Comparative in situ analysis of α-synuclein seeding activity versus pS129 pathology reveals cryptic, non-phosphorylated seeds

Introduction
Standard detection of pathological α-synuclein relies on markers like phosphorylation at serine 129. However, this static marker may not fully represent the functional, prion-like seeding activity that drives disease propagation. A method to map this biological activity in situ is needed to better understand the initial stages of pathology.

Objectives
This study aimed to directly compare the spatial distribution of functional αSyn seeding activity with pS129 immunoreactivity using a novel IF-QSAA, while validating an agarose gel overlay technique for spatial fidelity.

Methods
We applied an optimized IF-QSAA protocol to frozen brain sections from a synucleinopathy mouse model. A key refinement involved applying a low-concentration agarose gel overlay prior to the QSAA reaction to prevent diffusion of amplification products. Spatial patterns of QSAA and pS129 signals were then systematically compared.

Results/Discussion
The agarose overlay successfully constrained amplification products, enabling high-resolution mapping. At a regional level, the distribution of QSAA signals largely corresponded with pS129 pathology. However, at the cellular level, IF-QSAA revealed a crucial divergence: a distinct population of QSAA-positive signals was identified in areas lacking any detectable pS129 immunoreactivity. The QSAA+/pS129- population demonstrates a dissociation between functional seeding competency and the pS129 modification. This suggests seeding activity can precede S129 phosphorylation, highlighting limitations of relying solely on static markers to identify all active pathological species.

Conclusion
IF-QSAA provides functional information complementing traditional immunohistochemistry. By revealing a non-phosphorylated, seeding-active αSyn population, this technique is a valuable tool for investigating early pathology and assessing therapies aimed at inhibiting seed formation.