Fig. 4

Overexpression of miR-140 and miR-122 reduced microgliosis and microglia phagocytosis. (A-B). Representative confocal z-stack images showing results of immunohistochemical analysis in the DG subregions on coronal sections of the hippocampus of control C57 and APP/PS1 mice, which were transfected with AAV9-Scr-eGFP or AAV9-miR-140-miR-122-eGFP, respectively. Aβ deposition was stained with MOAB-2 (white) or ThioS (green). Microglia cells were stained using anti-Iba1 (red). Scale bar: 200 μm. (C-E). Quantitative analysis of the immunofluorescence intensities of three consecutive sections of three independent biological replicates, showing average number of MOAB-2⁺ Aβ plaques (C), ThioS⁺ fibrilar Aβ plaques (D) and percentage of Iba1 postive area (E). Results were presented as meam ± SEM. *p < 0.05. Statistical analyses were performed using One-way ANOVA. (F-G). Results of western blot analysis and densitometric quantifications of the relative expression of Iba1 (F) and IL-1β (G) in the hippocampi of APP/PS1 mice transfected with AAV9-Scr-eGFP or AAV9-miR-140-miR-122-eGFP, respectively. Untransfected APP/PS1 mice and wild type C57 mice were used as controls. The expression of β-actin was used as an internal control. The results were presented as means ± SD (n = 3). *p < 0.05, **p < 0.01. Statistical analyses were performed using One-way ANOVA. (H-J). Aβ phagocytosis analysis in BV2 cells co-cultured with untransfected HT22 cells or HT22 cells that were transfected with scramble miRNAs, mimics of miR-140 and miR-122, pcDNA3.1, or pcDNA3.1-miR-140-miR-122-TuD, respectively. Representative images of immunofluorescence showing Iba1 (red) labelled BV2 cells and FITC-Aβ (green) (H). Quantitative analysis of fluorescence intensities showing the average fluorescence intensity of FITC-Aβ in phagocytic BV2 cell (I) and proportion of Aβ phagocytic BV2 cells (J). The results were presented as means ± SEM (n = 9). *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analyses were performed using One-way ANOVA. Scale bar: 20 μm. (K-M). Western blot analysis and densitometric quantification of the relative expression of Cx3cl1 (K), Cx3cr1 (L) and Trem2 (M) in the hippocampi of APP/PS1 mice transfected with AAV9-Scr-eGFP or AAV9-miR-140-miR-122-eGFP, respectively. Untransfected APP/PS1 mice and wild type C57 mice were used as controls. The expression of β-actin was used as an internal control. The results were presented as means ± SD (n = 3). Statistical analyses were performed using One-way ANOVA