In Situ Amplification and Spatial Mapping of α -Synuclein Seeding Activity

The prion-like propagation of proteopathic α-synuclein (αSyn) "seeds" is a central pathogenic mechanism in synucleinopathies. However, methods to directly visualize this "seeding activity" within intact tissue are lacking. Conventional antibody-based techniques provide only static snapshots of pathological deposits, while sensitive seed amplification assays lose spatial information via mechanical fragmentation. Here, we introduce the αSyn Quiescent Seed Amplification Assay (QSAA), a method for specific, robust, in situ amplification of αSyn seeds directly within tissue sections. QSAA demonstrates superior sensitivity and earlier detection than pS129 and ThS staining, revealing widespread seeding in a PD mouse model, and successfully identifies endogenous seeds in archival human brains. By integrating with immunofluorescence, IF-QSAA uncovered microglial seeding activity and established that pS129 phosphorylation and seeding activity represent distinct pathological states. This offers unprecedented spatial insights into synucleinopathy pathogenesis, holding significant promises for early diagnosis and therapeutic evaluation.