Revealing the proteome of motor cortex derived extracellular vesicles isolated from amyotrophic lateral sclerosis human postmortem tissues

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the deposition of misfolded proteins in the motor cortex and motor neurons. Although a multitude of ALS-associated mutated proteins have been identified, several have been linked to small extracellular vesicles such as exosomes involved in cell-cell communication. This study aims to determine the proteome of extracellular vesicles isolated from the motor cortex of ALS subjects and to identify novel ALS-associated deregulated proteins. Motor cortex extracellular vesicles (MCEVs) were isolated from human postmortem ALS (n = 10) and neurological control (NC, n = 5) motor cortex brain tissues and the MCEVs protein content subsequently underwent mass spectrometry analysis, allowing for a panel of ALS-associated proteins to be identified. This panel consists of 16 statistically significant differentially packaged proteins identified in the ALS MCEVs. This includes several upregulated RNA-binding proteins which were determined through pathway analysis to be associated with stress granule dynamics. The identification of these RNA-binding proteins in the ALS MCEVs suggests there may be a relationship between ALS-associated stress granules and ALS MCEV packaging, highlighting a potential role for small extracellular vesicles such as exosomes in the pathogenesis of ALS and as potential peripheral biomarkers for ALS

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

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