Fig. 6

TLR2 mediates neurotoxic neuron-to-neuron α-synuclein transmission. a Overview diagram. Donor neuronal cells (V1S), expressing α-synuclein-conjugated with N-terminus of venus were plated in trans-well insert and the recipient neuronal cells (SV2), expressing α-synuclein conjugated with C-terminus of venus were seeded onto cover slips in the bottom well. Only SV2 cells were treated with pam3CSK4 (10 μg/ml), lentiviral vectors, or antibodies. Images were taken from SV2 cells after a 3-days co-culture. b–e Representative confocal images for BiFC fluorescence and caspase-3 activity in SV2 cells. Middle panels are enlargements of cropped regions outlined with dashed lines from upper panels. Lower panels are double-immunolabeling assay with active casepase-3. The average numbers of venus fluorescence intensity in each cell, the average size of the venus punctum diameters, and caspase-3 fluorescence intensity were analyzed. b V1S and SV2 cells were co-cultured in the presence or absence of pam3CSK4 (10 μg/ml) (n = 3). c V1S and SV2 cells were co-cultured with either LV-control or LV-TLR2 (n = 3). d V1S and SV2 cells were co-cultured with either LV-sh.control or LV-shTLR2 (n = 3). e V1S and SV2 cells were co-cultured with either IgG (5 μg/ml) or T2.5 (5 μg/ml) (n = 3). f The kinetics of α-synuclein internalization in the presence of antibodies. dSY5Y cells were incubated with αSCM for indicated hours in the presence of either IgG (5 μg/ml) or T2.5 (5 μg/ml). The kinetics was analyzed by immunolabeling assay (n = 3). g Neuronal internalization of α-synuclein in the presence of antibodies. Mouse primary cortical neurons were incubated with αSCM or LZCM for indicated hours in the presence of either IgG (5 μg/ml) or T2.5 (5 μg/ml). Neurons were double immunolabelled with human α-synuclein (Middle panels) and active form of caspase-3 (low panels) (n = 3). Data are mean ± SEM (n = 3 per group). *p < 0.05, **p < 0.01, and ***p < 0.001; unpaired t test for all analysis except (g) (one way ANOVA). Scale bar, 20 μm