Investigating the Role of Distinct α-synuclein Strains in Different α-synucleinopathies

Abstract

α-synuclein (α-Syn) is a structurally complex protein that is central to the pathogenesis of Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as α-synucleinopathies. How the pathogenic misfolding of this single protein precipitates three pathologically and clinically distinct disease phenotypes remains enigmatic. The prevailing hypothesis posits that α-Syn is capable of adopting a multitude of unique 3D conformations, which are known as strains. These distinct strains are thought to drive the disease-specific misfolding and propagation of α-Syn in different α-synucleinopathies, giving rise to their heterogeneity. This thesis sought to comprehensively interrogate the differential biochemical properties of patient-derived α-Syn from the human brain affected with PD and MSA. Multiplex immunohistochemistry with epitope-specific α-Syn antibodies revealed that α-Syn aggregate structures in pathologically burdened regions of the human brain with PD and MSA look dramatically different and exhibit distinct cell type-specific and subcellular arrangements. N-terminus immunolabelling identified novel populations of perinuclear, microglial, astrocytic, and neuronal lysosomal α-Syn aggregates in the human brain with PD, that are not detected by other canonical α-Syn antibodies. Multiplex immunolabelling revealed that antibodies targeting α-Syn phosphorylated at the serine 129 residue significantly underestimate the actual α-Syn pathology load across α-synucleinopathies. Biochemical analysis of α-Syn with proteinase K demonstrated that N-terminus immunoreactive inclusion structures in PD, and oligodendroglial inclusions in MSA, are more susceptible to proteolysis compared to mature PD Lewy pathology and MSA neuronal inclusions, respectively. High-resolution confocal microscopy was utilised to study the intracellular and intranuclear architecture of oligodendroglial and neuronal α-Syn inclusions in MSA and characterise their pathological maturation. Finally, seed amplification assays were used to demonstrate distinct aggregation kinetics in neuropathologically confirmed PD and MSA cases, affirming the ability of these assays to reliably discriminate between α-synucleinopathies. In addition, these data demonstrate the enhanced seeding potential of MSA patient-derived α-Syn compared to PD α-Syn. The multifaceted investigation of α-Syn in this thesis highlights that the pathological misfolding and propagation of this protein in PD and MSA is disease-specific. It subsequently demonstrates that disease-specific α-Syn species represent distinct conformational strains. Collectively, this thesis sets the stage for more refined diagnostic techniques and, with further ultrastructural analysis of these unique α-Syn strains, provides a platform for more targeted therapeutic strategies for treating α-synucleinopathies.