Preparing Preformed Fibrils of α-Synuclein

Recombinant full-length human WT α-syn with a N-terminal His-tag and a C-terminal Avi-tag was obtained as previously described [34 (link)]. To prepare PFFs, 0.9 mg/mL recombinant protein in PBS was incubated at 37°C with constant agitation for 4 days. The presence of cross-β-sheet structure and final plateau of aggregation was verified by Thioflavin T fluorescence. The reaction product was centrifuged (20000 g) at 4°C for 1 h and the supernatant was discarded. The pellet was resuspended in PBS (1.67 mg/mL) and sonicated with a Hielscher UP200St ultrasonic homogeniser (two cycles of 1 min in pulses of 10 s and 5 s rest). The resulting PFFs were aliquoted, snap frozen and stored at −80°C. Commercial untagged human WT α-syn PFFs (StressMarq) were also employed. Prior to use, PFFs were further diluted in PBS into the required concentration and re-sonicated (1 min in pulses of 10 s and 5 s rest). PFFs were directly added to the medium of WT neurons to reach a final concentration of 5 μg/mL. Equal volume of PBS was added as control condition.