Investigation of Endocytic Pathways for the Internalization of Exosome-Associated Oligomeric Alpha-Synuclein

Misfolding and aggregation of alpha-synuclein (alpha syn)resulting in cytotoxicity is a hallmark of Parkinson's disease (PD) and related synucleinopathies. The recent body of evidence indicates that alpha syn can be released from neuronal cells by nonconventional exocytosis involving extracellular vesicles (EVs) such as exosomes. The transfer of alpha syn between cells has been proposed to be an important mechanism of disease propagation in PD. To date, exosome trafficking mechanisms, including release and cell-cell transmission, have not been fully described. To gain insight into the mechanisms involved, exosomes were purified from conditioned media of stable cells secreting alpha syn oligomers. A novel bimolecular protein complementation assay was used to detect exosomes containing alpha syn oligomers. Recipient cells were treated with exosomes containing alpha syn oligomers or "free" non-exosome-associated alpha syn oligomers and internalization was monitored. We demonstrate that cell-derived exosome- associated alpha syn oligomers can be efficiently internalized by recipient cells. Interestingly exosome- free alpha syn oligomers isolated from conditioned medium were not internalized but remained bound to the extracellular surface. To investigate the endocytic pathway(s) required for the exosome uptake different pharmacological inhibitors of caveolin-dependent, clathrin-dependent, and macropinocytosis pathways were utilized. Surprisingly, none of these pathways appear to play a significant role in the internalization of exosome-associated alpha syn oligomers. Finally, the role of heparin sulfate proteoglycans (HSPGs) in exosome- associated alpha syn internalization was investigated using genetic approach. Despite previous studies showing HSPGs can modulate internalization of fibrillar alpha syn, genetic manipulations did not attenuate internalization of exosome- associated alpha syn oligomers in our hands, suggesting that exosome-associated a syn is internalized via an alternative endocytic pathway(s) that has yet to be elucidated.