Abstract
The misfolding and fibrillization of the protein, α‐synuclein (αsyn), is associated with neurodegenerative disorders referred to as the synucleinopathies. Understanding the mechanisms of αsyn misfolding is an important area of interest given that αsyn misfolding contributes to disease pathogenesis. While many studies report the ability of synthetic lipid membranes to modulate αsyn folding, there is little data pertaining to the mechanism(s) of this interaction. αSyn 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 αsyn 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 αsyn induced using the Protein Misfolding Cyclic Amplification (PMCA) assay. The biochemical and biophysical properties of misfolded αsyn 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 αsyn 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 αsyn fibrillization compared to untreated counterparts. This study reports the first comprehensive examination of αsyn:EV interactions and demonstrates that sEVs are powerful modulators of αsyn 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 αsyn misfolding in the synucleinopathy disorders.
Highlights
Neurodegenerative proteinopathies (NDPs) are a group of disorders where the aggregation of specific proteins in compartments of the central nervous system (CNS) occurs in conjugation with neuronal loss
Numerous data show misfolded αsyn can contribute to disease pathogenesis (Ugalde et al, 2016), the molecular triggers which underlie αsyn misfolding in sporadic disease are poorly elucidated
This was determined using small extracellular vesicles (sEVs), biologically-derived lipid-rich vesicles, which were found to cause dramatic changes to how αsyn misfolds. This is the first study to thoroughly characterise the effect sEVs have on the formation of misfolded αsyn and was performed using multiple technical approaches to enable strong experiment conclusions to be drawn from the data obtained
Summary
Neurodegenerative proteinopathies (NDPs) are a group of disorders where the aggregation of specific proteins in compartments of the central nervous system (CNS) occurs in conjugation with neuronal loss. The synucleinopathies are one of the most common NDP, which collectively include disorders associated with the intracellular accumulation of β-sheet rich fibrils composed on misfolded α-synuclein (αsyn). They include Parkinson’s disease, dementia with Lewy Body and Multiple System Atrophy, among others. Despite the strong link between familial mutations in SNCA and the aggregation of translated protein, the overwhelming majority of synucleinopathies are of sporadic origin. In these disorders, there is little understanding into the events that trigger or contribute to the misfolding of αsyn
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