Fig. 2: TNF-α regulates α-synuclein propagation.
a, b Effects of TNF-α on the cell-to-cell propagation of α-synuclein. a Representative images in which α-synuclein propagation was observed (treated dose: 50 ng/ml). BiFC-positive cells are indicated with arrows. Scale bar: 20 µm. b Quantification of the relative percentages of BiFC-positive cells. c, d Effects of MgCM from cultures of the WT (TNF-α+/+) or TNF-α−/− microglia treated with LPS (MgCM-LPS) or DMSO (MgCM-control) on the propagation of α-synuclein. Arrows: BiFC-positive puncta. Scale bar: 20 µm. d Percentage of BiFC-positive cells in (c). e–i Effects of TNF-α on the spreading of α-synuclein in vivo. e Representative images of phosphorylated α-synuclein (pS129) staining in mouse brain sections at 20 weeks post-injection of α-synucleinV40G multimers (or PBS) into the WT (TNF-α+/+) or TNF-α−/− mice. Scale bar: 100 μm. f–i Levels of pS129. Optical densities were measured in the motor cortex (f; WT + PBS, n = 8; KO + PBS, n = 6; WT + α-synuclein, n = 7; KO + α-synuclein, n = 8), cingulate cortex (g; WT + PBS, n = 8; KO + PBS, n = 6; WT + α-synuclein, n = 7; KO + α-synuclein, n = 8), rhinal cortex (h; WT + PBS, n = 8; KO + PBS, n = 7; WT + α-synuclein, n = 8; KO + α-synuclein, n = 8), and amygdala (i; WT + PBS, n = 8; KO + PBS, n = 7; WT + α-synuclein, n = 7; KO + α-synuclein, n = 8). j–l Effects of TNF-α on the accumulation of α-synuclein in neurons. j Representative western blot images of α-synuclein in Triton X-100-soluble (Tx-sol) and Triton X-100-insoluble (Tx-insol) fractions. The quantified region is indicated on the right side of the blot. The arrowhead indicates quantified α-synuclein in Tx-sol. The line includes the quantified area in Tx-insol. Quantification of α-synuclein in Tx-sol (k) and Tx-insol (l) fractions. m, n Effects of TNF-α on the secretion of α-synuclein in neurons. m Secreted α-synuclein in media, measured by ELISAs. n Effects of TNF-α-deficient microglia on the neuronal secretion of α-synuclein. Primary neurons were treated with conditioned media acquired from the WT or TNF-α-deficient microglia pretreated with LPS or vehicle. Data are expressed as the mean ± SEM (*P < 0.05, #P < 0.05 **P < 0.005, ##P < 0.005, ***P < 0.0005, ****P < 0.0001, ####P < 0.0001). Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc comparison between groups (b, k–m), two-way ANOVA with Bonferroni’s post hoc comparison between groups (d), and two-way ANOVA with Tukey’s post hoc comparison between groups (f–i, n).
