Parkinson's disease (PD) is the second most common neurodegenerative disorder, after Alzheimer's disease (AD), occurring at a rate of 0.1%-0.2% of the population. The incidence of PD increases with advancing age, affecting 1% of the population over the age of 65. Extensive loss of dopaminergic neurons and aggregation of the protein α-synuclein (α-syn) into ubiquitin-positive Lewy bodies (LBs) represents a major neuropathological hallmark of PD. The impact of LB pathology on the disease pathogenesis is still largely unknown, with evidence suggesting small soluble oligomeric assemblies that precede LB development are the causative agent in PD. At present, the generation of large nuclear-associated LBs from endogenous wildtype α-syn, translationally regulated under its own promoter in human cell culture models, requires costly and time-consuming protocols. The primary objective of this thesis was to develop a more physiologically relevant cell culture model of PD that recapitulates the development of LB inclusions. Using a cell culture model of fully differentiated human SH-SY5Y neuroblastoma cells grown in three-dimensions (3D), cells were shown to develop LB-like pathology upon exposure to exogenous α-syn species. In contrast to most cell- and rodent based PD models, which exhibit multiple diffuse α-syn aggregates throughout the cytoplasm, a single large nuclear inclusion that is immunopositive for α-syn and ubiquitin is rapidly obtained in our model. However, phosphorylation of α-syn within these inclusions was not observed. This was achieved without the need for overexpression of α-syn or genetic modification of the cell line. To further explore the mechanism of LB formation the recently discovered programmed cell death pathway ferroptosis was investigated. Ferroptosis is an irondependent cell death pathway that shares similar pathogenic features with PD including elevated iron concentration, GSH depletion, lipid peroxidation and increased ROS. However, there are currently no studies that have explored whether α-syn is involved in ferroptotic cell death. Viability assays within the 3D cell culture model following treatment with ferroptosis and apoptosis inducers and qPCR of ferroptotic targets demonstrated resistance to this mechanism of programmed cell death. Nevertheless, treatment with iron was associated with some features of ferroptosis including increased ROS, some lipid peroxidation and reduced levels of glutathione peroxidase 4 (GPX4). Phosphorylation of α-syn at serine 129 (S129) was increased upon iron treatment and reduced following treatment with a ferroptosis inhibitor, liproxstatin-1. These results demonstrate the potential implications of iron exposure, α-syn aggregation, and ferroptosis in the pathogenesis of PD. The system described in this thesis provides an ideal tool to screen compounds to intervene therapeutically in LB formation, and to investigate the mechanisms involved in disease progression in synucleinopathies.
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