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Melentijevic I et al. (2017) Nature "C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress."

Gabel CV, Melentijevic I, Parker JA, Nguyen KC, Toth ML, Hall DH, Neri C, Taub D, Guasp RJ, Driscoll M, Arnold ML, Harinath G

[

Nature,

2017]

The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4m) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neurons that did not produce exophers. The extruded exopher transits through surrounding tissue in which some contents appear degraded, but some non-degradable materials can subsequently be found in more remote cells, suggesting secondary release. Our observations suggest that exopher-genesis is a potential response to rid cells of neurotoxic components when proteostasis and organelle function are challenged. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.

Melentijevic, I. et al. (2017) International Worm Meeting "Neuronal Exophers: A Novel Mechanism for the Removal of Neurotoxic Cytoplasm Components."

Driscoll, M, Melentijevic, I., Taub, D, Arnold, M., Gabel, C, Toth, M, Guasp, R, Nguyen, K, Harinath, G, Hall, D

[

International Worm Meeting,

2017]

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 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 DNA1. We call these extrusions "exophers". The ability to jettison cell contents is first evident about day 2-3 of adult life, coincident with the documented changes to adult proteostasis2,3, is then minimal until 9-10 days of adult life. Extrusion is increased when protein turnover or autophagy is inhibited. Moreover, exophers can selectively incorporate aggregation-prone proteins and oxidized mitochondria. Exopher contents can appear later in remote cells, and their clearance is mediated by the ced-1, ced-6, and ced-7 engulfment pathway. Neurons that have made exophers appear to maintain functionality longer than non-exopher producing neurons. 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 diseases, this pathway may malfunction to promote spread of pathology. We will present the basic characterization of neuronal exopher production, structural findings as revealed through EM, and our latest data on genetic influences on exopher generation and clearance. 1 Melentijevic, I. et al. C. elegans neurons jettison protein aggregates and mitochondria Into the extracellular environment in response to proteotoxic stress, under revision. 2 Labbadia, J. & Morimoto, R. I. Repression of the Heat Shock Response Is a Programmed Event at the Onset of Reproduction. Mol Cell 59, 639-650, doi:10.1016/j.molcel.2015.06.027 (2015). 3 Labbadia, J. & Morimoto, R. I. Proteostasis and longevity: when does aging really begin? F1000prime reports 6, 7, doi:10.12703/P6-7 (2014).

Melentijevic, I. et al. (2019) International Worm Meeting "High throughput exopher whole genome RNAi screening with machine vision."

Wang, G., Szot, M., Driscoll, M., Guasp, R., Parekh, M., St. Ange, J., Melentijevic, I., Abbott, M., Anusionwu, I., Grant, B.

[

International Worm Meeting,

2019]

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 pathological 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 one week, fast enough to allow for replicated screens and epistasis analysis of hits. We will present our current genetic and computational approaches to detect rare exopher events in a crowded well.

Abbott, M. et al. (2017) International Worm Meeting "A novel high-throughput whole-genome RNAi screening technology utilized to investigate the molecular pathways of exopher production."

Abbott, M., Guasp, R., Driscoll, M., Melentijevic, I.

[

International Worm Meeting,

2017]

RNA interference is a powerful tool for the exploration of gene function in C. elegans. Past whole genome RNAi screens in C. elegans have provided fascinating insights into nematode biology, however, current screening protocols can be prohibitively resource and time intensive. We are developing 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. Multiple fluorescent channels and pattern recognition algorithms enable the analysis of multiple parameters simultaneously. We have developed methods for animal growth and imaging that should enable screening of the entire genome in approximately one week, fast enough to make epistatic studies feasible. Our initial experiments investigate genes that influence production of exophers, large extracellular vesicles that can mediate expulsion of protein aggregates and damaged organelles from neurons1. If conserved, exophers may play important roles in neurodegenerative diseases characterized by protein aggregation, including Parkinson's and Alzheimer's. In our model, we track an aggregating mCherry protein that is expelled in exophers. The mCherry protein exits touch neurons as exopher cargo, transits through the hypodermis, and can later be found concentrated in coelomocytes. When coelomocyte or hypodermal uptake is genetically disrupted, fluorescent exopher contents accumulate in the body in a phenotype we call "starry night." We will present preliminary screening data and images demonstrating the viability of the technology to screen suppressors and enhancers of the starry night phenotype. 1 Melentijevic, I. et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 542, 367-371, doi:10.1038/nature21362 (2017).

Ardeshna, S. et al. (2019) International Worm Meeting "The Role of Intermediate Filaments in Aggregate Collection and Exopher Ejection."

Melentijevic, I., Driscoll , M., Nguyen, K., Grant, B., Hall, D., Guasp, R., Arnold , M., Ardeshna, S.

[

International Worm Meeting,

2019]

A molecular marker of aging and neurodegeneration is cellular increase of aggregated proteins. Accumulation and spread of misfolded proteins causes toxicity that can induce loss of neurological function. In neurodegenerative disease cases, it is known that aggregates can accumulate in one part of the brain and subsequently spread across other brain tissues. Evidently, through some uncharacterized mechanism, aggregated species can physically travel to other regions of the brain and cause toxicity as part of disease pathology. Thus, it is important to understand both the transfer of aggregates from one cell to another, and how a neuron first handles aggregated species. In mammals, cells can eliminate toxic aggregates through multiple pathways including ubiquitin mediated proteolysis, the degradation through autophagy-lysosome system, or aggregate sequestration via aggresome formation. Our lab discovered that cells can also deal with toxic aggregate build up by collecting and selectively extruding toxic cellular components, through a budding process in a large vesicle called the exopher. The exopher contents are taken up by surrounding tissues to be degraded, potentially describing aggregate transfer from tissue to tissue. This is particularly exciting in light of current research on neurodegeneration, as it has been established that human disease-associated aggregates can transfer between neurons or between neurons and glia in brain, via a largely uncharacterized mechanism thought to promote pathology. We speculate that a mechanism analogous to exopher ejection could be responsible for the transfer of toxic aggregates in mammalian cells. While conducting a large scale targeted RNAi screen of C. elegans genes to probe for important exopher-related proteins, we found several hits that acted as either enhancers or suppressors of exophergenesis. Two particular genes that we found to be important for the exopher mechanism are ifd-1 and ifd-2, which are unessential intermediate filament proteins that have undescribed functions. In mammalian systems, both mammalian homologs of C. elegans intermediate filaments are implicated in cytoskeletal organization and aggregate management. These homologs are known to express in neurons and have been associated with neurodegenerative disease aggregated plaques and Lewy Bodies. My studies focus on characterizing the role of IFD-1 and IFD-2 in exophergenesis and will address the relationship between and pathway ordering of IFD-1 and IFD-2. Work will address a novel role for intermediate filaments in C. elegans neurons and shed light on the exopher mechanism. 1) Melentijevic, I, Toth, ML, Arnold, ML, et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature 2017; 542( 7641): 367? 371.

Abbott M et al. (2020) J Biol Methods "A simplified design for the C. elegans lifespan machine."

Driscoll M, Phillips PC, Coleman-Hulbert AL, Sedore CA, Melentijevic I, Falkowski R, Banse SA, Abbott M, Ange JS, Blue BW, Guo M, Jarrett CM, Johnson E, Lithgow GJ

[

J Biol Methods,

2020]

Caenorhabditis elegans (C. elegans) lifespan assays constitute a broadly used approach for investigating the fundamental biology of longevity. Traditional C. elegans lifespan assays require labor-intensive microscopic monitoring of individual animals to evaluate life/death over a period of weeks, making large-scale high throughput studies impractical. The lifespan machine developed by Stroustrup et al. (2013) adapted flatbed scanner technologies to contribute a major technical advance in the efficiency of C. elegans survival assays. Introducing a platform in which large portions of a lifespan assay are automated enabled longevity studies of a scope not possible with previous exclusively manual assays and facilitated novel discovery. Still, as initially described, constructing and operating scanner-based lifespan machines requires considerable effort and expertise. Here we report on design modifications that simplify construction, decrease cost, eliminate certain mechanical failures, and decrease assay workload requirements. The modifications we document should make the lifespan machine more accessible to interested laboratories.

Souza ACR et al. (2018) Genes (Basel) "Pathogenesis of the Candida parapsilosis Complex in the Model Host Caenorhabditis elegans."

Colombo AL, Fuchs BB, Jayamani E, Mylonakis E, Souza ACR, Alves VS

[

Genes (Basel),

2018]

Caenorhabditiselegans is a valuable tool as an infection model toward the study of Candida species. In this work, we endeavored to develop a C. elegans-Candidaparapsilosis infection model by using the fungi as a food source. Three species of the C. parapsilosis complex (C.parapsilosis (sensustricto), Candidaorthopsilosis and Candidametapsilosis) caused infection resulting in C. elegans killing. All three strains that comprised the complex significantly diminished the nematode lifespan, indicating the virulence of the pathogens against the host. The infection process included invasion of the intestine and vulva which resulted in organ protrusion and hyphae formation. Importantly, hyphae formation at the vulva opening was not previously reported in C. elegans-Candida infections. Fungal infected worms in the liquid assay were susceptible to fluconazole and caspofungin and could be found to mount an immune response mediated through increased expression of cnc-4, cnc-7, and fipr-22/23. Overall, the C. elegans-C. parapsilosis infection model can be used to model C. parapsilosis host-pathogen interactions.

Fourie R et al. (2021) Front Cell Infect Microbiol "Candida albicans SET3 Plays a Role in Early Biofilm Formation, Interaction With Pseudomonas aeruginosa and Virulence in Caenorhabditis elegans."

Sebolai O, Fourie R, Albertyn J, Pohl CH, Gcilitshana O

[

Front Cell Infect Microbiol,

2021]

The yeast Candida albicans exhibits multiple morphologies dependent on environmental cues. Candida albicans biofilms are frequently polymicrobial, enabling interspecies interaction through proximity and contact. The interaction between C. albicans and the bacterium, Pseudomonas aeruginosa, is antagonistic in vitro, with P. aeruginosa repressing the yeast-to-hyphal switch in C. albicans. Previous transcriptional analysis of C. albicans in polymicrobial biofilms with P. aeruginosa revealed upregulation of genes involved in regulation of morphology and biofilm formation, including SET3, a component of the Set3/Hos2 histone deacetylase complex (Set3C). This prompted the question regarding the involvement of SET3 in the interaction between C. albicans and P. aeruginosa, both in vitro and in vivo. We found that SET3 may influence early biofilm formation by C. albicans and the interaction between C. albicans and P. aeruginosa. In addition, although deletion of SET3 did not alter the morphology of C. albicans in the presence of P. aeruginosa, it did cause a reduction in virulence in a Caenorhabditis elegans infection model, even in the presence of P. aeruginosa.

Zhu Q et al. (2020) Oxid Med Cell Longev "A Dihydroflavonoid Naringin Extends the Lifespan of C. elegans and Delays the Progression of Aging-Related Diseases in PD/AD Models via DAF-16."

Zhou XG, Qu Y, Luo HR, Zhu Q, Wu GS, Chen JN

[

Oxid Med Cell Longev,

2020]

Naringin is a dihydroflavonoid, which is rich in several plant species used for herbal medicine. It has a wide range of biological activities, including antineoplastic, anti-inflammatory, antiphotoaging, and antioxidative activities. So it would be interesting to know if naringin has an effect on aging and aging-related diseases. We examined the effect of naringin on the aging of Caenorhabditis elegans (C. elegans). Our results showed that naringin could extend the lifespan of C. elegans. Moreover, naringin could also increase the thermal and oxidative stress tolerance, reduce the accumulation of lipofuscin, and delay the progress of aging-related diseases in C. elegans models of AD and PD. Naringin could not significantly extend the lifespan of long-lived mutants from genes in insulin/IGF-1 signaling (IIS) and nutrient-sensing pathways, such as daf-2, akt-2, akt-1, eat-2, sir-2.1, and rsks-1. Naringin treatment prolonged the lifespan of long-lived glp-1 mutants, which have decreased reproductive stem cells. Naringin could not extend the lifespan of a null mutant of the fox-head transcription factor DAF-16. Moreover, naringin could increase the mRNA expression of genes regulated by daf-16 and itself. In conclusion, we show that a natural product naringin could extend the lifespan of C. elegans and delay the progression of aging-related diseases in C. elegans models via DAF-16.

Coomansingh-Springer C et al. (2019) Heliyon "Internal parasitic burdens in brown rats (Rattus norvegicus) from Grenada, West Indies."

Sharma RN, Armstrong E, Vishakha V, Acuna AM, Coomansingh-Springer C

[

Heliyon,

2019]

This study identified the endoparasites in Brown rat (Rattus norvegicus) during May to July 2017 in Grenada, West Indies. A total of 162 rats, 76 females and 86 males were trapped from St. George and St. David parishes in Grenada. The collected fecal samples were examined for parasitic eggs and/or oocysts using simple fecal flotation technique. Adult parasites found in the intestinal tract were examined for identification. The overall prevalence of intestinal parasites among rats was 79 %. Ten helminth species were recovered, several of which were reported for the first time in rodents in Grenada. The internal parasites consist of seven nematodes (Angiostrongylus spp., Nippostrongylus braziliensis, Heterakis spumosa, Strongyloides ratti, Aspiculuris tetraptera, Syphacia spp. and Protospirura spp.), one cestode (Hymenolepsis diminuta), one acanthocephalan (Moniliformis moniliformis) and one protozoa species (Eimeria spp.). The most prevalent zoonotic species were Angiostrongylus spp. (35.2%), Hymenolepsis diminuta (7.4%) and Moniliformis moniliformis (3.1%). Several nonzoonotic endoparasites; which included Nippostrongylus braziliensis (50.6%), Heterakis spumosa (15.4%), Strongyloides ratti (43.2%), Aspiculuris tetraptera (2.5%), Syphacia spp. (1.9%), Protospirura spp. (1.2%) and Eimeria spp. (4.7%) were also identified. The most prevalent parasites were Nippostrongylus brasiliensis (50.6%), Strongyloides ratti (43.2%) and Angiostrongylus spp. (35.2%). Co-infections occurred with up to six species per rat showing different combinations of parasitic infections.

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