Stools and stool-derived extracellular vesicles from patients with Parkinson`s disease show alpha-4 synuclein seeding activity

Civitelli, L.; Stafford-Dorlandt, P.; Jovanoski, K. D.; Begum, A.; Lee, S. S.; Dellar, E. R.; Mertsalmi, T.; Kainulainen, V.; Arkkila, P.; Levo, R.; Ortiz, R.; Kaasinen, V.; Scheperjans, F.; Parkkinen, L.

doi:10.64898/2026.03.12.709633

bioRxiv · 10.64898/2026.03.12.709633

Stools and stool-derived extracellular vesicles from patients with Parkinson`s disease show alpha-4 synuclein seeding activity

Civitelli, L.·Stafford-Dorlandt, P.·Jovanoski, K. D.·Begum, A.·Lee, S. S.·Dellar, E. R.·Mertsalmi, T.·Kainulainen, V.·Arkkila, P.·Levo, R.·Ortiz, R.·Kaasinen, V.·Scheperjans, F.·Parkkinen, L.

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Abstract

BackgroundParkinsons 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 (Syn) 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 Syn species in the cerebrospinal fluid (CSF) of PD patients. However, Syn 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). MethodsStool samples from PD patients (n=45) and healthy controls (n=35) were analysed for the presence of Syn species using slot blot assays with a panel of six Syn 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. ResultsProtein extracts from both PD and HC stool samples revealed pathological Syn species in the slot blot assay using the phosphorylated Syn antibody, pS129 and conformation-specific antibodies, MJFR-14 and 5G4. ELISA showed significantly elevated total Syn levels in PD samples compared to HC, although no differences in aggregated Syn 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 Syn achieved 100% sensitivity and 100% specificity. ConclusionThese findings suggest that SAA applied to EVs isolated from stool samples, particularly after pre-incubation with recombinant monomeric Syn, may serve as a valuable, non-invasive screening tool for the diagnosis of PD.

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Civitelli, L., Stafford-Dorlandt, P., Jovanoski, K. D., Begum, A., Lee, S. S., Dellar, E. R., Mertsalmi, T., Kainulainen, V., Arkkila, P., Levo, R., Ortiz, R., Kaasinen, V., Scheperjans, F., Parkkinen, L.. 2026-03-16. Stools and stool-derived extracellular vesicles from patients with Parkinson`s disease show alpha-4 synuclein seeding activity. https://doi.org/10.64898/2026.03.12.709633

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