo may play a role in a daf-2/InR animal's life-span extension

While this study represents the first comprehensive spatiotemporal analysis of autophagic activity in a living organism, we identified a number of critical technical issues to be cautious of in future studies using autophagy reporters. To confirm that GFP::LGG-1-positive puncta represent APs, we used a mutant GFP::LGG-1^G116A, that is incapable of being lipidated and inserted into the phagophore membrane, and should result in no observable puncta (Fig. 1A). However, in seam cells, a specialized hypodermal cell type, of glp-1/Notch animals, we observed fluorescent puncta using this mutant reporter, suggesting that these structures may not represent APs. While the identity of such GFP::LGG-1^G116A structures remains to be determined, these findings highlight the necessity of using lipidation-deficient reporters to verify that puncta represent APs in every tissue and genetic background monitored.

Using a tandem-LGG-1 reporter, we observed two new outcomes following BafA treatment. First, we observed no change in AP number but a change in AL number in the pharynx of wild-type, daf-2/InR and glp-1/Notch animals, which is consistent with active autophagy, and indicates that the tandem reporter provides additional information over the GFP reporter. Second, we found no change in AP or AL number following BafA treatment in the muscle of glp-1/Notch animals, which is consistent with a lack of autophagic activity. These two scenarios are not often observed, and additional experiments, including further analysis of LGG-1^G116A mutant reporters, are required to verify these observations (Fig. 1A). We also note that interpretation of changes in steady-state quantification of autophagic puncta can be difficult and quantification of the rates of each step is needed to convincingly solve this issue. Indeed, in this study we propose that steady-state puncta quantification may at times not be sufficient to infer autophagic activity, as increases in APs and ALs are consistent with multiple scenarios, i.e., no change, an increase, or a decrease in autophagic activity. Further characterization of the rates of autophagic degradation is critical to accurately measure autophagic flux and is necessary to confirm our conclusions.

Overall, our results are consistent with a decline in autophagic activity during wild-type C. elegans aging, whereas long-lived animals appear capable of maintaining autophagic activity in critical tissues, thereby increasing life span. Enhanced clearance of damaged organelles and proteins may be possible mechanisms by which long-lived animals utilize autophagy for life-span extension, and further investigation into this important notion is required. Identifying the regulation of autophagic activity provides a basis for understanding autophagy's role during aging and age-related disorders and will be important to guide therapeutic developments against such diseases.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Additional information

Funding

Glenn Foundation for Medical Research MH was supported by NIH/NIA [grant number R01 AG038664], [grant number R01 AG039756], and by a Julie Martin Mid-Career Award in Aging Research supposed by the Ellison Medical Foundation and AFAR.