Giorgi, Victoria et al. (2021) International Worm Meeting "C. elegans as a model for studying Cannabis sativa extracts"

Garcia, Carlos, Carrera, Ines, Fernandez, Santiago, Castro, Rosina, Giorgi, Victoria, Vila, Agustina, Pazos, Mariana

[International Worm Meeting, 2021]

The Cannabis genus includes a variety of plants that are a valuable source of secondary bioactive metabolites, including flavonoids, terpenes, and cannabinoids. These metabolites are responsible for a wide variety of biological activities such as anti-inflammatory, anti-oxidant, psychotropic and others. The long term objective of this project is to develop C. elegans as a model for studying the bioactive potential of different extract types from cannabis plants. Previous studies showed that aqueous extracts from Cannabis sativa leaves exhibit nematicidal activity on plant parasitic nematodes, and suggested that phytocannabinoids present in the extract are responsible for this effect. However, little is known about the nematicidal activity from extracts of other parts of cannabis plants, including female flowers where phytocannabinoids accumulate in highest amounts. C.elegans has proven to be an excellent model for the discovery of new anthelmintic drugs and its mechanism of action. In this sense extracts from flowers of two Cannabis plant varieties which differ in its ratio of tetrahydrocannabinolic acid (THCA)/ cannabidiolic acid (CBDA) content were prepared and its activity on C. elegans motility assessed. The phytocannabinoids profile of the extracts was studied and the main phytocannabinoids were identified and quantified by HPLC-DAD. Automated motility assays were conducted with N2 worms at 10 microM and 100 microM in the main cannabinoid (CBDA or THCA). Failure to see a motility effect in this background prompted us to use mutants with a compromised cuticle. C.elegans has a robust cuticle that functions as a physical barrier against chemicals which may represents a problem for compounds testing, such as acidic phytocannabinoids. In this sense, motility assays were performed using mutants in the bus-8 gene, which encodes a predicted glycosyltransferase required for cuticle integrity. Cannabis flower extracts caused severe reduction in motility in bus-8 mutants at 50 microM and 100 microM in the main cannabinoid. We are currently analyzing purified compounds to dissect the complex effect of these extracts and using mutant analysis to understand its mechanism of action.

Smart, Joelle et al. (2021) International Worm Meeting "Neuronal mitochondria utilize a novel fission mechanism during extrusion into exophers"

Driscoll, Monica, Nguyen, Ken, Park, Eunchan, Hall, Dave, Smart, Joelle, Rongo, Chris

[International Worm Meeting, 2021]

The Driscoll lab discovered a previously unknown capacity of C. elegans adult neurons to extrude large (~5 microm) vesicles that include cytoplasmic contents (Melentijevic, 2017). These vesicles, which we call "exophers", are a microcosm of the originating soma: they can contain almost anything from the cell, including aggregated proteins, cytoskeletal components, and organelles. Importantly, I have found mitochondria are extruded into the majority of C. elegans touch neuron exophers, and recent work in mouse cardiomyocytes demonstrates elimination of defective mitochondria through exopher-genesis plays a critical role in cardiac homeostasis (Nicolás-Ávila, 2020). Mitochondrial morphology is thought to influence cell function and age-associated decline. In all animals, FZO-1/MFN1/2 and EAT-3/OPA1 respectively mediate outer and inner mitochondrial membrane fusion, while the dynamin motor DRP-1 is required for canonical mitochondrial fission. Several interesting questions arise: Are small, hyper-fragmented mitochondria (produced by inhibiting mito fusion) more amenable to trafficking into exophers? Does abolishing mitochondrial fission (knocking out DRP-1) prevent mitochondrial severance and extrusion into exophers? I have made the surprising discovery that portions of the mitochondrial network are severed and ejected into exophers even when DRP-1-mediated mitochondrial fission is abolished. Furthermore, I have shown mitochondria contained within exophers are typically intact. In fact, in a small fraction of cases, exophers and their mitochondria can remain morphologically intact for weeks following separation from the soma, suggesting mitochondrial fragmentation during exopher-genesis is not irreparably traumatic. Most extruded mitochondria, however, appear to be degraded by the surrounding hypodermis within 3 hours of exopher-genesis. I will be presenting my findings on the proteins, organelles, and physical factors governing this non-canonical mechanism of mitochondrial fission, as well as the biochemical and functional properties of both extruded and retained mitochondria. This research documents a novel method of mitochondrial sculpting and distribution that likely has important implications for mitochondrial homeostasis, trafficking, and membrane repair. Melentijevic I, et al. C. elegans neurons jettison protein aggregates and mitochondria under neurotoxic stress. Nature. 2017 Feb 16;542(7641):367-371. doi: 10.1038/nature21362. Epub 2017 Feb 8. PMID: 28178240; PMCID: PMC5336134. Nicolás-Ávila JA, et al. A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart. Cell. 2020 Oct 1;183(1):94-109.e23. doi: 10.1016/j.cell.2020.08.031. Epub 2020 Sep 15. PMID: 32937105.