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Autophagy protein ATG-16.2 and its WD40 domain mediate the beneficial effects of inhibiting early-acting autophagy genes in C. elegans neurons

January 2024
Nature Aging 4(2):1-15

Authors:

Rutgers, The State University of New Jersey

While autophagy genes are required for lifespan of long-lived animals, their tissue-specific roles in aging remain unclear. Here, we inhibited autophagy genes in Caenorhabditis elegans neurons, and found that knockdown of early-acting autophagy genes, except atg-16.2, increased lifespan, and decreased neuronal PolyQ aggregates, independently of autophagosomal degradation. Neurons can secrete protein aggregates via vesicles called exophers. Inhibiting neuronal early-acting autophagy genes, except atg-16.2, increased exopher formation and exopher events extended lifespan, suggesting exophers promote organismal fitness. Lifespan extension, reduction in PolyQ aggregates and increase in exophers were absent in atg-16.2 null mutants, and restored by full-length ATG-16.2 expression in neurons, but not by ATG-16.2 lacking its WD40 domain, which mediates noncanonical functions in mammalian systems. We discovered a neuronal role for C. elegans ATG-16.2 and its WD40 domain in lifespan, proteostasis and exopher biogenesis. Our findings suggest noncanonical functions for select autophagy genes in both exopher formation and in aging.

Neuronal expression of SID-1, an RNA channel protein, leads to RNAi-competent neurons (a) Mean GFP fluorescence intensity in head region of rgef-1p::gfp animals on day 1 to day 12 of adulthood, relative to day 1. Error bars are s.d. of n = 3 experiments, with n = 29, 34, 32, 24, 28, 19 animals over 3 independent experiments. ns P = 0.78, P = 0.05, P = 0.116, ****P < 0.0001, **P = 0.005, by one-way ANOVA with Dunnett’s multiple comparisons test. (b-f) Wild-type animals (N2, WT), sid-1; rgef-1p::sid-1 + rgef-1p::gfp, and sid-1 mutants after whole-life RNAi against the indicated gene with tissue-specific functions, compared to control (CTRL). (b) Representative animals are shown with WT animals displaying paralysis (Prz) on unc-112 RNAi, larval arrest (Lva) on rpl-2 RNAi, blister formation (Bli) and larval arrest (Lva) on bli-1 RNAi, and clear (Clr) and larval arrest on elt-2 RNAi. Mean percent phenotypic penetrance after knockdown of genes with functions in (c) body-wall muscle; unc-22 – twitching and uncoordinated movement (Unc) (n = 8 experiments, ****P < 0.0001) and unc-112 – paralysis (n = 7 experiments, ****P < 0.0001), (d) a ubiquitous manner; rpl-2 – larval arrest (n = 8 experiments, ****P < 0.0001), (e) hypodermis; tsp-15 – blisters (n = 8 experiments, ****P < 0.0001) and bli-1 – blisters and larval arrest; (f) intestine; elt-2 – clear and larval arrest. Error bars are s.d. (g) Mean GFP fluorescence intensity in head region of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp rgef-1p::gfp animals after whole-life gfp RNAi compared to control (CTRL). Error bars are s.d. with n = 30 over 3 independent experiments. ****P < 0.0001, **P = 0.005, by two-tailed Student’s t-test. Scale bar: 100 µm. (h) Mean GFP fluorescence intensity in head region of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp rgef-1p::gfp animals after whole-life lgg-1 RNAi (n = 32) compared to control (CTRL) (n = 35). Error bars are s.d. over 3 independent experiments. ****P < 0.0001 by two-tailed Student’s t-test. Scale bar: 100 µm. (i) Mean percent of shrinker phenotype in day 2 WT, rde-1; unc-47p::rde-1::SL2::sid-1 (capable of GABA neuron-specific RNAi), sid-1; rgef-1p::sid-1 + rgef-1p::gfp, or sid-1 animals after two generations of whole-life snb-1 or unc-13 RNAi compared to control (CTRL). Error bars are s.d. with ****P < 0.0001 and ns P > 0.99 by one-way ANOVA with Dunnett’s multiple comparisons test. Source data

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Healthspan and neuronal phenotypes of animals after neuronal inhibition of atg-7 and lgg-1/ATG8 (a) Mean body bends per 20 s of sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after whole-life atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Error bars are s.e.m. of one representative experiment, each with n = 16 animals. Experiment was performed three times with similar results. Linear regression comparison versus CTRL: atg-7 RNAi: Pslope = 0.5; Py-intercept = 0.02; lgg-1/ATG8 RNAi: Pslope = 0.3; Py-intercept = 0.01. (b) Mean number of contractions in the terminal pharyngeal bulb per 30 s of sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after whole-life atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Error bars are s.d. of n = 30 animals over 3 independent experiments. Linear regression comparison versus CTRL: atg-7 RNAi: Pslope = 0.4; Py-intercept = 0.4; lgg-1 RNAi: Pslope = 0.3; Py-intercept = 0.5. (c) Mean number of progeny produced per day in sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after atg-7 (n = 19 animals), or lgg-1/ATG8 RNAi (n = 18 animals) compared to control (CTRL) (n = 22 animals) over 2 independent experiments. Error bars are s.d. CTRL versus atg-7 RNAi: ns P = 0.72, 0.89, 0.91, 0.82, >0.99; CTRL versus lgg-1/ATG8 RNAi: ns P = 0.95, 0.96,0.60, 0.70, >0.99, by two-way ANOVA with Dunnett’s multiple comparisons test. (d) Analysis of integrity of sensory neurons in day 5 sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Sensory mutants daf-10(e1387) and osm-6(p811) are negative controls. Shown are representative images of n = 10 animals. Experiment was performed three times with similar results. Scale bar, 20 μm. (e) Representative image of neuronal branch (arrowhead) from ALM neuron in day 15 sid-1; rgef-1p::sid-1+ rgef-1p::gfp animals. Scale bar: 20 µm. Mean percent of animals with branches after whole-life atg-7, or lgg-1/ATG8 dsRNA compared to control (CTRL) of n = 4 experiments. Error bars are s.d. ***P = 0.0002, ****P < 0.0001 by Cochran-Mantel-Haenszel test. (f) Mean chemotaxis index of day 5 sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after whole-life atg-7, or lgg-1/ATG8 RNAi using the chemoattractant butanone. Error bars are 95% C.I. of n = 4 experiments. *P = 0.048, **P = 0.0094, by one-way ANOVA with Dunnett’s multiple comparisons test. Source data

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Autophagy status is unchanged in animals expressing sid-1 and in non-neuronal tissues after neuronal knockdown of early-acting autophagy genes (a) Mean neuronal GFP::LGG-1 and GFP::LGG-1(G116A) punctae in day 1 wild-type (sid-1 + /+) (n = 28, 29) and sid-1(qt9) (sid-1-/-) animals (n = 27, 27) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 26, 29). Error bars are s.d. over 3 independent experiments. Comparison between strains: LGG-1: ns P = 0.75, P = 0.97, G116A: ns P = 0.98, P > 0.99. Comparison of lipidated and unlipidated structures: ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparisons test. (b) Mean sqst-1p::sqst-1::gfp fluorescence intensity in head region of day 1 wild-type (sid-1 + /+), and sid-1(qt9) (sid-1(-/-)) animals with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) on day 1 of adulthood. Error bars are s.d. of n = 31 animals over 3 independent experiments. ns P = 0.18 and P = 0.59 by one-way ANOVA with Dunnett’s multiple comparisons test. (c) GFP::LGG-1 punctae in intestinal cells of day 1 wild-type (sid-1 + /+) (n = 59) and sid-1(qt9) (sid-1-/-) animals (n = 62) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 62). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.79, P = 0.99 by one-way ANOVA with Dunnett’s multiple comparisons test. (d) GFP::LGG-1 punctae in body-wall muscle areas of day 1 wild-type (sid-1 + /+) (n = 48) and sid-1(qt9) (sid-1-/-) animals (n = 45) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 52). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.64, P = 0.70 by one-way ANOVA with Dunnett’s multiple comparisons test. (e) GFP::LGG-1 punctae in intestinal cells of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; lgg-1p::gfp::lgg-1 animals after whole-life atg-7 (n = 65), or lgg-1/ATG8 (n = 48) RNAi compared to control (CTRL) (n = 81). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.98, P = 0.71 by one-way ANOVA with Dunnett’s multiple comparisons test. (f) GFP::LGG-1 punctae in body-wall muscle areas of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; lgg-1p::gfp::lgg-1 animals after whole-life atg-7 (n = 54), or lgg-1/ATG8 (n = 53) RNAi compared to control (CTRL) (n = 49). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.34, P = 0.96 by one-way ANOVA with Dunnett’s multiple comparisons test. Source data

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Neuronal PolyQ aggregation, exopher formation and lifespan extension are correlated (a) Number of neuronal PolyQ aggregates in day 7 rgef-1::Q40::yfp wild-type (sid-1 + /+) (n = 41) and sid-1(qt9) (sid-1-/-) animals (n = 48 with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 42). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.79, P = 0.82 by one-way ANOVA with Dunnett’s multiple comparisons test. (b) Mean percent of ALMR neurons with exophers of day 2 mec-4p::mCherry wild-type (sid-1 + /+) (n = 228 animals) and sid-1(qt9) (sid-1-/-) (n = 262 animals) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 295 animals). Error bars are s.d. of n = 7 experiments, ns P = 0.62, P = 0.54 by two-sided Cochran-Mantel-Haenszel test. (c) Mean percent of ALMR neurons with exophers of day 2 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; mec-4p::mCherry animals (n = 247 animals) after adult-only atg-7 (n = 271 animals), or lgg-1/ATG8 (n = 285 animals) RNAi compared to control (CTRL). Error bars are s.d. of n = 6 experiments, *P = 0.028, P = 0.00006 by two-sided Cochran-Mantel-Haenszel test. (d) Mean percent of ALMR neurons with exophers of day 2 mec-4p::mCherry animals (n = 151 animals) after whole-life atg-7 (n = 157 animals), or lgg-1/ATG8 (n = 180 animals) RNAi compared to control (CTRL). Error bars are s.d. of n = 5 experiments, ns P = 0.39, P = 0.53 by two-sided Cochran-Mantel-Haenszel test. (e-g) Percent mean lifespan (LS) change (Fig. 1b), number of neuronal PolyQ aggregates (Fig. 3b), and mean percent of AMLR neurons with exophers (Fig. 4b) plotted against each other with simple linear regression (solid line with 95% C.I. as dashed lines). Numbers refer to specific RNAi treatment; ¹unc-51/ATG1, ²atg-13, ³bec-1/BECN1, ⁴atg-9, ⁵atg-16.2, ⁶atg-7, ⁷atg-4.1, ⁸lgg-1/ATG8, ⁹cup-5, ¹⁰epg-5, ¹¹vha-13, ¹²vha-15, ¹³vha-16. P values determined by two-sided Spearman correlation test. Source data

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Atg-16.2 and atg-4.1 mutants display similar RNAi phenotypes (a-d) Phenotypes of day 2 WT, atg-16.2(ok3224), atg-4.1(bp501), and sid-1(qt9) animals after whole-life RNAi against a specific gene expressed in different tissues. Mean percent phenotypic penetrance after knockdown of genes with functions in (a) body-wall muscle; unc-22 – twitching and uncoordinated movement (Unc) and unc-112 – paralysis, (b) a ubiquitous manner; rpl-2 – larval arrest, (c) hypodermis; tsp-15 – blisters and bli-1 – blisters and larval arrest; (d) intestine; elt-2 – clear and larval arrest. Error bars are s.d. of n = 4 experiments. (a) unc-22 RNAi: ns P = 0.59, P = 0.87, ****P < 0.0001. unc-112 RNAi: ns P = 0.92, P = 0.96, ****P < 0.0001. (b) rpl-2 RNAi: ns P = 0.82, P = 0.96, ****P < 0.0001. (c) tsp-15 RNAi: ns P = 0.99, P = 0.64, ****P < 0.0001. bli-1 RNAi: ns P = 0.24, P = 0.99, ****P < 0.0001. (d) elt-2 RNAi: ns P = 0.76, P = 0.18, ****P < 0.0001 by one-way one-way ANOVA with Dunnett’s multiple comparisons test. Source data

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Nature Aging | Volume 4 | February 2024 | 198–212 198

nature aging

Article

https://doi.org/10.1038/s43587-023-00548-1

Autophagy protein ATG-16.2 and its WD40

domain mediate the beneficial effects of

inhibiting early-acting autophagy genes in

C. elegans neurons

Yongzhi Yang 1,5, Meghan Lee Arnold 2, Caitlin M. Lange1, Ling-Hsuan Sun3,4,

Michael Broussalian3, Saam Doroodian3, Hiroshi Ebata 3, Elizabeth H. Choy1,

Karie Poon1, Tatiana M. Moreno 1, Anupama Singh 1, Monica Driscoll2,

Caroline Kumsta 1 & Malene Hansen 1,3

While autophagy genes are required for lifespan of long-lived animals, their

tissue-specic roles in aging remain unclear. Here, we inhibited autophagy

genes in Caenorhabditis elegans neurons, and found that knockdown of

early-acting autophagy genes, except atg-16.2, increased lifespan, and

decreased neuronal PolyQ aggregates, independently of autophagosomal

degradation. Neurons can secrete protein aggregates via vesicles called

exophers. Inhibiting neuronal early-acting autophagy genes, except atg-

16.2, increased exopher formation and exopher events extended lifespan,

suggesting exophers promote organismal tness. Lifespan extension,

reduction in PolyQ aggregates and increase in exophers were absent in

atg-16.2 null mutants, and restored by full-length ATG-16.2 expression in

neurons, but not by ATG-16.2 lacking its WD40 domain, which mediates

noncanonical functions in mammalian systems. We discovered a neuronal

role for C. elegans ATG-16.2 and its WD40 domain in lifespan, proteostasis

and exopher biogenesis. Our ndings suggest noncanonical functions for

select autophagy genes in both exopher formation and in aging.

Macroautophagy (hereafter autophagy) is an intracellular recycling

process by which cytosolic cargo is subjected to lysosomal degradation,

referred to here as canonical autophagy. Autophagy plays important

roles in numerous late-onset diseases including neurodegenerative

disorders and has been directly linked to aging in multiple model organ-

isms including the nematode C. elegans1. In this organism, RNA inter-

ference (RNAi) of multiple autophagy (Atg) genes during adulthood

abrogates lifespan extension in long-lived mutants, which together

with data from other organisms suggest that autophagy is required for

longevity

. In contrast, RNAi inhibition of autophagy genes in wild-type

(WT) C. elegans typically has limited effects on lifespan1, indicating that

basal autophagy is not restricting normal aging. Still, the tissue-specific

contributions of autophagy to organismal fitness and longevity remain

unclear. The role of autophagy genes in neurons is of special interest

because neuronal signaling plays key roles in several longevity para-

digms

. Moreover, neuronal overexpression of Atg1 or Atg8 extends

Drosophila lifespan3,4, whereas loss of either the autophagy gene Atg5

or Atg7 specifically in neurons causes neurodegeneration in mice

5,6

Received: 13 March 2023

Accepted: 27 November 2023

Published online: 4 January 2024

Check for updates

1Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA. 2Rutgers, The State University of New Jersey, Nelson Biological Labs, Piscataway,

NJ, USA. 3Buck Institute for Aging Research, Novato, CA, USA. 4Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA,

USA. 5Present address: Scripps Research Institute, La Jolla, CA, USA. e-mail: ckumsta@sbpdiscovery.org; mhansen@buckinstitute.org

Content courtesy of Springer Nature, terms of use apply. Rights reserved

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Full-text available

Dec 2021

Maintenance of cellular homeostasis is critically important for the survival of cells and organisms. Degradation and recycling of biomolecules and whole organelles is an essential mechanism for maintaining cellular homeostasis. The main systems responsible for these processes are the ubiquitin-proteasome system and autophagy-lysosome pathway. Another mechanism was reported in c. elegans--formation of large, membrane-enclosed projections called exophers, into which cells direct debris and toxic protein aggregates. Exophers were shown to act as large, temporary disposal compartments that disconnected from the cells within several hours. Here, we report the discovery of exophers in the mammalian brain, including the brains of humans and mice. Similar to those described in nematodes, the mammalian exophers appear to emanate from the cell body, initially connected by a nanotube, and eventually disconnect. Rare, innate exophers were found in healthy human brain and primary neurons from wild-type mice, presumably mediating transfer of large cargo between cells. The number of exophers increased as an adaptive response under proteostatic stress, e.g., in Alzheimer's disease brain or in primary neurons from two tauopathy mouse models, where the exophers likely facilitated expulsion of proteotoxic material. Our findings suggest that exopherogenesis is a rare, innate house-keeping process that is elevated adaptively in response to proteostatic pressure and is a conserved mechanism from nematodes to humans.

Muscle-derived exophers promote reproductive fitness

Article

Full-text available

Jul 2021
EMBO REP

Organismal functionality and reproduction depend on metabolic rewiring and balanced energy resources. However, the crosstalk between organismal homeostasis and fecundity and the associated paracrine signaling mechanisms are still poorly understood. Using Caenorhabditis elegans, we discovered that large extracellular vesi-cles (known as exophers) previously found to remove damaged subcellular elements in neurons and cardiomyocytes are released by body wall muscles (BWM) to support embryonic growth. Exopher formation (exopheresis) by BWM is sex-specific and a non-cell autonomous process regulated by developing embryos in the uterus. Embryo-derived factors induce the production of exophers that transport yolk proteins produced in the BWM and ultimately deliver them to newly formed oocytes. Consequently, offspring of mothers with a high number of muscle-derived exophers grew faster. We propose that the primary role of muscular exopheresis is to stimulate reproductive capacity, thereby influencing the adaptation of worm populations to the current environmental conditions.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Article

Full-text available

Apr 2021
MOL CELL

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway.

Cell non-autonomous regulation of health and longevity

Article

Full-text available

Dec 2020
eLife

As the demographics of the modern world skew older, understanding and mitigating the effects of aging is increasingly important within biomedical research. Recent studies in model organisms demonstrate that the aging process is frequently modified by an organism’s ability to perceive and respond to changes in its environment. Many well-studied pathways that influence aging involve sensory cells, frequently neurons, that signal to peripheral tissues and promote survival during the presence of stress. Importantly, this activation of stress response pathways is often sufficient to improve health and longevity even in the absence of stress. Here, we review the current landscape of research highlighting the importance of cell non-autonomous signaling in modulating aging from C. elegans to mammals. We also discuss emerging concepts including retrograde signaling, approaches to mapping these networks, and development of potential therapeutics.

Individual cell types in C. elegans age differently and activate distinct cell-protective responses

Article

Aug 2023

Aging is characterized by a global decline in physiological function. However, by constructing a complete single-cell gene expression atlas, we find that Caenorhabditis elegans aging is not random in nature but instead is characterized by coordinated changes in functionally related metabolic, proteostasis, and stress-response genes in a cell-type-specific fashion, with downregulation of energy metabolism being the only nearly universal change. Similarly, the rates at which cells age differ significantly between cell types. In some cell types, aging is characterized by an increase in cell-to-cell variance, whereas in others, variance actually decreases. Remarkably, multiple resilience-enhancing transcription factors known to extend lifespan are activated across many cell types with age; we discovered new longevity candidates, such as GEI-3, among these. Together, our findings suggest that cells do not age passively but instead react strongly, and individualistically, to events that occur during aging. This atlas can be queried through a public interface.

Emerging roles of extracellular vesicles in polyglutamine diseases: Mutant protein transmission, therapeutic potential, and diagnostics

Article

May 2022
NEUROCHEM INT

Polyglutamine (PolyQ) diseases are a group of inherited neurodegenerative diseases including Huntington's disease and several types of spinocerebellar ataxias, which are caused by aggregation and accumulation of the disease-causative proteins with an abnormally expanded PolyQ stretch. Extracellular vesicles (EVs) are membrane particles that are released from cells, including exosomes, microvesicles, and other extracellular particles. Recent studies have suggested that the PolyQ proteins, which are the disease-causative proteins of PolyQ diseases, and its aggregates are secreted via EVs, similar to the aggregation-prone proteins associated with other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The PolyQ proteins that are secreted from cells can transmit intercellularly, which may contribute to pathological propagation of the PolyQ protein aggregates in patient brain, and therefore, the pathological roles of EVs in the onset and progression of PolyQ diseases has attracted much attention. EVs may also mediate intercellular transfer of heat shock proteins and other neuroprotective factors, which are beneficial for protein homeostasis and cell survival, and thus, have therapeutic potential for the neurodegenerative diseases including PolyQ diseases. Furthermore, because EVs contain not only the disease-associated proteins, but also various proteins, miRNAs and other components, and changes in the levels of these contents might reflect pathological changes, EVs derived from blood, cerebrospinal fluid, and urine would be a potential source of minimally invasive diagnostic biomarkers that report disease-associated changes in PolyQ diseases. In this review, we summarize the current understanding of the pathological roles of EVs in PolyQ diseases, and therapeutic and diagnostic potential of EVs for these diseases. Elucidation of the pathological and physiological roles of EVs would lead to identification of a proper therapeutic target that would not interfere the protective roles of EVs for cell survival but suppress pathological propagation of the disease-causative proteins in PolyQ disease.

Beyond Autophagy: The Expanding Roles of ATG8 Proteins

Article

Feb 2021
TRENDS BIOCHEM SCI

The ATG8 family proteins are critical players in autophagy, a cytoprotective process that mediates degradation of cytosolic cargo. During autophagy, ATG8s conjugate to autophagosome membranes to facilitate cargo recruitment, autophagosome biogenesis, transport, and fusion with lysosomes, for cargo degradation. In addition to these canonical functions, recent reports demonstrate that ATG8s are also delivered to single-membrane organelles, which leads to highly divergent degradative or secretory fates, vesicle maturation, and cargo specification. The association of ATG8s with different vesicles involves complex regulatory mechanisms still to be fully elucidated. Whether individual ATG8 family members play unique canonical or non-canonical roles, also remains unclear. This review summarizes the many open molecular questions regarding ATG8s that are only beginning to be unraveled.

Autophagy protein ATG-16.2 and its WD40 domain mediate the beneficial effects of inhibiting early-acting autophagy genes in C. elegans neurons

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