Melentijevic, Ilija et al. (2019) International Worm Meeting "Exophers colocalize with hypodermal lysosomes via endocytic trafficking."

Melentijevic, Ilija, St. Ange, Jonathan, Grant, Barth, Wang, Yu, Parekh, Manan, Driscoll, Monica

[International Worm Meeting, 2019]

A general hallmark of neurodegenerative disease is pathology associated with aberrant aggregates. Moreover, it has recently come to be appreciated that neurodegenerative disease proteins/aggregates can be found outside of mammalian neurons, and when outside can actually be taken up by neighboring cells. Transfer of offending molecules has been suggested to be a mechanism of pathogenesis spread for multiple neurodegenerative diseases, including the prevalent Alzheimer's and Parkinson diseases. Understanding of the basic mechanisms by which aggregates are extruded and handled by their cellular neighbors is thus a critical problem is neurodegenerative disease. We discovered a novel capacity of young adult C. elegans neurons to extrude large membrane-bound packets of cellular contents called exohers, which can include aggregated human neurodegenerative disease proteins, mitochondria, or lysosomes, but no nuclear DNA. Interestingly, exophers can selectively incorporate aggregation-prone proteins and oxidized mitochondria. Exophers extruded from touch neurons are initially taken up by the hypodermis, after which their fluorescent contents are either degraded, persist in a networked form we have called the "starry night" phenotype, or are thrown about again into the pseudocoloem and appear later in remote cells. The extruded exopher clearance in the hypodermis appears to be mediated by the ced-1, ced-6, and ced-7engulfment pathway, but how exopher content is handled by the hypodermis is unknown. We have begun to track the molecular details by which the hypodermis addresses invading exopher contents. Our investigations into the hypodermal processing of exopher mCherry aggregates shows that the aggregates will colocalize with the hypodermal lysosome network, as visualized by the lysosome membrane protein SCAV-3::GFP. We also find that this touch neuron-derived cargo colocalizes with late endosome components RAB-7 and RAB-10. We will present these findings as well as current data on other phagocytosis, endocytosis and autophagic machinery that influence our working models of the fates of extruded cargoes derived from neurons that end up in other cells.

Melentijevic, Ilija et al. (2013) International Worm Meeting "Neuronal Exophers: a Novel Mechanism for Removal of Neurotoxic Cytoplasm Components."

Driscoll, Monica, Melentijevic, Ilija, Toth, Marton, Neri, Christian

[International Worm Meeting, 2013]

We discovered a new, previously unknown feature of young adult C. elegans neurons-neurons can extrude substantial packets of cellular contents, which can include aggregated human neurodegenerative disease proteins or mitochondria, but no nuclear DNA. We currently call these extrusions "exophers". The ability to jettison cell contents appears to change with age, and extrusion is increased when protein turnover or autophagy is inhibited. Thus, this pathway may constitute a novel neuronal protection mechanism that serves to maintain protein/organelle homeostasis when other systems are compromised. We propose that the neuronal extrusion phenomenon constitutes a significant but currently unknown conserved pathway by which healthy neurons maintain their functions, and speculate that, in disease, this pathway may malfunction to promote spread of pathology. We will present the basic characterization of neuronal exopher production and our latest data on genetic influences on exopher generation.

Melentijevic, Ilija et al. (2015) International Worm Meeting "Neuronal Exophers: a novel mechanism for the removal of neurotoxic cytoplasm components."

Driscoll, Monica, Arnold, Meghan, Parker, Alex, Guasp, Ryan, Toth, Marton, Neri, Christian, Harinath, Girish, Melentijevic, Ilija

[International Worm Meeting, 2015]

Combating late-onset neurodegenerative disease and age associated functional decline in brain are major health challenges of our time. For the effective design of interventions that protect the nervous system from disease-induced and/or age-associated deterioration, we must fully understand endogenous mechanisms for neuronal protection and how they might fail to enable disease promotion. Recently, it has come to be appreciated that neurodegenerative disease proteins/aggregates can be found outside of mammalian neurons, and when outside, can actually be taken up by neighboring cells. Transfer of offending molecules has been suggested to be a mechanism of pathogenesis spread for multiple neurodegenerative diseases, including the prevalent Alzheimer's and Parkinson's diseases.We discovered a novel capacity of young adult C. elegans neurons - neurons can extrude substantial packets of cellular contents, which can include aggregated human neurodegenerative disease proteins, mitochondria, or lysosomes, but no nuclear DNA. We currently call these extrusions "exophers". The ability to jettison cell contents appears to change with age, and extrusion is increased when protein turnover is impaired, autophagy is inhibited, or mitochondria are compromised. Moreover, exophers can selectively incorporate aggregation-prone proteins and mitochondria with elevated levels of an oxidized reporter. Thus, exopher-mediated extrusion may constitute a novel neuronal protection mechanism that serves to maintain protein/organelle homeostasis when other systems are compromised or overloaded. We propose that the neuronal extrusion phenomenon constitutes a significant but currently unknown conserved pathway by which healthy neurons maintain their functions, and speculate that, in neurodegenerative diseases, this pathway may malfunction to promote spread of pathology. We will present the basic characterization of neuronal exopher production and our latest data on genetic influences on exopher generation.

Melentijevic, Ilija et al. (2021) International Worm Meeting "High Throughput Exopher Whole Genome RNAi Screening with Machine Vision and Machine Learning Approaches"

St. Ange, Jonathan, Abbott, Mark, Guasp, Ryan, Driscoll, Monica, Melentijevic, Ilija

[International Worm Meeting, 2021]

It has recently come to be appreciated that neurodegenerative disease proteins/aggregates can be found outside of mammalian neurons, and when outside can actually be taken up by neighboring cells. Transfer of offending molecules has been suggested to be a mechanism of pathogenesis spread for multiple neurodegenerative diseases, including the prevalent Alzheimer's and Parkinson diseases. We discovered a novel capacity of young adult C. elegans neurons to extrude substantial membrane-bound packages of cellular contents via exohers, which can include aggregated human neurodegenerative disease proteins, mitochondria, or lysosomes, but no nuclear DNA. We speculate that the mechanism of exopher formation in C. elegans is analogous to that used in the transfer of aggregated proteins in human neurodegenerative disease. If so, it will be absolutely critical for us to identify genes contributing to the recognition/sorting of cellular trash and to the expulsion of this material. Although we have identified some exopher mechanism players in candidate gene RNAi screens, measurement rates are slow and important players are likely hard to predict, necessitating an unbiased candidate approach and faster methods in order to elucidate a genetic mechanism. To accomplish genome-wide screens for exopher-genesis modifier genes, we have developed a highly automated, high-throughput whole-genome RNAi screening platform that can be implemented at a fraction of the time and cost of manual screens. Our screening protocol employs robotic dispensers and aspirators, coupled with a high content imaging system for animals grown and measured in a 96-well plate format that mimics a standard solid media plate environment. We are developing several machine vision approaches to allow for automated scoring of animals with an exopher to expedite the analysis. Our screen protocol allows for an entire genome to be screened in about two weeks, fast enough to allow for replicated screens and epistasis analysis of hits. We have developed approaches to store a 3D digital library and physical library of prepared samples for later re-imaging at higher resolution and re-analysis. We will present our current genetic approaches to establish a high exopher baseline, and computational approaches to detect exopher events in a crowded well.

Smart, Joelle et al. (2017) International Worm Meeting "Touch neurons can toss out mitochondria."

Arnold, Meghan, Toth, Marton, Melentijevic, Ilija, Driscoll, Monica, Harinath, Girish, Smart, Joelle, Guasp, Ryan

[International Worm Meeting, 2017]

Mitochondria provide energy, execute key steps of metabolism, control calcium, and modulate cellular decisions for life/death. Given these critical functions in cell, tissue, and organism health, it is not surprising that mitochondrial functionality plays an essential role in neuronal maintenance in everyday biology, aging, and late-onset neurodegenerative disease. Mitochondrial quality control is thought to be primarily executed through cell-internal elimination via mitophagy and lysosome degradation. However, the Driscoll lab has discovered, and recently published (Melentijevic, 2017 Nature 542:367) that mitochondria can be thrown out of neurons in large membrane bound vesicles we call exophers. Mitochondria in exophers budding from C. elegans touch neurons tend to have elevated oxidation of mitoROGFP reporters localized to the matrix. Genetic and pharmacological treatments that compromise mitochondria can increase numbers of exophers produced by touch neurons, suggesting that throwing away defective mitochondria may be a mechanism for neuronal quality control. Indeed, some mammalian neurons can throw out their mitochondria for degradation by neighboring astrocytes (Davis, PNAS 11:9633), suggesting relevance across phyla. In C. elegans, beautiful work on degradation of sperm mitochondria upon fertilization have been published (Sato, Science 334:1141). We will present data on our initial efforts to characterize mito-exopher production and the factors that prompt neurons to extrude mitochondria. Our hope is that our findings will be relevant to understanding neuronal maintenance and neuronal degeneration, especially as associated with perturbed mitochondrial quality as may occur in Parkinson's disease and many other human disorders.

Guasp, Ryan et al. (2019) International Worm Meeting "Trans-tissue signals from developing embryos instruct distant neurons to throw out their trash."

Guasp, Ryan, Salam, Sangeena, Harinath, Girish, Wang, Guoqiang, Driscoll, Monica, Melentijevic, Ilija

[International Worm Meeting, 2019]

The maintenance of proteostasis is critical for cell and organism function. We have reported that, in addition to internal protein quality control functions of chaperone action, proteasome-mediated degradation, and autophagy, certain C. elegans cells have the capacity to select, package and throw out their trash (Melentijevic et al., 2017). Large membrane-bound vesicles that contain protein aggregates and damaged organelles can be extruded by touch receptor neurons into the neighboring hypodermis, which attempts exopher degradation. The frequency of exopher production can be greatly exacerbated by internal proteo-stresses or by factors that disrupt mitochondrial quality. We have shown that in hermaphrodites, exophers produced by multiple neurons are generated in a distinct bi-modal pattern during adult life. We observe an initial peak in exopher production on adult day 2-3, followed by relatively low levels until around adult days 9-11 when a second peak can occur. The young adult exopher peak appears at about the time of a reconfiguration of proteostasis strategies (Ben-Zvi et al., 2009), and thus might reflect a developmental trash-elimination period in which deleterious materials accumulated during early life are collected and thrown away at one designated moment to clean house. What triggers this "trash day" is unclear. We have found that genetic and pharmacological interventions that disrupt embryogenesis can abrogate the early adult peak of exopher production. FuDR can inhibit the early peak; disruption of sperm maturation using the auxin-inducible spe-44 degron system blocks virtually all early exopher production; and genetic mutants in glp-4 and gld-1 that lack oocytes have very few early exophers. Similarly, the small-molecule drug C22, which causes embryonic lethality via increasing eggshell permeability (Weicksel et al., 2016) prevents early exopher generation. These data suggest signals associated with housing developing embryos act to ultimately increase neuronal exopher production in distant cells. Male C. elegans display a temporal and spatial distribution of exopher production distinct from hermaphrodites. In hermaphrodite touch neurons the highest rate of exopher production is from ALMR neurons with almost no production in the PLM neurons. This pattern is reversed in males. Furthermore, exophers in males increase progressively with age rather than occurring with the temporal peak pattern characteristic of hermaphrodites. These data suggest that signals associated with housing fertilized embryos modulate exopher production in hermaphrodites. We will discuss these studies with regard to understanding the molecular nature of the trans-tissue signaling.

Guasp, Ryan et al. (2017) International Worm Meeting "Structural Components and Genetic Requirements of Exophers and their Formation."

Arnold, Meghan, Driscoll, Monica, Guasp, Ryan, Taub, Daniel, Toth, Marton, Nyguen, Ken, Xue, Jian, Hall, David, Melentijevic, Ilija, Gabel, Christopher, Harinath, Girish

[International Worm Meeting, 2017]

Combating late-onset neurodegenerative disease and age-associated cognitive decline are major world health challenges. A striking commonality between neurodegenerative pathology and age-associated decline is the aggregation of proteins. Protein aggregates can move from neurons to surrounding cells, potentially promoting pathology spread, however the mechanism is largely a mystery. We previously found that some C. elegans neurons can dramatically extrude aggregates within large released vesicles, which we called exophers (Melentijevic 2017). To form an exopher, cytoplasmic materials such as aggregates, damaged mitochondria, and lysosomes become concentrated at the periphery of the soma. The material that will be exported is included as the membrane buds outward, forming a vesicle that moves away from the soma. The large vesicle can remain attached to the soma via a thin filament that can transfer tagged protein and calcium into the exopher compartment. Genetic conditions that compromise proteostasis enhance exopher production and sensitized neurons that produce exophers function better than those that do not. Thus, exophers appear neuroprotective. Exophers derived from touch neurons must traverse the hypodermal syncytium that surrounds the touch neurons, and EM images support that the hypodermis responds vigorously to exopher content. Electron microscopy images reveal that extruded ALMR exophers have a very heterogeneous, multilamellar, and multicompartmental structure within the hypodermis. It is likely that hypodermal lysosomes degrade the exopher content, however some non-digestible material such as mCherry appears to be later released into the pseudocoelom where it can be taken up by coelomocytes. Exopher formation and expulsion may shed light on how aggregated proteins can be released into neighboring cells, and how those cells react to transcellular cargo. We will present data on screens intended to define the genetic components needed for exopher formation and the cytoskeletal components needed for exopher extrusion. 1) Melentijevic, I. et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature (2017)DOI: 10.1038/nature21362

Guasp, Ryan et al. (2015) International Worm Meeting "Genetic dissection of mechanisms that contribute to extrusion of neurotoxic aggregates from neurons."

Driscoll, Monica, Harinath, Girish, Guasp, Ryan, Melentijevic, Ilija, Arnold, Meghan

[International Worm Meeting, 2015]

The central challenges that face aging neurons are protein aggregates and dysfunctional mitochondria. We have found that specific neurons have the capacity to sort and extrude aggregates and mitochondria. We speculate that the mechanism involved is a conserved process that plays a critical role in adult neuronal proteostasis. Here we address the molecular basis of this directed extrusion. Extracellular vesicles have been shown to be important in processes as diverse as inflammatory signalling, angiogenesis, and cancer invasion. We discovered a novel extracellular vesicle jettisoned by C. elegans neurons that is distinct from exosomes and microvesicles in both morphology and cargo. We term this vesicle the "exopher." Exophers bud outwardly from the soma and can remain attached via a thin filament. Exophers can contain whole organelles (mitochondria and lysosomes) and protein aggregates, and their frequency of generation is increased by proteotoxic or mitochondrial stress. What is the cellular machinery that directs selection of compromised cell components, localizes them, and extrudes them inside vesicles? We examined roughly 300 candidate genes that could potentially contribute to the process of exopher-genesis. We selected genes that are involved in vesicular trafficking and docking, as well as genes that encode motor proteins, polarity proteins, and cytoskeletal proteins. Out of the original screen, we found 12 RNAi interventions that were consistent suppressors of exopher-genesis and 2 RNAi interventions that were consistent enhancers. Three actin isoforms proved to be among the strongest suppressors along with mec-12, a mechanosensory neuron-specific tubulin isoform. Knockdown of two kinesin-like protein genes, klp-6 and klp-18, also reduce exopher release. Two nematode-specific small GTPases with no known mutant phenotype, rab-Y2 and rab-Y3, may also contribute to exopher formation. The gene knockdowns that had no significant effects, are also likely to be informative. For example, only two genes among all encoding components of the four ESCRT complexes seem to have a role. Overall, our data define novel functions for cytoskeletal proteins and motors in the process of exopher-genesis. We will present our working model for the molecular extrusion process that may correspond to the mechanism by which mammalian neurons eliminate toxic aggregates and dysfunctional mitochondria.

Smart, Joelle et al. (2019) International Worm Meeting "Touch and dopaminergic neurons eject mitochondria under native and genetic stress conditions."

Rongo, Chris, Wang, Guoqiang, Driscoll, Monica, Smart, Joelle, Brennan, Rachel, Melentijevic, Ilija, Grant, Barth, Park, Eunchan, Vergalasov, Edward

[International Worm Meeting, 2019]

Mitochondrial quality control is thought to be primarily executed through cell-internal elimination via mitophagy and lysosome degradation. However, we discovered, and recently published (Melentijevic, Nature 542:367) that mitochondria can be thrown out of neurons in large membrane-bound vesicles we call exophers. Genetic and pharmacological treatments that compromise mitochondria can increase numbers of exophers produced by touch neurons, suggesting that throwing away defective mitochondria may be a mechanism for neuronal quality control. Some mammalian neurons can throw out their mitochondria for degradation by neighboring astrocytes (Davis, PNAS 11:9633), suggesting relevance across phyla. Mitochondrial dysfunction is considered a central factor in the progressive loss of substantia nigra pars compacta dopaminergic neurons in Parkinson's disease. Mutations in genes encoding the mitophagy proteins PINK1 and Parkin are directly linked to autosomal recessive PD. The major cause of late-onset autosomal dominant PD is a gain-of-kinase function mutation in LRRK2, which is involved in mitochondrial dynamics, turnover, and DA neuron oxidative stress response (Saha, J Neurosci 29:9210). Furthermore, chemical disruption of mitochondrial electron transport chain complexes can selectively kill dopaminergic neurons in rats and C. elegans, with mutations in PINK1 and Parkin enhancing this vulnerability. We have reported that pink RNAi disruption and Parkin deletion in C. elegans can increase exopher production. However, our initial studies involved strains in which aggregating proteins were expressed in neurons studied via high copy number transgenes. We have found that mitochondria can still be extruded in exophers in the absence of foreign aggregating proteins, and that genetic manipulation of mitochondrial fission/fusion and PD-related genes can significantly impact mito-exopher rates. We will present data on our initial efforts to characterize mito-exopher production from touch and DA neurons and the factors that prompt neurons to extrude mitochondria. Our hope is that our findings will be relevant to understanding neuronal maintenance and neuronal degeneration, especially as associated within relation to the perturbed mitochondrial quality as may occurobserved in Parkinson's disease and many other human disorders.

Wang, Yu et al. (2021) International Worm Meeting "Phagocytosis and processing of neuron-derived exophers by the C. elegans hypodermis."

Smart, Joelle, Driscoll, Monica, Nguyen, Ken C., Melentijevic, Ilija, Morera, Andres, Wang, Yu, Hall, David H., Grant, Barth D., Cooper, Jason, Wang, Guoqiang, Arnold, Meghan L., Guasp, Ryan J.

[International Worm Meeting, 2021]

Previous work showed that adult C. elegans neurons are capable of ejecting a large portion their cell bodies as a giant vesicle (average size 3.8 micron) loaded with aggregated protein and mitochondria, a process that becomes much more frequent upon challenge by several forms of cellular stress (Melentijevic et al., 2017). Current models posit that such ejection removes toxic materials from the neuron, enhancing neuronal stress recovery. Here we have focused on the fate of exophers ejected by the mechanosensory neuron ALMR, a neuron whose soma is completely surrounded by the syncytial skin cell hyp7. By following exophers within individual animals over time we found a characteristic pattern in which a single large ALMR exopher typically moves within hyp7 in a posterior direction, away from the neuron, but initially maintains a long thin connection to the neuronal soma. Within 3 hours of ejection most intact exophers have disappeared, with remaining material derived from the exopher broken up into many smaller vesicles within the hypodermis that we have termed "starry night". Over the next few days most starry night vesicles diminish in intensity, and in many cases fluorescent material derived from exophers appears outside of the hypodermis in coelomocyte cells. This pattern suggested that ejected exophers are phagocytosed by the much larger hypodermal cell, with the hypodermal endolysosomal machinery activated to degrade toxic exopher materials via phagosome maturation. Undigested material may then be resecreted by the hypodermis, allowing uptake and another chance at degradation by the coelomocyte scavenger cells. Work in progress supporting this model will be presented, with an emphasis on current results analyzing hypodermal uptake and phagosome processing, and the role of ARF-6, CNT-1, and RAB-35 in this process.