Capturing the heterogeneity of alpha-synuclein pathology in synucleinopathies

Synucleinopathies such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy (MSA) are detrimental neurodegenerative diseases. Alpha-synuclein (aSyn), the main aggregating protein found in the pathological inclusions of these ailments, exists in numerous modified forms and conformations, leading to neuropathological heterogeneity in synucleinopathies. The functional, pathological and clinical implications of this heterogeneity remain unclear. Responding this knowledge gap requires the development of novel toolsets capable of capturing the biochemical and structural diversity of aSyn pathology in the brain and peripheral tissues.To address this challenge, we describe in Chapter 2 the development and characterisation of antibodies that cover all three regions and post-translational modifications (PTMs) of aSyn. We assess these antibodies through a comprehensive validation pipeline, and select the best performing antibody subset to capture the neuropathological diversity of aSyn across Lewy body diseases (LBDs). We demonstrate that these antibodies could be used to achieve an in-depth characterization of the biochemical and morphological diversity of aSyn aggregates in the neuronal and in vivo models of aSyn seeding. Our results suggest that aSyn pathology is rich in Serine 129 phosphorylation (pS129) and in previously under-described PTMs, including nitration and phosphorylation at Tyrosine 39 (nY39, pY39) and C-terminal tyrosine phosphorylations (pY133, pY136) across neuronal and glial populations in LBDs. We also report that aSyn is hyperphosphorylated in the cellular and in vivo seeding models. In Chapter 3, we demonstrate the power of our antibodies to achieve an unprecedented analysis of astrocytic aSyn pathology in LBDs. We characterise the biochemical properties, post-translational modifications and the aggregation state of astrocytic aSyn accumulations in the limbic regions across sporadic and familial LBDs. The astrocytic aSyn is revealed distinctively by antibodies against the late amino-terminus (N-terminus) and non-amyloid-beta component (NAC) region of aSyn, but not by the extreme N-terminal or carboxy-terminal (C-terminal) antibodies, suggesting that these aSyn species are truncated at both ends. The astrocytic aSyn accumulations are negative for the key markers of Lewy bodies (aSyn pS129, Amytracker, p62- and ubiquitin-positivity and proteinase K resistance), and positive for aSyn nY39 and pY39. These findings suggest that the astrocytic accumulations may be composed of non-fibrillar, possibly oligomeric conformers of aSyn. Chapter 4 focuses on deciphering the biochemical and morphological signatures of aSyn pathology in MSA. The antibodies described in Chapter 2 are applied to high and low pathology regions of MSA brains by immunohistochemistry (IHC). We also biochemically profile the aSyn species, and report an abundance of aSyn pS129-positive glial and neuronal inclusions that correspond to insoluble high molecular weight species by Western blot. We detect an enrichment of insoluble and truncated aSyn in the MSA cerebellum. By IHC, there is an enhancement of aSyn nY39, pY136 and of other aSyn PTMs.Collectively, the new tools and findings described here may pave the way for future studies elucidating the role of these neuronal and glial aSyn proteoforms in disease pathogenesis and propagation in synucleinopathies.