Fig. 6

Effect of human AD-tau triggered endogenous mouse tau aggregation on synapses. A a Western blots of two pre-synaptic markers (synaptophysin (SYN) and Bassoon) and two post-synaptic markers (PSD95 and Homer) of lysed neurons after 3, 7, and 14 days of inoculation with AD1-6 samples and controls. b The linear regression indicates the overall effect on the post-synaptic compartment (p < 0.001, n = 18 per time point, three independent experiments for each AD-tau inoculum, six AD-tau samples). There is no statistically significant difference in pre-synaptic marker levels at different time points (p > 0.05, n = 18 per time point). The data were expressed as densities of target protein bands normalized to GAPDH and then as n-fold change to medium-treated cells and combined from three independent experiments. B The levels of synapses in primary neurons inoculated with AD-tau samples. a Representative images of maximum intensities from 0.35 µm z-stacks of control (medium) and AD1-inoculated culture stained for actin (grey), Bassoon (cyan), and Homer (magenta) display disruption in actin pattern staining, a slightly elevated number of Bassoon dots (red) and overall decreased fluorescence of Homer staining (green) in AD-tau treated cells. Scale bars: 30 µm. b Neurons treated with various AD-tau show statistically significant differences in levels of both markers, the presynaptic Bassoon and the postsynaptic Homer, expressed as a corrected total cell intensity (CTCF) in individual neurons. One-way ANOVA was applied (mean ± SD; p < 0.0001, n = 30–35 per AD-tau case summarized from two independent experiments). c, d The overlap of Homer and Bassoon signal was quantified by colocalization of the color threshold of RGB image combined from red and green channels and the threshold was set as a scale of 0–255 for the total signal area (red, green, and overlapped combined) and 37–48 window for merged of green and red signal to yellow spectrum. The percentage of colocalization was calculated as a ratio of the merged signal area to the total signal area and multiplied by 100. When compared to untreated neurons, cultures with aggregated mouse tau triggered by all AD-tau samples had significantly reduced levels of synapses (One-way ANOVA with multiple comparisons, p < 0.0001, n = 32 per treatment from two independent experiments)