Transduction of α-Synuclein into Hippocampal Neurons

αSyn transduction into mouse primary hippocampal neurons was performed as described previously^{32 (link)}. hPFF, LB-αSyn, ampLB, and αSyn aggregates extracted from Thy1:SNCA/Snca^–/– mouse brains were diluted in DPBS and sonicated with a Diagenode Biorupter sonicator (20 min, 30 s on, 30 s off, 10 °C, high setting). Neurons were then treated with a noted dose of the αSyn preparations at 7 days in vitro (DIV), fixed, and immunostained at 14 days post-treatment (21 DIV). The amounts of αSyn transduced in Fig. 1f, Supplementary Fig. 1, and in Supplementary Fig. 4e were 25 and 50 ng/well, respectively. For the treatment with αSyn aggregates extracted from Thy1:SNCA/Snca^–/– mouse brains in Fig. 7b, the brain lysates containing 25 ng of αSyn or up to 6.4 μg of total protein per well were transduced to avoid significant toxicity of contaminants for cultured neurons^{32 (link)}. The resulting amounts of αSyn were 2.4 ng/well (Thy1:SNCA/Snca^–/– mice without αSyn pathology), 19 ng/well (ampLB-injected Thy1:SNCA/Snca^–/– mice), and 25 ng/well (hPFF-injected Thy1:SNCA/Snca^–/– mice and Thy1:SNCA/Snca^–/– mice with spontaneous αSyn pathology).