Using ultra-sensitive single-molecule assays and super-resolution microscopy, the authors find similar total alpha-synuclein aggregate counts in PD and control brains but detect larger aggregates and distinct diffusible/membrane-bound subpopulations enriched in PD, implying slow, cell-restricted…

By defining and detecting specific alpha-synuclein aggregate subtypes with highly sensitive methods, this work provides tools and candidate biomarkers to improve target engagement assays, stratify pathology-relevant species, and inform timing/targets for therapies directed at aggregate clearance or…

Nanoscopic aggregates of alpha-synuclein (ɑSyn) have been observed in Parkinson's disease (PD). However, the processes that occur in vivo leading to the formation of these small aggregates are not well understood. We used ultra-sensitive single-molecule methods, including single molecule array (SIMOA), and super-resolution microscopy to quantify and characterize ɑSyn aggregates harvested from human brain samples, alongside a mouse model of synucleinopathy, using different tissue processing methods. While aggregate numbers did not differ between PD and control samples, larger aggregates were detected in PD brain samples. Moreover, different sub-populations of aggregates were obtained by different extraction methods, with diffusible and membrane-bound aggregates producing a more pronounced difference between disease and control samples. Our data suggest that ɑSyn aggregates slowly in the brain, leading to formation of larger aggregates in a sub-set of cells.