Cellular Mechanisms of Neuropathology in Parkinson's Disease

Birmingham Va Medical Center, Birmingham AL

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Abstract

DESCRIPTION (provided by applicant): The accumulation of alpha-synuclein (ASYN)-containing Lewy bodies and associated degeneration of dopamine (DA) neurons are major pathological hallmarks of Parkinson's disease (PD). The autophagy- lysosome pathway (ALP) is an important intracellular degradation and recycling pathway and a compromise in ALP function promotes the accumulation of toxic ASYN species. As the ALP is altered in PD, identification and validation of novel ALP targets which promote ASYN clearance is a timely approach in the development of novel PD therapeutics. ATP6V0C forms the proton translocating channel of V-ATPase, an enzyme complex that regulates lysosomal acidification and clearance of ALP substrates. High concentrations of bafilomycin, a macrolide antibiotic that binds ATP6V0C with high affinity, inhibits V-ATPase and produces neurotoxicity related to its inhibition of lysosome function and ASYN clearance. However, concentrations of bafilomycin too low to inhibit V-ATPase attenuate cell death and endogenous ASYN accumulation resulting from lysosome dysfunction, and inhibit DA neuron death resulting from ASYN over-expression in vivo. While bafilomycin has a narrow therapeutic index which limits its long-term use in human PD patients, our data point to ATP6V0C as a novel target for promoting ASYN clearance and cell survival. However, whether ATP6V0C mediates these protective effects of bafilomycin and if over-expression of ATP6V0C itself can provide neuroprotective benefit in the absence of bafilomycin has not been tested. We hypothesize that ATP6V0C mediates the bafilomycin- dependent clearance of toxic ASYN species and attenuates ASYN-associated neurotoxicity. In Aim 1, we will genetically over-express ATP6V0C in cultured mammalian cells to determine its relative importance in regulating the clearance of endogenous ASYN, ALP function and cell death. We will also determine if the over-expression of human ATP6V0C in mouse substantia nigra (SN) attenuates ASYN accumulation and associated neurodegeneration resulting from over-expression of wild-type human ASYN. In Aim 2, we will genetically knockdown ATP6V0C or introduce point mutations in ATP6V0C that confer resistance to bafilomycin-mediated inhibition of V-ATPase, to determine the requirement of ATP6V0C in regulating such neuroprotective functions of bafilomycin in cultured mammalian cells. We will also develop bafilomycin analogs with reduced V-ATPase inhibitory activity to determine if bafilomycin mediated neuroprotection is enhanced with a reduction in V-ATPase inhibitory activity. These studies will validate the utility of ATP6V0C as a novel ALP-dependent target for the future generation of PD therapeutics which promotes ASYN clearance and DA neuron survival.