Exosome release and neuropathology induced by α-synuclein: new insights into protective mechanisms of Drp1 inhibition

Targeting alpha-synuclein (alpha-syn) as a therapeutic strategy for Parkinson's disease (PD) has been intensively pursued largely due to its well-recognized pathogenic role. Since its discovery as the first familial link to PD over two decades ago, this protein has been associated with multiple neurotoxic mechanisms, such as mitochondrial dysfunction and impaired autophagic flux. We report here that blocking dynamin-related protein 1 (Drp1) improved both mitochondrial function and autophagic flux in experimental models of alpha-syn. Using rat dopaminergic neuronal cells with inducible wild-type human alpha-syn, we observed excessive mitochondrial fragmentation and increased Drp1 levels 48 h after gene induction. Functionally, these cells exhibited lower mitochondrial membrane potential, reduced ATP production rate and mitochondrial spare respiratory capacity, as well as increased levels of mitochondrial reactive oxygen species. To evaluate the protective role of Drp1 inhibition, we used three complementary approaches: gene silencing mediated by siRNA, overexpression of Drp1-dominant negative and the small molecule mitochondrial division inhibitor-1 (mdivi-1). Both morphological and functional defects induced by alpha-syn were attenuated by these strategies. Importantly, Drp1 inhibition reduced proteinase K-resistant alpha-syn aggregates. Based on that observation, we investigated the involvement of autophagy. Through a combination of stable autophagy reporter cells and immunoreactivity for LC3 and p62 in neuronal cells with either alpha-syn overexpression or treatment of human alpha-syn preformed fibrils (PFF), we observed that Drp1 inhibition abolished autophagic impairment induced by alpha-syn. Consistent with its role in improving autophagy function, Drp1 inhibition reduced exosome release and spread of alpha-syn pathology from neurons to neurons and from microglia to neurons. In summary, this study highlights new insights that Drp1 inhibition confers neuroprotection through both mitochondrial and autophagy-lysosomal pathways, further strengthening the therapeutic potential of targeting Drp1.