An intact membrane is essential for small extracellular vesicle‐induced modulation of α‐synuclein fibrillization

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.

Beyond coincidence in Hong-Ou-Mandel interferometry

“…Our results with EV using protein aggregation assay also strongly implicate alpha-synuclein fibrillisation upon EV membrane binding. Intact EV membranes are essential for the process of EV-mediated aggregation of alpha-synuclein [89]. It is unclear if this effect is exerted by membrane lipids, membrane curvature, membrane-associated proteins, or other factors such as those identified in this study.…”

Exosomes, microvesicles, and other extracellular vesicles—a Keystone Symposia report

“…Hill's group has shown that small EVs can accelerate the misfolding of ɑ‐synuclein and promote the production of higher‐order, neurotoxic species. They confirmed that an intact EV membrane is important to support and enhance ɑ‐synuclein misfolding 62,63 . Hill also discussed the potential of EVs as diagnostic biomarkers for neurodegenerative diseases.…”

“…They confirmed that an intact EV membrane is important to support and enhance ɑ-synuclein misfolding. 62,63 Hill also discussed the potential of EVs as diagnostic biomarkers for neurodegenerative diseases. His lab has identified a panel of nine miRNAs that are dysregulated in exosomes from prion-infected neuronal cells in mice.…”

miR-6869-5p Transported by Plasma Extracellular Vesicles Mediates Renal Tubule Injury and Renin-Angiotensin System Activation in Obesity

“…The EV morphology was evaluated using transmission electron microscopy (TEM) (JEM-1400, Japan), as previously described ( 26 ). The particle size distribution and EV concentrations were measured by nanoparticle tracking analysis (NTA) ( 27 – 29 ).…”