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

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.

Intermediate filaments associate with aggresome-like structures in proteostressed C. elegans neurons and influence large vesicle extrusions as exophers.

Toxic protein aggregates can spread among neurons to promote human neurodegenerative disease pathology. We found that in C. elegans touch neurons intermediate filament proteins IFD-1 and IFD-2 associate with aggresome-like organelles and are required cell-autonomously for efficient production of neuronal exophers, giant vesicles that can carry aggregates away from the neuron of origin. The C. elegans aggresome-like organelles we identified are juxtanuclear, HttPolyQ aggregate-enriched, and dependent upon orthologs of mammalian aggresome adaptor proteins, dynein motors, and microtubule integrity for localized aggregate collection. These key hallmarks indicate that conserved mechanisms drive aggresome formation. Furthermore, we found that human neurofilament light chain (NFL) can substitute for C. elegans IFD-2 in promoting exopher extrusion. Taken together, our results suggest a conserved influence of intermediate filament association with aggresomes and neuronal extrusions that eject potentially toxic material. Our findings expand understanding of neuronal proteostasis and suggest implications for neurodegenerative disease progression.

Large vesicle extrusions from C. elegans neurons are consumed and stimulated by glial-like phagocytosis activity of the neighboring cell.

Caenorhabditis elegans neurons under stress can produce giant vesicles, several microns in diameter, called exophers. Current models suggest that exophers are neuroprotective, providing a mechanism for stressed neurons to eject toxic protein aggregates and organelles. However, little is known of the fate of the exopher once it leaves the neuron. We found that exophers produced by mechanosensory neurons in C. elegans are engulfed by surrounding hypodermal skin cells and are then broken up into numerous smaller vesicles that acquire hypodermal phagosome maturation markers, with vesicular contents gradually degraded by hypodermal lysosomes. Consistent with the hypodermis acting as an exopher phagocyte, we found that exopher removal requires hypodermal actin and Arp2/3, and the hypodermal plasma membrane adjacent to newly formed exophers accumulates dynamic F-actin during budding. Efficient fission of engulfed exopher-phagosomes to produce smaller vesicles and degrade their contents requires phagosome maturation factors SAND-1/Mon1, GTPase RAB-35, the CNT-1 ARF-GAP, and microtubule motor-associated GTPase ARL-8, suggesting a close coupling of phagosome fission and phagosome maturation. Lysosome activity was required to degrade exopher contents in the hypodermis but not for exopher-phagosome resolution into smaller vesicles. Importantly, we found that GTPase ARF-6 and effector SEC-10/exocyst activity in the hypodermis, along with the CED-1 phagocytic receptor, is required for efficient production of exophers by the neuron. Our results indicate that the neuron requires specific interaction with the phagocyte for an efficient exopher response, a mechanistic feature potentially conserved with mammalian exophergenesis, and similar to neuronal pruning by phagocytic glia that influences neurodegenerative disease.

Stress increases in exopher-mediated neuronal extrusion require lipid biosynthesis, FGF, and EGF RAS/MAPK signaling.

In human neurodegenerative diseases, neurons can transfer toxic protein aggregates to surrounding cells, promoting pathology via poorly understood mechanisms. In Caenorhabditis elegans, proteostressed neurons can expel neurotoxic proteins in large, membrane-bound vesicles called exophers. We investigated how specific stresses impact neuronal trash expulsion to show that neuronal exopher production can be markedly elevated by oxidative and osmotic stress. Unexpectedly, we also found that fasting dramatically increases exophergenesis. Mechanistic dissection focused on identifying nonautonomous factors that sense and activate the fasting-induced exopher response revealed that DAF16/FOXO-dependent and -independent processes are engaged. Fasting-induced exopher elevation requires the intestinal peptide transporter PEPT-1, lipid synthesis transcription factors Mediator complex MDT-15 and SBP-1/SREPB1, and fatty acid synthase FASN-1, implicating remotely initiated lipid signaling in neuronal trash elimination. A conserved fibroblast growth factor (FGF)/RAS/MAPK signaling pathway that acts downstream of, or in parallel to, lipid signaling also promotes fasting-induced neuronal exopher elevation. A germline-based epidermal growth factor (EGF) signal that acts through neurons is also required for exopher production. Our data define a nonautonomous network that links food availability changes to remote, and extreme, neuronal homeostasis responses relevant to aggregate transfer biology.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans.

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease. C. elegans adult neurons that express aggregating proteins can extrude large (~4 µm) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called "exophers" and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases. While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons. Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Q&A: Trash talk: disposal and remote degradation of neuronal garbage.

Caenorhabditis elegans neurons have recently been found to throw out cellular debris for remote degradation and/or storage, adding an "extracellular garbage elimination" option to known intracellular protein and organelle degradation pathways. This Q&A describes initial insights into the biology of seemingly selective protein and organelle elimination by challenged neurons, highlighting mysteries of how garbage is distinguished and sorted in the sending neuron, how the garbage-filled "exophers" appear to elicit degradative responses as they transit neighboring tissue, and how non-digestible materials get thrown out of cells again via processes that may be highly relevant to human neurodegenerative disease mechanisms.