Amyloidogenicity of Rationally-Designed and Naturally-Occurring Sequence Variants of α-Synuclein

Aggregation and amyloid-formation of α-synuclein (αSyn) is central to the
pathogenesis of synucleinopathies. Despite decades of research into the
mechanisms underlying the behaviour of αSyn, there remains no cure for these
diseases. Changes to the amino acid sequence of αSyn can occur naturally, for
example in familial Parkinson’s disease, or can be rationally designed to probe
changes to the behaviour of the protein. These sequence changes can drastically
alter αSyn as an intrinsically disordered protein in solution, as a helical lipid-bound
protein, or as a cross-β amyloid fibril. The work presented here first explores a
novel variant of αSyn lacking residues 2 to 7, αSynΔN7, which was rationally
designed based on in silico predictions of aggregation-prone regions. Experiments
are also described that explore three naturally-occurring alternative splicing
variants of αSyn. Using thioflavin T assays, the kinetics of amyloid assembly of
αSynΔN7 and the alternative splicing variants of αSyn are investigated. Other
biophysical methods such as circular dichroism, electron microscopy, and nuclear
magnetic resonance are also used to characterise the variants in solution, at the
membrane, and in the formation of amyloid fibrils. Finally, experiments in new
strains of the Caenorhabditis elegans model organism are used to corroborate
findings in vivo. Key findings confirm that the N7 region regulates amyloid
formation both with and without lipid membranes, as well as aggregation in vivo.
The alternative splicing variants of αSyn exhibit distinct rates of amyloid formation,
with deletion of exon 5 increasing the amyloidogenicity in the absence of lipid
membranes. In vivo, the variants all display different profiles of aggregation
propensity and proteotoxicity. Overall, the work described in this thesis enhances
understanding of the regions critical to the aggregation behaviour of αSyn. By
integrating these findings, the potential for targeting specific regions of αSyn in the
development of novel therapeutics is revealed.