Enrichment and amplification of secretedα-synuclein from cellular model and saliva of patients with synucleinopathiesOA

The aggregation and propagation of pathologicalα-synuclein(α-Syn)are central to the pathogenesis of synucleinopathies,including Parkinson''s disease(PD),multiple system atrophy(MSA),and dementia with Lewy bodies(DLB).This study focuses on the enrichment and amplification of secretedα-Syn from cellular models and the saliva of PD patients,utilizing trichloroacetic acid(TCA)precipitation to retain the post-translational modifications(PTMs)and seeding properties of extracellular proteins.By applying TCA precipitation,we successfully concentratedα-Syn from PD patient saliva and cell culture media while preserving critical PTMs(e.g.,truncation and phosphorylation)that are essential to amyloidogenicity.Subsequent Western blot(WB)and real-time quaking-induced conversion(RT-Qu IC)assays revealed that TCA-enrichedα-Syn retained robust prion-like seeding activity,enabling the cross-cellular propagation of pathologically misfolded species.This workflow establishes TCA precipitation as a unique tool for capturing pathologically modifiedα-Syn from biofluids while maintaining their native seeding capacity.By integrating enrichment and amplification strategies,our findings advance the utility of saliva-based biomarkers for synucleinopathies and provide a translational platform to evaluate therapeutics targetingα-Syn transmission.