Aggregation, Propagation and Pathological Implications of α-synuclein Amyloid Fibrils in Parkinson’s Disease

Abstract

Parkinson’s disease (PD) is a severe, progressive neurodegenerative disorder characterized by aggregation of the protein α-synuclein into amyloid fibrils, leading to the formation of Lewy bodies and Lewy neurites and degeneration of dopaminergic neurons in the substantia nigra. Although α-synuclein aggregation is a defining feature of PD, fundamental questions remain regarding how fibrils form, how aggregation pathways encode distinct structural polymorphs, and how these polymorphs influence prion-like propagation and neurodegeneration. This knowledge gap continues to challenge the development of effective therapeutics. This thesis addresses several of these knowledge gaps by investigating the aggregation and prion-like propagation of α-synuclein amyloid fibrils using wild-type and familial PD-associated variants. First, to understand how assembly conditions shape α-synuclein fibril formation and structure, I examined the effects of monovalent cations. I show that the physiologically abundant cations Na+ and K+, although often treated as interchangeable, produce distinct effects on aggregation kinetics and fibril morphology, demonstrating that changes in ionic conditions can reshape α-synuclein fibril assembly pathways. I next investigated consequences of fibril fragmentation, a mechanism that rarely occurs in vitro but can be significant in vivo, showing that early-onset PD variants underwent pronounced structural evolution following fragmentation, reflected in altered Thioflavin-T binding. This shows that α-synuclein fibrils are more structurally heterogenous than previously believed and that they may continue to remodel their architecture over the course of PD. To resolve such heterogeneity, I established a Thioflavin-T fluorescence-lifetime spectroscopy-based methodology for phenotyping of α-synuclein polymorphs. Cross-seeding experiments revealed variant-specific structural trajectories, including faithful templating, monomer-driven restructuring, and emergence of de novo polymorphs, highlighting the vast structural landscape accessible to α-synuclein fibrils. Finally, I investigated whether these in vitro traits influenced prion-like propagation in cells, showing that α-synuclein variants differed markedly in uptake efficiency, induction of endosomal enlargement, and ability to seed endogenous α-synuclein aggregation. These cellular outcomes aligned with the structural and kinetic behaviors observed in vitro, demonstrating that propagation efficiency is strongly shaped by intrinsic fibril properties.Altogether, this work advances the mechanistic understanding of α-synuclein aggregation and propagation in PD and highlights fibril assembly pathways and resulting fibril structure as critical targets for future therapeutic strategies.