Regulation of alpha-synuclein accumulation by the autophagy-lysosome pathway in a preclinical model of Parkinson Disease

Finding novel therapeutics that delay or halt the progression of Parkinson Disease (PD) is key to providing symptomatic relief of debilitating symptoms. A defining pathological feature of PD is characterized by the formation of Lewy bodies, intraneuronal inclusions comprised mainly of α-synuclein (ASYN) (4). Vacuolar ATPase (V-ATPase), an enzyme complex comprised of two multi-subunit-containing domains, critically regulates the acidification of vesicles such as lysosomes and thus proper function of the autophagy-lysosome pathway (ALP), which is critical for the degradation of ASYN. Compromises to V-ATPase have been shown to negatively affect lysosome function and its ability to clear toxic forms of ASYN (9-14). ATP6V0C forms the proton translocating channel of V-ATPase. Bafilomycin A1 (BafA1), a plecomacrolide antibiotic, binds ATP6V0C with high affinity, and at high concentrations blocks V-ATPase activity and produces toxicity related to its inhibition of autophagy and ASYN clearance (15, 16). However at low concentrations, BafA1 does not inhibit V-ATPase. Rather, it enhances macroautophagy and significantly attenuates ASYN accumulation and neuron death resulting from lysosome dysfunction (16-18). This finding highlights the potential utility of ATP6V0C itself, the principal bafilomycin binding site, as a therapeutic target in promoting ASYN clearance and preventing ASYN-associated neurodegeneration, as it is ubiquitously expressed in the human brain (19, 20). In this study, we investigated whether knockdown of ATP6V0C influences the accumulation of ASYN in a manner consistent with changes in markers of the ALP. We hypothesized that knockdown of ATP6V0C would increase the stress-induced accumulation of ASYN in association with an alteration of autophagy-related markers. Our data suggest that knockdown of ATP6V0C exacerbates both basal and stress-induced accumulation of ASYN species after treatment with rotenone and BafA1. We also observed increases in the autophagy markers LC3-II and p62, along with the lysosome marker LAMP-1, in cells receiving siRNA silencing ATP6V0C versus control cells containing non-target siRNA after treatment with low concentration BafA1, suggesting that ATP6V0C mediates the protective effects of a low concentration BafA1 by influencing the activity of the ALP. Together, these findings support our hypothesis that ATP6V0C regulates the intracellular accumulation of ASYN in an in vitro model of PD.