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.

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.

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.

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.

Toth, Marton et al. (2015) International Worm Meeting "Novel gene interactions that modulate morphological aging of neurons in C. elegans."

Harinath, Girish, Guasp, Ryan, Toth, Marton, Xue, Jian, Patel, Khushboo, Gaul, Kelli, Zhang, Wenying, Driscoll, Monica, Ganihong, Ivana

[International Worm Meeting, 2015]

Aging neurons in C. elegans and humans can experience dramatic morphological restructuring. In a recent screen for genes encoding proteins that maintain adult neuronal integrity we discovered a group of relatively poorly characterized EGF-like motif proteins. Systemic RNAi knockdown of these predicted calcium binding protein genes limited age-associated dendritic branching in touch neurons as much as 4 fold. Interestingly, for some genes, cell autonomous inactivation exacerbated morphological neuronal aging. Furthermore, we found some genes with cell non-autonomous actions on touch neuron maintenance. We found that systemic inactivation of certain EGF-like motif genes increase life- and healthspan, via a mechanism distinct from dietary restriction. These genes have conserved functions in cell adhesion and can thus serve as models with which to investigate crosstalk between the regulation of aging and tissue integrity, potentially assigning an entirely new function to this protein family.

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

Kumar, Sandeep et al. (2021) International Worm Meeting "Enhanced functional restoration through axon regeneration by swimming exercise"

Ghosh-Roy, Anindya, Behera, Sibaram, Dey, Shirshendu, Kumar, Sandeep, Basu, Atrayee

[International Worm Meeting, 2021]

Axonal regeneration is a promising approach to overcome impaired functionality due to axonal injury. In mammals, central nervous system has poor regenerative capacity due to both extrinsic and intrinsic factors. The regenerative capacity also declines significantly with ageing. Therefore, functional axon regeneration in adulthood is challenging and needs more understanding. The pharmacological manipulations are not very successful for functional restoration whereas rehabilitation and physical activity shows improvement. As physical exercise has complex systemic effects, understanding the downstream effectors of physical exercise that is relevant for axon regeneration might be useful. Studying this using simple model organism has several advantages. Using posterior gentle touch circuit neuron (PLM) of Caenorhabditis elegans, we are studying effect of swimming exercise on functional restoration after laser assisted axotomy. We found that a single swimming exercise session of 90 minutes, which is an established paradigm of exercise in worm (Laranjeiro et al., 2017; Laranjeiro et al., 2019) improves functional recovery irrespective of age. However multiple swimming session is required for older worms (A5 stage). Anatomical correlation showed that swimming session improves regrowth initiation, regrowth length and functional connections. We found that the energy sensor kinase AMPK/AAK-2 plays an essential role mediating swimming benefits. Characterizing tissue specific requirement, we found that it has both cell autonomous (PLM neuron) and non-autonomous (muscle) requirement. Pharmacological activation of AMPK/AAK-2 showed enhanced functional restoration similar to swimming. We are studying the downstream molecules and their specific roles in various tissues for swimming mediated functional enhancement which will be helpful for better implementation of this approach. References Laranjeiro R, Harinath G, Burke D, Braeckman BP, Driscoll M (2017) Single swim sessions in C. elegans induce key features of mammalian exercise. BMC Biology 15. Laranjeiro R, Harinath G, Hewitt JE, Hartman JH, Royal MA, Meyer JN, Vanapalli SA, Driscoll M (2019) Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration. Proc Natl Acad Sci U S A 116:23829-23839.

Pokharel DR et al. (2006) Vaccine "Vaccination with Setaria cervi 175 kDa collagenase induces high level of protection against Brugia malayi infection in jirds."

Nandakumar Kodumudi K, Rai R, Pokharel DR, Rathaur S, Reddy MV

[Vaccine, 2006]

A zinc containing metalloprotease, 175 kDa collagenase, purified from adult female Setaria cervi showed strong cross-reactivity with sera from putatively immune (PI) individuals (unpublished observation) and induced cytotoxicity to B. malayi L3 larvae and microfilariae by ADCC mechanism [Srivastava Y, Bhandari YP, Reddy MVR, Harinath BC, Rathaur S. An adult 175 kDa collagenase antigen of Setaria cervi in immunoprophylaxis against Brugia malayi. J Helminth 2004;78:347-52]. These preliminary observations suggested the immunoprotective nature of collagenase. To confirm the vaccine potential of this protease, a vaccine trial was conducted in jirds (Meriones unguiculatus) against human filarial parasite B. malayi. The vaccination resulted into a mean protection level of 75.86% and produced high level of protease neutralizing antibodies. Cytokine analysis in immune jirds sera suggested a mixed Th1/Th2 type cellular immune response whereas ELISA, immunoblotting and enzyme antibody inhibition assay revealed the presence of specific anti-collagenase antibodies. Taken together, all these results suggest that S. cervi 175 kDa collagenase could form the basis of an effective molecular vaccine against human lymphatic filariasis.

Onken, Brian et al. (2019) International Worm Meeting "Assessing health vs. longevity in the Caenorhabditis Intervention Testing Program."

Guo, Suzhen, Johnson, Erik, Lucanic, Mark, Morshead, Mackenzie, Bhaumik, Dipa, Xue, Jian, Huynh, Phu, Sedore, Christine, Inman, Delaney, Harinath, Girish, Lithgow, Gordon, Phillips, Patrick, Driscoll, Monica, Hall, David, Foulger, Anna, Guo, Max, Coleman-Hulbert, Anna, Plummer, Todd, Onken, Brian, Chen, Michelle

[International Worm Meeting, 2019]

One of the promises of aging research is to extend the quality of life and limit the onset of age-related disease by promoting/extending youthful physiology. The Caenorhabditis Intervention Testing Program (CITP) is a multi-institution effort, sponsored by the National Institute on Aging, that screens bioactive compounds for their ability to enhance healthy aging and extend lifespan. Critical to the design of this collaborative effort is the testing of interventions across a genetically diverse collection of three Caenorhabditis species - C. elegans, C. briggsae and C. tropicalis - to enable identification of compounds that improve health and longevity across varied genetic backgrounds. A second distinctive feature of our effort is the coordinated reproduction of datasets in three different laboratories. Overall, we anticipate robust and reproducible interventions will be identified in the CITP effort (1, 2). Here we report on the healthspan effects of three compounds that we previously found to extend lifespan in some - but not all - of our tested strains: resveratrol and NP1, both potential dietary restriction mimetics, and propyl gallate, which exhibits antioxidant properties. We measured relative health in aging animals by using automated lifespan machines (3) to test for resistance to oxidative and heat stress, and by measuring locomotory vigor using CeleST computational analsysis (4, 5). Our data show variation in healthspan benefits, depending on the assay used and the strain examined. Often we found that how a compound affects lifespan extends to effects on healthspan. For example, NP1 increases lifespan in four of the six strains we tested, and decreases lifespan in one strain - we saw similar NP1 outcomes in healthspan assays. In some cases, however, we found healthspan effects that did not parallel lifespan outcomes. Overall, we present reproducible methods for measuring healthspan across nematode species that have the potential to reveal health benefits of various compounds that might be missed through traditional longevity assays. 1. Lucanic et al (2017) Nat Commun 8:14256 2. Lithgow et al (2017) Nature 548:387-388 3. Stroustrup et al (2013) Nat Methods 10:665-670 4. Restif et al (2014) PLoS Comput Biol 10:e1003702 5. Ibanez-Ventoso et al (2016) J Vis Exp 118

Onken, Brian et al. (2021) International Worm Meeting "Metformin Treatment of Diverse Caenorhabditis Species Reveals the Importance of Genetic Background in Longevity and Healthspan Extension Outcomes"

Phillips, Patick, Chen, Esteban, Foulger, Anna, Inman, Delaney, Morshead, Mackenzie, Bhaumik, Dipa, Lithgow, Gordon, Coleman-Hulbert, Anna, Guo, Max, Plummer, W. Todd, Driscoll, Monica, Johnson, Erik, Chao, Elizabeth, Jones, E. Grace, Sedore, Christine, Onken, Brian, Guo, Suzhen, Banse, Stephen, Hope, June, Xue, Jian, Hall, David, Harinath, Girish, Huynh, Phu

[International Worm Meeting, 2021]

Metformin, the most commonly prescribed anti-diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti-aging drug - the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalisstrains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose-dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations.