The Mechanistic Role of Alpha-synuclein Hyperphosphorylation in Parkinson’s Disease Pathogenicity

Protein misfolding and aggregation is a cellular hallmark of neurodegenerative disorders. Alpha synuclein (α-syn) is a protein expressed at the presynaptic region of dopaminergic neurons and is found to misfold and aggregate in Parkinson’s disease (PD). α-Syn aggregates under cellular stress conditions and forms inclusion bodies called Lewy bodies (LBs). Nearly 90% of the α-syn found in LBs is phosphorylated as opposed to ~4% in healthy conditions. This increase in the phosphorylation of α-syn suggests its role in PD pathology.   This thesis focuses on the understanding of the mechanistic role of α-syn phosphorylation in α-syn induced toxicity. Chapter 1 is a comprehensive review on aggregation of α-syn in PD, its clearance mechanisms and role of post translational modifications in neurodegenerative disorders. This chapter elaborates on the role of phosphorylation in neurodegenerative disorders. Chapter 2 enlists the techniques and methods used in this study. Chapter 3 reports the effect of phosphorylation of α-syn on its aggregation, nucleation, and cytotoxic properties in vitro.   Chapter 4 replicates the toxic effects of α-syn phosphorylation in vivo, emphasizing on its role in neurodegeneration and phospho-proteomic perturbations in substantia nigra (SN) of rat brain. This chapter entails the changes observed in increased aggregation and spread of pathology by phosphorylated α-syn aggregates. Chapter 5 describes the possible role of phosphorylated α-syn in causing damage to lipid membranes as a mechanism of cytotoxicity.   The study of in vitro and in vivo models of PD-like pathology revealed an increase in toxic potential of α-syn phosphorylation. Phosphorylation of α-syn increased its aggregation and induced structural alterations in the protein. The kinases used in this study phosphorylated α-syn at multi-serine residues shedding light on the role of multi-serine phosphorylation in PD. The increased nucleation capacity of phosphorylated α-syn in vitro supports the increased spread of α-syn aggregates and neurodegeneration observed in vivo. The phospho-proteomic profiling of the SN of rat brains showed significant downregulation of phosphoproteins by phosphorylated α-syn, shedding light on its role in affecting key cellular mechanisms. The observed binding and deformation of lipid vesicle morphology by phosphorylated α-syn in vitro is proposed to hint on a mechanism of cytotoxicity.    Overall, the results obtained from this study contribute to understanding the role of phosphorylated α-syn in neurodegeneration. These findings could further be explored as mechanisms of cytotoxicity and as early detection of PD, by targeting phosphorylated α-syn as a biomarker of PD.