Fig. 3
TNAP hemizygosity influences microglial response in APP/PS1+/− mice. (A) Representative immunofluorescence images of hippocampal sections stained with the microglial marker Iba-1 (green channel) used to assess microglial density in the vicinity of senile plaques (WO2, red channel) in APP/PS1+/− and APP/PS1+/−/TNAP+/− mice. Distances of 40 and 80 μm from the periphery of senile plaques are identified with white dashed lines. Scale bar: 50 μm. (B, C) The graphs represent the distribution of values per group (median, IQR, maximum, and minimum values) of the density of hippocampal microglia per plaque area (µm²) (B) and the percentage of microglia cells found in every delimitated area (C) (n ≧ five mice per genotype). ** P ≤ 0.01; using an unpaired two-tailed Student’s t-test or * P ≤ 0.05 or *** P ≤ 0.001; using a one-way ANOVA followed by Tukey’s post hoc test (D) Representative single confocal microscopy and orthogonal views showing CD68 positive-phagocytic microvesicles (red channel) phagocytosing Aβ peptides (Aβ, grey channel) within microglia (Iba-1, green channel) surrounding hippocampal senile plaques. (E) Graph represents the distribution of values per group (median, IQR, maximum, and minimum values) of the percentage of lysosomes containing amyloid-β of the total microglial lysosomes. ** P ≤ 0.01; using an unpaired two-tailed Student’s t-test (F) Representative immunoblot of NLRP3, P2X7, and IL-1β in homogenates from the hippocampus of WT, APP/PS1+/− and APP/PS1+/−/TNAP+/− mice. (G-I) Quantification of NLRP3 (G), P2X7 (H), and IL-1β (I) protein levels in the hippocampus of WT (n = 10), APP/PS1+/− (n = 10 or 11), and APP/PS1+/−/TNAP+/− (n = 8) mice. (J) Representative immunoblot using an antibody recognizing cleaved and full-length OPN in homogenates from the hippocampus of WT, APP/PS1+/− and APP/PS1+/−/TNAP+/− mice. (K and l) Quantification of the ratio between cleaved OPN and full-length OPN (K) and total OPN (L) protein levels in the hippocampus of WT (n = 11), APP/PS1+/− (n = 11), and APP/PS1+/−/TNAP+/− (n = 8) mice. (M) Representative immunoblot of LRP1 and MMP9 in homogenates from the hippocampus of WT, APP/PS1+/− and APP/PS1+/−/TNAP+/− mice. Quantification of LRP1 (M) and MMP9 (P) protein levels in the hippocampus of WT (n = 10), APP/PS1+/− (n = 10 or 12), and APP/PS1+/−/TNAP+/− (n = 8) mice. Levels of α-tubulin were used as loading control for normalization purposes. Data are given as a percentage relative to WT mice, and graphs show the distribution of values per group (median, IQR, maximum, and minimum values). #P ≤ 0,08; * P ≤ 0.05 or ** P ≤ 0.01; using a one-way ANOVA followed by Tukey’s post hoc test. O. Aβ1−40 levels in serum samples from APP/PS1+/− (n = 8) and APP/PS1+/−/TNAP+/- (n = 8) mice. Serum samples were analyzed via Aβ1−40-detecting ELISA. * P ≤ 0.05, using an unpaired two-tailed Student’s t-test