Fig. 6: Seeding of aggregation of recombinant and neuronally expressed αSyn by pathogenic αSyn species exocytosed from neurons.

a–c Recombinant purified myc-tagged αSyn (myc-αSyn) was shaken at 37 °C in presence of concentrated extracellular medium from mouse cortical neuron cultures collected over 48 h (DIV 47–49), generated either from wild-type (WT) mice, or from Tg^x2-αSyn^A53T mice with or without lentiviral expression of VAMP7 dominant-negative fragment (VAMP7^DN; infected at DIV7). Aggregation of myc-αSyn was analyzed at the indicated days of incubation by the following assays: a Congo-red derivative, amyloid-binding dye K114 fluorescence at 390/535 nm (n = 4). b Amyloid-binding dye Thioflavin-T fluorescence at 450/485 nm (n = 4). c Quantitative immunoblotting for the myc epitope-tag, where aggregation is measured as disappearance of monomeric myc-αSyn (top; n = 4); dot-blotting for filamentous myc-αSyn aggregates using αSyn^Fila antibody (middle; n = 4); and dot-blotting for amyloid-type myc-αSyn aggregates using αSyn^Amyl A11 antibody (bottom; n = 4). d Concentrated media collected over 48 h (DIV 47–49) from WT neurons, or from Tg^x2-αSyn^A53T neurons with or without lentiviral expression of VAMP7^DN was added to the medium of Tg^x2-αSyn^A53T neurons at DIV14 and removed 48 h later by replacing the medium. Neurons were harvested at indicated times, and αSyn aggregation was measured by immunoblotting, and normalized to synaptogyrin-1 (Syngr-1) levels. Membrane-matched dot blots (β-Actin = loading control) are separated by dashed lines. (n = 3). All data represent means ± SEM, where each “n” is an independent media collection and aggregation experiment. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-way ANOVA.