UCLA

Alpha-synuclein (α-syn) is a protein with the capacity to form beta sheet rich structures called amyloid fibrils. These fibrils have been found in cytoplasmic inclusions of patient brains with Lewy body diseases (LBD) such as Parkinson’s (PD) and are thought to directly and/or indirectly contribute to disease pathology. Clinical signs of LBD typically appear in the later stages of life; the disease can be sporadic or inherited through point mutations in the α-syn gene. Recent studies have also discovered the presence of α-syn enriched inclusions in the brain of patients with Krabbe disease, an infantile lysosomal storage disorder. The presence of these inclusions implicates dysregulation of metabolic processes as a potential molecular mechanism driving α-syn aggregation.α-syn fibrils have been hypothesized to spread like prions, transferring from cell to cell in the central nervous system, and templating naïve proteins to grow aggregates. That process by which amyloid fibrils seed monomer to form nascent aggregates in cells remains poorly understood. Studies have sought to gain clarity on this mechanism by tracking signals from endogenous fluorophores genetically fused to an amyloid protein expressed in cells. Those cells (biosensors) detect the formation of dense aggregates that arise as fluorescence resonance energy transfer (FRET) positive puncta.In this dissertation, two systems of interest have been selected for structural and functional characterization: (1) aggregates of a familial sequence variant of α-syn that harbors a point mutation (T72M) and (2) α-syn aggregate formation in the presence of lipid-containing environments that mimic aspects of Krabbe disease. We found that, in each case, α-syn adopts a distinct fibril polymorph. We employed cryo-electron microscopy (cryoEM) to inform on the structures of these fibrils with the goal of assessing conformation differences between their cores and those of previously determined recombinant α-syn fibrils. We also assessed the seeding potential of these fibrils in biochemical assays and with HEK293T biosensor cells. In these assays, we are able to surveil incident fibrils by covalently labeling them with NHS ester dyes or through genetic code expansion. We could track fluorescence from these fibrils alongside the FRET signal from newly formed aggregates in biosensor cells. We find that the modified fibrils retain their structure, and visible and persist in cells, and can induce FRET signals. These results help inform on the relationship between the structure of an α-syn polymorph and their templating behavior in cells. Probing structural α-syn variation and its effects on pathogenic activity will further our understanding of familial forms of alpha-synucleinopathies.