Enrichment and amplification of secreted α-synuclein from cellular model and saliva of patients with synucleinopathies

The aggregation and propagation of pathological α-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 secreted α-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 concentrated α-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-QuIC) assays revealed that TCA-enriched α-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 modified α-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 targeting α-Syn transmission.