Single molecule fingerprinting reveals different growth mechanisms in seed amplification assays for different polymorphs of alpha Synuclein fibrils.

Alpha-synuclein (Syn) aggregates, detected in the biofluids of patients with Parkinsons disease, have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinsons disease and turned the presence of Syn aggregates into a biomarker of the disease. It has become evident that Syn can form fibrils with slightly different structures, called "strains" or polymorphs, but little is known about their differential reactivity in diagnostic assays. Here we compared the properties of two well-described Syn polymorphs. Using single molecule techniques, we observed that one of the polymorphs had an increased tendency to undergo secondary nucleation and we showed that this could explain the differences of reactivity observed in in vitro seed amplification assay and cellular assays. Simulations and high-resolution microscopy suggest that a 100-fold difference in apparent rate of growth can be generated by a surprisingly low number of secondary nucleation "points" (1 every 2,000 monomers added by elongation). When both strains are present in the same seeded reaction, secondary nucleation displaces proportions dramatically and causes a single strain to dominate the reaction as the major end-product.