Tracking Oligomerization Of Alpha-Synuclein Demonstrates Pivotal Role Of Mitochondria In Seeding

Aggregation of α-synuclein, which normally exists as an unfolded monomer, in human brain contributes to the major pathogenesis of the synucleinopathies, Parkinson's disease and Lewybody dementia. However, the initial triggers to form early aggregates are unknown. Of note, visualizing the dynamic process of aggregation in neurons has been limited due to the inability to detect the early soluble toxic intermediates, oligomers, inside cells. Here we report a Foster resonance energy transfer (FRET) based method that enables the kinetics of intracellular oligomerization to be tracked in live cells. We first visualized that neurons uptake externally applied monomers and intracellularly form early and late oligomers, dependent on the initial monomer concentration. Then we utilized mutant α-synuclein carrying A53T point mutation that causes early onset progressive Parkinson's disease to investigate triggers of aggregation. We demonstrated that the A53T mutant monomer progressively oligomerizes due to the fast intracellular uptake than WT and other α-synuclein mutants. In addition, correlative light and electron microscopy (CLEM) allowed us to identify cellular regions in human neurons in which oligomerization is detected, such as mitochondria and lysosome. Importantly, A53T monomer produces excess reactive oxygen species (ROS) and opens mitochondria permeabilization pore (PTP) prior to cell death unlike the WT monomer. Importantly prevention of mitochondrial ROS inhibits the oligomerization of the A53T monomer and maintains the protein in its monomeric state, and this subsequently prevents cell death. In summary, our study provides evidence that mitochondria seed the aggregation of α-synuclein in neurons and mitochondrial stress in the form of enhanced ROS production is a major trigger for oligomerization.