In Situ Detection of α -Synuclein Seeding Activity Using Quiescent Seed Amplification Assay

Accurate detection of α-synuclein (αSyn) seeding activity is crucial for understanding synucleinopathies like Parkinson’s disease. Traditional seed amplification assays lack spatial resolution, while antibody-based methods only capture pre-formed aggregates. To overcome this limitation, we developed the Quiescent Seed Amplification Assay (QSAA), enabling the in situ amplification and visualization of αSyn seeding activity directly within tissue sections. Using QSAA, we generated the first brain-wide map of seeding activity in mice injected with αSyn pre-formed fibrils and identified endogenous αSyn aggregates in human brain samples. Additionally, by combined QSAA with immunofluorescence, IF-QSAA revealed that a portion of αSyn seeding occurs independently of pathological aggregates detected by antibodies, such as pS129. This highlights the complementary value of seeding activity mapping alongside antibody-based assessments of Lewy body pathology. Overall, QSAA and IF-QSAA provide valuable tools for investigating αSyn seeding and propagation, offering new insights into synucleinopathy pathogenesis and potential therapeutic strategies.