Stabilization of α-synuclein oligomers using formaldehyde

The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type alpha-synuclein (alpha-syn) aggregates within degenerating brain cells. alpha-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating alpha-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized alpha-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific alpha-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked alpha-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked alpha-syn oligomers could serve as important calibrators to facilitate comparative and standardized alpha-syn biomarker studies going forward.