An intact membrane is essential for small extracellular vesicle-induced modulation of ?-synuclein fibrillization.

ABSTRACT: The misfolding and fibrillization of the protein, ?-synuclein (?syn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of ?syn misfolding is an important area of interest given that ?syn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate ?syn folding, there is little data pertaining to the mechanism(s) of this interaction. ?Syn has previously been shown to associate with small lipid vesicles released by cells called extracellular vesicles (EVs) and it is postulated these interactions may assist in the spreading of pathological forms of this protein. Together, this presents the need for robust characterisation studies on ?syn fibrillization using biologically-derived vesicles. In this study, we comprehensively characterised the ability of lipid-rich small extracellular vesicles (sEVs) to alter the misfolding of ?syn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded ?syn were examined using a range of techniques including: Thioflavin T fluorescence, transmission electron microscopy, analytical centrifugation and western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation. We show that sEVs cause an acceleration in ?syn fibrillization and provide comprehensive evidence that this results in an increase in the abundance of mature insoluble fibrillar species. In order to elucidate the relevance of the lipid membrane to this interaction, sEV lipid membranes were modified by treatment with methanol, or a combination of methanol and sarkosyl. These treatments altered the ultrastructure of the sEVs without changing the protein cargo. Critically, these modified sEVs had a reduced ability to influence ?syn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of ?syn:EV interactions and demonstrates that sEVs are powerful modulators of ?syn fibrillization, which is mediated by the sEV membrane. In doing so, this work provides strong evidence for a role of sEVs in contributing directly to ?syn misfolding in the synucleinopathy disorders.

SUBMITTER: Ugalde CL 

PROVIDER: S-EPMC7726797 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature