The goal of this application is to investigate the impact of extracellular alpha-synuclein on neurodegeneration and disease progression in Parkinson's disease (PD). There are several reasons to target extracellular alpha-synuclein for the development of novel therapeutic agents for PD including the fact that Lewy bodies containing alpha-synuclein have been found in healthy grafted neurons in PD patients several years after transplant suggesting a possible role for alpha-synuclein transmission in disease propagation and progression. We have proposed complementary approaches to elucidate not only the fundamental mechanisms and species involved in alpha-synuclein release, but to develop unique therapeutic interventions that target extracellular alpha- synuclein oligomers with a view that understanding extracellular alpha-synuclein biology can lead to a translation of new or different therapeutic approaches. We will examine 3 major aims: The first aim will define the types of alpha-synuclein released from neurons using a series of biophysical and molecular tools that allow characterization of released oligomeric forms of alpha-synuclein. We will also investigate the mechanisms involved in the release and uptake of alpha-synuclein and the impact of extracellular alpha-synuclein on cell viability .The second aim will expand on our preliminary studies and on recently published studies to investigate pathways involved in alpha-synuclein release and uptake and in particular the role of macroautophagy in alpha-synuclein release and uptake. Finally, the third aim extends these observations in vivo, utilizing in vivo microdialysis and AAV introduced forms of alpha-synuclein that are neurotoxic in vitro, and furthermore extends the hypothesis that macroautophagy can modulate alpha-synuclein release and uptake in vivo. Together, we will be able to determine which species of alpha-synuclein are released from cells and ultimately taken up by neighboring cells; the mechanisms associated with release and uptake; and if manipulations affecting alpha-synuclein release and uptake affect alpha-synuclein-induced toxicity in cells and animal models with the goal of translation into the clinic.