Tissue-specific autophagy responses to aging and stress in C. elegans

Abstract

Cellular function relies on a balance between protein synthesis and breakdown. Macromolecular breakdown through autophagy is broadly required for cellular and tissue development, function, and recovery from stress. While Caenorhabditis elegans is frequently used to explore cellular responses to development and stress, the most common assays for autophagy in this system lack tissue-level resolution. Different tissues within an organism have unique functional characteristics and likely vary in their reliance on autophagy under different conditions. To generate a tissue-specific map of autophagy in C. elegans we used a dual fluorescent protein (dFP) tag that releases monomeric fluorescent protein (mFP) upon arrival at the lysosome. Tissue-specific expression of dFP::LGG-1 revealed autophagic flux in all tissues, but mFP accumulation was most dramatic in the intestine. We also observed variable responses to stress: starvation increased autophagic mFP release in all tissues, whereas anoxia primarily increased intestinal autophagic flux. We observed autophagic flux with tagged LGG-1, LGG-2, and two autophagic cargo reporters: a soluble cytoplasmic protein, and mitochondrial TOMM-7. Finally, an increase in mFP in older worms was consistent with an age-dependent shift in proteostasis. These novel measures of autophagic flux in C. elegans reveal heterogeneity in autophagic response across tissues during stress and aging.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans

“…Consistent with our observation that tald-1(RNAi) induces nuclear localization of HLH-30, we found that components of the autophagy pathway [62] were upregulated in a HLH-30-dependent fashion, including lgg-1 ( LC3 homolog), sqst-1 (p62/ SQSTM1 homolog), lmp-1 ( LAMP1 homolog), and lysosomal subunit vha-17 (Fig 6E and 6F). In addition, autophagic flux is increased by tald-1(RNAi) (Fig 6G and 6H), as measured by a recently described LGG-1 reporter of lysosomal protease activity [67]. The reporter consists of LGG-1 tagged with two fluorescent proteins containing a flexible protease-sensitive linker.…”

Disrupted Autophagy and Neuronal Dysfunction in C. elegans Knock-in Models of FUS Amyotrophic Lateral Sclerosis

“…Differences in neuronal and muscular autophagy in ALS FUS models may arise in part from tissue-specific variations in basal autophagy. Indeed, two previous studies showed marked differences in C. elegans neuronal and muscular autophagy [35,36].…”

“…LGG LGG-1 protein levels by Western Blot permits the quantitation of neuronal autophagy flux [36]. We found that ALS fust-1 alleles decreased the relative ratio of neuronal mFP to fulllength dFP::…”

“…To assess neuronal autophagy flux by Western Blot, we integrated a tandem-tagged neuronal LGG-1 dual fluorescent reporter (strain DLM11 from [36]) and crossed it into ALS fust-1 models. To synchronize different genetic populations, 100 gravid adults were allowed to lay eggs for 5 hours at 20 o C on large 60 ml NGM plates seeded with 2.4 ml of OP50.…”

“…LGG-1, mFP and actin from ALS fust-1 and fust-1WT C control animals, after overnight chloroquine treatment. Chloroquine inhibits lysosomal activity [36], allowing for visualization of neuronal mFP proteins in autolysosomes. Without chloroquine treatment, the mFP protein band was invisible.…”