Stools and stool-derived extracellular vesicles from patients with Parkinson’s disease contain alpha-synuclein species with seeding capacity

Abstract Background Parkinson’s disease (PD) is a neurodegenerative disorder for which there is currently no cure or reliable biomarker for early detection or for evaluating the effectiveness of potential treatments. PD pathology is driven by misfolding and subsequent accumulation of alpha-synuclein (αSyn) protein into pathological aggregates within neurons and glial cells. Seed amplification assay (SAA) is a highly sensitive and specific diagnostic tool developed to detect pathological αSyn species in the cerebrospinal fluid (CSF) of PD patients. However, αSyn aggregates are present in multiple tissues and biosamples, including stools. In this study, we aimed to investigate the potential diagnostic value of SAA using stool samples from PD patients and healthy controls (HC). Methods Stool samples from PD patients (n=45) and healthy controls (n=35) were analysed for the presence of αSyn species using slot blot assays with a panel of six αSyn antibodies, and ELISA assays. Samples were subjected to SAA, and the end-point products (SAA EP) were characterised using transmission electron microscopy (TEM). Extracellular vesicles (EVs) were isolated from the subset of samples (n=5 per group) using size exclusion chromatography and characterized by TEM. The seeding activity of isolated EVs was evaluated using SAA, followed by TEM analysis of SAA EP. Results Protein extracts from both PD and HC stool samples revealed pathological αSyn species in the slot blot assay using the phosphorylated αSyn antibody, pS129 and conformation-specific antibodies, MJFR-14 and 5G4. ELISA showed significantly elevated total αSyn levels in PD samples compared to HC, although no differences in aggregated αSyn levels were detected. In stool protein extracts, SAA demonstrated 55.6% sensitivity and 60% specificity. When applied to stool-derived EVs from PD patients and controls, sensitivity increased to 100%, while specificity remained at 60%. Notably, SAA applied to stool-derived EVs pre-incubated with recombinant monomeric αSyn achieved 100% sensitivity and 100% specificity. Conclusion These findings suggest that SAA applied to EVs isolated from stool samples, particularly after pre-incubation with recombinant monomeric αSyn, may serve as a valuable, non-invasive screening tool for the diagnosis of PD.