Extracellular vesicle α-synuclein topology reveals disease-associated conformers in brain and blood

Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are unified by pathological α-synuclein (αSyn) aggregation, yet diverge in terms of the cellular distribution of pathology and clinical phenotype. Extracellular vesicles (EVs) have emerged as potential mediators or biomarkers of αSyn pathology, but whether their molecular constituents in the brain reflect disease-specific processes or conformations remains unknown. Here, we combined EV isolation, electrochemiluminescence, quantitative mass spectrometry and nanoscale single-vesicle imaging by direct stochastic optical reconstruction microscopy (dSTORM) to map the topology, epitope exposure and cellular origin of αSyn in EVs isolated from post-mortem PD, DLB, MSA, and healthy control (HC) brains and assessed the tractability of these molecular signatures in blood. Proteomics and immunoblotting of EV lysates isolated from human brains confirmed that they were highly purified and derived from neurons and glial cell subtypes. Across diseases, αSyn association with EVs exhibited distinct patterns: In PD and DLB, both total αSyn levels normalised to CD81 and the percentage of neuronal (L1CAM+) EVs with aggregated αSyn as detected by aggregate-preferring antibodies, were increased, whereas in MSA, aggregated αSyn was detected primarily in neuronal and to a lesser extent glial EV subpopulations without an increase in total EV-associated αSyn. Further epitope mapping by dSTORM identified a distinct αSyn conformational arrangement, as detected by antibodies against an epitope spanning αSyn residues 34-45, that was enriched on the surface of L1CAM+ EVs in MSA compared to PD. This N-terminal immunoreactive conformer was tractable in serum and enriched on the surface of L1CAM+ EVs from MSA compared to PD patients or controls. Collectively, our findings suggest that the epitope accessibility and topology of EV-associated αSyn proteoforms, rather than their abundance alone, discriminate synucleinopathy subtypes, offering a framework for a blood-based conformational stratification of these disorders.