The role of SOD2 and autophagy in a Drosophila model of Machado-Joseph Disease. Natalie M. Clark, John M. Warrick. Department of Biology, University of Richmond, Richmond, VA.

   Spinocerebellar ataxia 3 (SCA3), also known as Machado-Joseph Disease (MJD), is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the ataxin-3 (ATX3) protein. Research has suggested that MJD potentially increases the amount of reactive oxidative species within the body, accelerating the cell aging process and increasing neural death. It is hypothesized that the increase of naturally occurring antioxidant gene products such as Superoxide Dismutase 2 (SOD2) could decrease the severity of this disease and serve as a possible treatment. UAS-ATX 3 alleles of mutant and normal MJD as well as UAS-SOD2 were expressed in the fly eye using the gmrGal4 driver. Fly heads were fixed and embedded in epon blocks. Semi-thin sections of fly retinas were evaluated using light microscopy. We found flies expressing both MJD and increased levels of SOD2 had greater eye degeneration and faster progression of disease than flies with MJD and endogenous SOD2 levels. Other research has implicated superoxide in the autophagy pathway, and autophagy has been suggested to reduce the degeneration caused by MJD by removing aggregates. We propose that the increase in SOD2 levels interfered with the autophagy pathway causing the increase in degeneration. To test this hypothesis, flies were aged and their heads were frozen in OCT. 12 micron-thick frozen sections were taken using a cryostat, and the sections were stained with antibodies to ATX3 and Autophagy Protein 12 (ATG12). Viewing the sections using confocal fluorescence microscopy revealed that flies with MJD have strong expression of ATG12 protein co-aggregated with ATX3 in the nucleus at 2 and 7 days, while flies expressing normal ATX3 do not show significant expression of ATG12. Likewise, flies with MJD and increased SOD2 have co-aggregation at 2 days and 7 days, yet the staining appears weaker. Since ATG12 is not able to function in these nuclear aggregates, these results suggest a decrease in autophagy in MJD due to SOD2 overexpression.