Seed amplification assays for diagnosing synucleinopathies: The issue of influencing factors

Background: The prion-like misfolding and aggregation of α-synuclein (α-syn) is involved in the pathophysiology of Parkinson’s disease and other synucleinopathies. Seed amplification assays (SAAs) are biophysical tools that take advantage on the peculiar properties of prion proteins by amplifying small amounts of aggregates in biological fluids at the expense of recombinant monomeric protein added in solution. SAAs have emerged as the most promising tools for the diagnosis of synucleinopathies in vivo. However, the diagnostic outcome of SAAs depends on the aggregation kinetics of α-syn, which in turn is influenced by several experimental variables. Methods: In our work, we analysed the impact on SAAs of some of the most critical experimental factors by considering models that describe the aggregation kinetics of α-syn. Results: We started our analysis by making simulations to understand which kinetic models could explain the aggregation kinetics of α-syn during incubation/shaking cycles. Subsequently, under shaking/incubation cycles similar to the ones commonly used in SAAs, we tested the influence of some analytical variables such as monomer concentration, presence/absence of glass beads, pH, addition of human cerebrospinal fluid, and use of detergents on α-syn aggregation. Conclusions: Our investigation highlighted how optimization and standardization of experimental procedures for αsyn SAAs is of utmost relevance for the ultimate goal of applying these assays in clinical routine. Although these aspects have been evaluated with specific SAA protocols, most of the experimental variables considered influenced very general aggregation mechanisms of α-syn, thus making most of the results obtained from our analyses extendable to other protocols.