Impact of α-synuclein fibril structure on seeding activity in experimental models of Parkinson's disease

The central pathogenesis of Parkinson's disease involves the misfolding and aggregation of α-synuclein (α-syn). There is a widespread belief that α-syn can propagate in a prion-like manner, and α-syn preformed fibrils (PFFs) have been widely used to establish α-syn propagation models. However, achieving standardized protocols for generating PFFs is challenging due to the influence of various factors on propagation efficiency, resulting in inter-laboratory and inter-experimental variability. Among these factors, the size of the PFFs is considered the most influential as unsonicated PFFs exhibit limited seeding and propagation abilities. Therefore, the objective of our research is to examine the impact of the size and conformation of sonicated PFFs on seeding activity. PFFs were sonicated under various conditions using a conventional water bath sonicator and a high-power sonicator, which is commonly used for DNA shearing in next-generation sequencing. Each sonicated PFF was analyzed for in vitro/in vivo seeding activities, after size confirmation by electron microscopy and a conformational analysis by Fourier Transform Infrared (FTIR) spectroscopy. Strong sonication for 30 min generated extremely short fibrils with the highest seeding activity, which is the optimal condition for the propagation model, whereas sonication for 60 minutes or more led to a reduction in seeding activity. FTIR spectroscopy suggested that sonication disrupted the aggregated strands and generated new fibril ends, thereby accounting for the increased seeding activity; however, prolonged sonication for 60 min or more released monomers with disrupted β-sheet structure from PFFs and reduced the seeding activity. In conclusion, the balance between size reduction and preservation of the β-sheet structure in PFFs plays a critical role in seeding activity. Optimizing these parameters of α-syn PFFs can help improve reproducible preclinical animal models based on α-syn propagation.