α-Synuclein purification significantly impacts seed amplification assay performance and consistency
Zaid A.M. Al‐Azzawi, Nicholas R. G. Silver, Surabhi Mehra, Simeng Niu, Christopher Situ, Wen Luo, Irina Shlaifer, Martin Ingelsson, Bradley T. Hyman, Jean‐François Trempe, Thomas M. Durcan, Joel C. Watts, Edward A. Fon
Abstract
α-Synuclein seed amplification assays are a promising diagnostic tool for synucleinopathies such as Parkinson's disease and multiple system atrophy. Standardized conditions are required to ensure a high degree of inter- and intra-laboratory reproducibility when performing these assays. A significant issue that hinders the utility of seed amplification assays is the de novo aggregation propensity of the α-synuclein substrate as well as inter-batch heterogeneity. While much work has focused on determining appropriate seed amplification assay buffer compositions as well as the type and amount of seed used, a robust comparison of α-synuclein substrate purification methods has not been reported. We therefore compared the utility of recombinant α-synuclein purified using four different methods as seed amplification assay substrates across two laboratories. Osmotic shock-purified α-synuclein monomer substrate showed the lowest propensity for de novo aggregation, which translated into being the best substrate for seed amplification assay reactions seeded with α-synuclein preformed fibrils or patient brain homogenates. Furthermore, osmotic shock α-synuclein monomer showed the best inter-batch reproducibility compared to all other substrates tested. As α-synuclein seed amplification assays continue to evolve and move towards adoption in the clinical realm, this work showcases the vital importance of standardizing the production and characterization of recombinant α-synuclein substrate. We encourage the widespread adoption of osmotic shock α-synuclein monomer as the universal substrate for seed amplification assays to maximize intra- and inter-laboratory reproducibility.