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

journal contribution
posted on 07.01.2021, 02:50 by Cathryn Ugalde, Shane Gordon, Mitch Shambrook, Amirmohammad Nasiri Kenari, Bradley M Coleman, Matthew Perugini, Victoria A Lawson, David I Finkelstein, Andrew Hill
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.

History

Publication Date

10/12/2020

Journal

Journal of Extracellular Vesicles

Volume

10

Issue

2

Article Number

e12034

Pagination

14p. (p. 1-14)

Publisher

Wiley

ISSN

2001-3078

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The Author reserves all moral rights over the deposited text and must be credited if any re-use occurs. Documents deposited in OPAL are the Open Access versions of outputs published elsewhere. Changes resulting from the publishing process may therefore not be reflected in this document. The final published version may be obtained via the publisher’s DOI. Please note that additional copyright and access restrictions may apply to the published version.

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