Investigating the effects of autophagic perturbations on mitochondrial function in Parkinson’s Disease

Parkinson’s disease is among the fastest growing neurodegenerative disease, projected to impact over 12 million individuals worldwide by 2040. Though extensive research has identified that alpha-synuclein, a canonical Parkinson’s disease gene, impacts proper autophagy and mitophagy, its exact role in the pathogenesis of the disease remains contested. This thesis aims to develop an assay for measuring rates of mitophagy in vivo and to use that assay to understand how alpha-synuclein impacts rates of mitophagy. The fluorescent protein MitoTimer is trafficked to mitochondria where it displays a time-dependent fluorescent profile. Though this temporal profile was robust, a single pulse of expression did not recapitulate prior reports of sensitivity to oxidative stress; rather, observed changes correlated more strongly with cell death. An alternative double-pulse protocol did not detect any changes in mitochondria following FCCP treatment. Together, these data suggest that MitoTimer is not ideal for measuring rates of mitophagy. mt-mKeima revealed accumulation of acidic puncta over time, indicating active basal mitophagy. This basal mitophagy was inhibited by autophagic inhibitors (wortmannin, VPS34IN1) and increased by autophagic activators (rapamycin, torin). Overexpression of alpha-synuclein appeared to decrease rates of mitophagy, though the effect did not reach statistical significance. Assays in patient-derived, iPSC dopaminergic neurons carrying a triplication in alpha-synuclein revealed decreased levels of MFN2, indicative of increased rates of mitophagy. Furthermore, treatments with MLi2, a LRRK2 inhibitor, appeared to recover this phenotype. Overall, these data indicate that alpha-synuclein impacts strongly on rates of mitophagy, though the effect varies by model, and that LRRK2 inhibition may have therapeutic potential for Parkinson’s disease.