Conformational changes and the self-assembly of alpha-synuclein

Parkinson's disease is the second most common neurodegenerative disease, affecting 0.1 - 0.2% of the population. Incidence of this debilitating disorder rises to 1% of the population over the age of 65, posing a substantial socioeconomic burden on the UKs aging population. There are currently no disease altering treatments for PD, in part due to the incomplete knowledge of the disease mechanism. The misfolding and aggregation of the protein alpha-synuclein is associated with a range of neurological disorders, including Parkinson's disease. Alpha-synuclein has been irrefutably linked to Parkinson's disease through both genetic and pathological data, with increasing evidence suggesting prefibrillar oligomeric forms of the protein are the toxic species. However, the precise molecular mechanisms through which this protein is linked to the disease are currently unknown. Consequently, increasing knowledge of alpha-synuclein is of great importance, as new discoveries will potentially further the development of new therapeutics for the disease. In this thesis the primary aim was to conduct investigations into the structural and functional aspects of N-terminally acetylated alpha-synuclein and its oligomers through a combination of Electrospray Ionisation – Ion Mobility Spectrometry – Mass Spectrometry, biochemical and cell culture assays. Alpha-synuclein is a known metal binding protein and the copper binding and subsequent conformational changes and aggregation of modified and mutated forms of the protein were investigated. A novel loss of metal binding function was found for the N-terminally acetylated H50Q form of the protein. The conformational effects of N-terminal acetylation on the oligomeric forms of the protein were also investigated. It was demonstrated that this co-translational modification of alpha-synuclein did not affect oligomer formation or function. Low order oligomers were found to be dynamic during the course of aggregation, by the use of isotopically labelled forms of the protein. Lastly the response of SH-SY5Y cells treated with alpha-synuclein oligomers with the ability to seed intracellular aggregation was investigated, demonstrating that this procedure evokes a stress response in these cells, highlighting a potential mode of action. Together, investigations presented in this thesis have demonstrated the role of a constitutive co-translational modification in ligand binding and oligomer assembly. Furthermore, the cellular stress response to treatment with these oligomers was also determined giving insights into disease progression. Results have highlighted the importance of using the biologically relevant form of a protein when performing in vitro experiments, validated the cellular effects of previously characterised alpha-synuclein oligomers when produced from N-terminally acetylated protein, and extended knowledge of the cellular response of the SH-SY5Y cell line to treatment with a specific class of oligomers.