Stochastic misfolding drives the emergence of distinct α-synuclein strains.

α-Synuclein conformational strains provide a potential explanation for the clinical and pathological differences among synucleinopathies such as Parkinson's disease and multiple system atrophy. However, how distinct α-synuclein strains arise remains unknown. Here, we observed conformational heterogeneity between individual preparations of α-synuclein pre-formed fibrils (PFFs) generated by polymerizing wild-type or A53T-mutant human α-synuclein under identical conditions. Moreover, we found that α-synuclein aggregates formed spontaneously in the brains of a transgenic synucleinopathy mouse model are conformationally diverse. Propagation of stochastically formed PFF- and brain-derived α-synuclein strains in mice initiated several distinct synucleinopathies. The conformational diversity of α-synuclein aggregates across PFF preparations and between individual mice demonstrates that α-synuclein can spontaneously form multiple self-propagating strains within an identical environment. This suggests that stochastic misfolding into distinct aggregate structures drives the emergence of α-synuclein strains and reveals that the intrinsic variability of common synucleinopathy research tools must be considered when designing and interpreting experiments.