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bioRxiv · 10.1101/2022.11.25.517910

Kinetic control in amyloid polymorphism: Different agitation and solution conditions promote distinct amyloid polymorphs of alpha-synuclein

Devi, S.·Garg, D. K.·Bhat, R.
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Abstract

Aggregation of neuronal protein -synuclein is implicated in synucleinopathies, including Parkinsons disease. Despite abundant in vitro studies, the mechanism of -synuclein assembly process remains ambiguous. In this work, -synuclein aggregation was induced by its constant mixing in two separate modes, either by agitation in a 96-well microplate reader (MP) or in microcentrifuge tubes using a shaker incubator (SI). Aggregation in both modes occurred through a sigmoidal growth pattern with a well-defined lag, growth, and saturation phase. The end-stage MP- and SI-derived aggregates displayed distinct differences in morphological, biochemical, and spectral signatures as discerned through AFM, proteinase-K digestion, FTIR, Raman, and CD spectroscopy. The MP-derived aggregates showed irregular morphology with a significant random coil conformation, contrary to SI-derived aggregates, which showed typical {beta}-sheet fibrillar structures. The end-stage MP aggregates convert to {beta}-rich SI-like aggregates upon 1) seeding with SI-derived aggregates and 2) agitating in SI. We conclude that end-stage MP aggregates were in a kinetically trapped conformation, whose kinetic barrier was bypassed upon either seeding by SI-derived fibrils or shaking in SI. We further show that MP-derived aggregates that form in the presence of sorbitol, an osmolyte, displayed a {beta}-rich signature, indicating that the preferential exclusion effect of osmolytes helped overcome the kinetic barrier. Our findings help in unravelling the kinetic origin of different -synuclein aggregated polymorphs (strains) that encode diverse variants of synucleinopathies. We demonstrate that kinetic control shapes the polymorphic landscape of -synuclein aggregates, both through de novo generation of polymorphs, and by their interconversion.

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Devi, S., Garg, D. K., Bhat, R.. 2022-11-25. Kinetic control in amyloid polymorphism: Different agitation and solution conditions promote distinct amyloid polymorphs of alpha-synuclein. https://doi.org/10.1101/2022.11.25.517910

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