Urine‐Derived Neuronal Extracellular Vesicle α ‐Synuclein Seed Amplification Assay for Synucleinopathies: A Non‐Invasive Biomarker Study

Abstract Background α ‐Synuclein aggregation is a pathological hallmark of synucleinopathies, including Parkinson's disease (PD) and multiple system atrophy (MSA). Although pathogenic α ‐synuclein seeds can be detected by real‐time quaking‐induced conversion (RT‐QuIC), current assays primarily rely on cerebrospinal fluid, whose invasiveness limits repeated and large‐scale applications. We therefore evaluated the feasibility of detecting pathogenic α ‐synuclein using urine‐derived neuronal extracellular vesicles (NEVs). Methods Urine‐derived NEVs were enriched by direct immunoprecipitation using L1CAM‐conjugated magnetic beads. The RT‐QuIC assay was performed in 26 patients with PD, 13 with MSA, and 25 disease controls, including 16 healthy controls (HC), 4 with progressive supranuclear palsy (PSP), and 5 with normal pressure hydrocephalus (NPH). Results Urine‐derived NEVs exhibited characteristic neuronal and exosomal markers and a typical extracellular vesicle size distribution. The urine‐based RT‐QuIC assay distinguished PD from disease controls with 73.1% sensitivity and 84% specificity, and MSA from disease controls with 76.9% sensitivity and 84% specificity. When compared with HC alone, sensitivity and specificity were 73.1% and 93.8% for PD, and 76.9% and 93.8% for MSA, respectively. Kinetic analysis showed higher maximum fluorescence intensity and area under the curve (AUC) in PD compared with both disease controls and HC ( P < 0.001 in all). MSA also demonstrated higher maximum fluorescence intensity than HC and increased AUC compared with both disease controls and HC ( P < 0.05 in all). Conclusions We developed and optimized a completely non‐invasive urine‐derived NEV α ‐synuclein RT‐QuIC assay that demonstrates high specificity and moderate sensitivity for synucleinopathies. This approach warrants further validation in larger cohorts. © 2026 International Parkinson and Movement Disorder Society.