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Inhibition of early-acting autophagy genes in C. elegans neurons improves protein homeostasis, promotes exopher production, and extends lifespan via the ATG-16.2 WD40 domain

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Abstract

While autophagy is key to maintain cellular homeostasis, tissue-specific roles of individual autophagy genes are less understood. To study neuronal autophagy in vivo, we inhibited autophagy genes specifically in C. elegans neurons, and unexpectedly found that knockdown of early-acting autophagy genes, i.e., involved in formation of the autophagosome, except for atg-16.2, decreased PolyQ aggregates and increased lifespan, albeit independently of the degradation of autophagosomal cargo. Neuronal aggregates can be secreted from neurons via vesicles called exophers, and we found that neuronal inhibition of early-acting autophagy genes atg-7 and lgg-1/Atg8, but not atg-16.2 increased exopher formation. Moreover, atg-16.2 mutants were unable to form exophers, and atg-16.2 was required for the effects of early autophagy gene reduction on neuronal PolyQ aggregation, exopher formation, and lifespan. Notably, neuronal expression of full-length ATG-16.2 but not ATG-16.2 without a functional WD40 domain, important for non-canonical functions of ATG16L1 in mammalian cells, restored these phenotypes. Collectively, we discovered a specific role for C. elegans ATG-16.2 and its WD40 domain in exopher biogenesis, neuronal proteostasis, and lifespan determination, highlighting a possible role for non-canonical autophagy functions in both exopher formation and in aging.

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... We speculate that young adult physiology might be temporally tweaked such that some tissues have optimized capacity to manage/degrade large aggregates and organelles at an early adult developmental 'clean up' time, possibly analogous to how a town service for bulky oversized garbage pick-up might be limited to particular days during the year. As exopher production appears generally beneficial for neuronal function and survival (Melentijevic et al., 2017;Yang et al., 2022), the early life extrusion phase appears a positive feature of reproductive life. More broadly, proteome 'clean up' phases may be programmed as key steps at specific transitions during development and homeostasis, for example, as occurs in the temporal lysosome activation that clears aggregate debris in C. elegans maturing oocytes (Bohnert and Kenyon, 2017) or in the maturation of mouse adult neuronal stem cells via vimentin-dependent proteasome activity during quiescence exit (Morrow et al., 2020). ...
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Preprint
Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.
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Autophagy is an intracellular bulk degradation system, through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Microtuble-associated protein light chain 3 (LC3), a mammalian homolog of yeast Atg8, has been used as a specific marker to monitor autophagy. Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes. Therefore, changes in LC3 localization have been used to measure autophagy. However, this method has some limitations. In this report, we show that LC3 protein tends to aggregate in an autophagy-independent manner when it is transiently overexpressed by transfection. In addition, LC3 is easily incorporated into intracellular protein aggregates, such as inclusion bodies induced by polyQ expression or formed in autophagy-deficient hepatocytes, neurons, or senescent fibroblasts. These findings demonstrate that punctate dots containing LC3 do not always represent autophagic structures. Therefore, LC3 localization should be carefully interpreted, particularly if LC3 is overexpressed by transient transfection or if aggregates are formed within cells.
Enhanced neuronal 836 RNAi in C. elegans using SID-1
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The complete cell atlas of an aging multicellular organism
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Atg16L1, an essential factor 898 for canonical autophagy, participates in hormone secretion from PC12 cells 899 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted
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Ishibashi, K., Uemura, T., Waguri, S. & Fukuda, M. Atg16L1, an essential factor 898 for canonical autophagy, participates in hormone secretion from PC12 cells 899 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted December 14, 2022. ; https://doi.org/10.1101/2022.12.12.520171 doi: bioRxiv preprint independently of autophagic activity. Mol Biol Cell 23, 3193-3202, 900 doi:10.1091/mbc.E12-01-0010 (2012).
Loss of autophagy in the central nervous system causes 911 neurodegeneration in mice
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