Distinct Mechanisms Determine α-Synuclein Fibril Morphology during Growth and Maturation

Abstract

Amyloid polymorphs have become one of the focal points of molecular studies of neurodegenerative diseases like Parkinson's disease. Due to their distinct biochemical properties and prion-like characteristics, insights into the molecular origin and stability of amyloid polymorphs over time are crucial for understanding the potential role of amyloid polymorphism in these diseases. Here, we systematically study the fibrillization of recombinantly produced human α-synuclein (αSyn) over an extended period of time to unravel the origin and temporal evolution of polymorphism. We follow morphological changes in the same fibril sample with atomic force microscopy over a period of 1 year. We show that wild-type (wt) αSyn fibrils undergo a slow maturation over time after reaching the plateau phase of aggregation (as detected in a Thioflavin-T fluorescence assay). This maturation, visualized by changes in the fibril periodicity over time, is absent in the disease mutant fibrils. The β-sheet content of the plateau phase and matured fibrils, obtained using Fourier transform infrared spectroscopy, is however similar for the αSyn protein sequences, suggesting that the morphological changes in wt αSyn fibrils are tertiary or quaternary in origin. Furthermore, results from a reversibility assay show that the plateau phase fibrils do not disassemble over time. Together, the observed changes in the periodicity distributions and stability of the fibrillar core over time point toward two distinct mechanisms that determine the morphology of wt αSyn fibrils: competitive growth between different polymorphs during the fibrillization phase followed by a process wherein fibrils undergo slow maturation or annealing.

Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils

“…A30P and A53T fibrils [30]. Here, A30P and A53T samples contained several micron long fibrils (Figure 3(A,B): inset).…”

“…The two mutants have been shown to exhibit distinct aggregation kinetics and fibril morphologies [17][18][19][28][29][30]. In earlier studies, we have shown that these disease mutants form stable fibrils of distinct morphology and that cross seeded aggregation between these mutants is sensitive for seed morphology [19,30]. Based on these observations, we here study the interaction of morphologically distinct but chemically identical polymorphs of aSyn with the standard amyloid probe ThT, to understand the contribution of fibril morphology in ThT readouts.…”

“…A30P and A53T aSyn are two disease associated mutants of aSyn implicated in familial forms of Parkinson's disease. The two mutants have been shown to exhibit distinct aggregation kinetics and fibril morphologies [17][18][19][28][29][30]. In earlier studies, we have shown that these disease mutants form stable fibrils of distinct morphology and that cross seeded aggregation between these mutants is sensitive for seed morphology [19,30].…”

Characterization and Delivery of DNA Aptamers Selected for the Prevention of Alpha-Synuclein Aggregation in Parkinson's Disease

“…After several months of fibril maturation, wild type α-Syn fibrils display decreased homogeneity suggesting that the fibrils present in the plateau phase are in a quasi-equilibrium state. Furthermore, the ß-sheet content of fibrils at the plateau phase and matured fibrils is similar, implying that any change in the morphology of fibrils over time is due to change in the tertiary or quaternary structure 29 . modification of this residue is strongly linked to the pathology of synucleinopathies 32 .…”

“…Some research hypothesizes that α-Syn behaves as a prion, a misfolded protein that negatively impacts cell physiology and functionality. The ability for α-Syn to form amyloids with a variety of conformations when aggregating is similar to the cellular prion protein (PrP C ); a change in the α-helical conformation of PrP C to an insoluble ß-sheet aggregate scrapie prion protein (PrP Sc ) is observed 29,42,43 . Neurodegeneration in patients due to PrP aggregation has been noted to affect the electrophysiology of neuronal cells and are generally neurotoxic.…”