Modeling and Mechanistic Investigation of α-synuclein aggregation

Our understanding of the α-synuclein aggregation process and the consequences thereof is currently limited, which in turn prevents the development of targeted therapeutic interventions. The work presented here, as a part of this thesis, is focused on expanding our understanding of the molecular events involved in α-synuclein aggregation. Towards this goal we have studied the impact of pathologically relevant forms of α-synuclein, namely A53T mutant α-synuclein and fibrillar α-synuclein, and characterized their impact on N-methyl-D-aspartate receptor (NMDAR) diffusion and function. We found both mutant and fibrillar α-synuclein, decreased the NMDAR diffusion and expression at the post-synapse. Moving further towards the mechanistic investigations we investigated the effect of two neuroprotective compounds on α-synuclein aggregation and found both compounds capable of clearing α-synuclein in cell and animal models potentially through autophagy related functions. In our efforts to scale mechanistic investigations we developed a high-throughput screening (HTS) capable FRET-based reporter for detection of α-synuclein aggregation in cells. Using this model, we performed a proof-of-concept screen of kinase inhibitors from which we identified three inhibitors with potent protective effects on α-synuclein aggregation. We further showed through mechanistic investigation that the protective effects likely involved lysosomal changes. Finally, in an effort to advance our knowledge of α-synuclein aggregation, we performed a genome-wide knockout screen to identify genes in the human genome with an impact on α-synuclein aggregation. This study also highlighted among other pathways the importance of the endolysosomal system in relation to α-synuclein aggregation. Many questions remain in regard to the molecular mechanisms involved in α-synuclein aggregation, but we hope our insights and models presented here will assist in the elucidation of the underlying mechanisms of α-synuclein aggregation.