Fig. 2: Deletion of RHBDL4 in APPtg mice leads to higher APP production and amyloidogenic processing.
From: Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer’s disease mouse model
A Schematic representation of experimental design and analysis timeline for APPtg J20 mice crossed to the R4−/− model. B RHBDL4 expression in brain, pancreas and liver lysates of WT mice. Lysates from R4−/− mice are used as a negative control for RHBDL4 immunoblot. β-actin as loading control. β-actin shows variations in molecular weight depending on the tissue, which has been observed previously [86]. We speculate it is due in part to differential isoform expression, as the β-actin antibody cross-reacts with γ-actin, and to differential post-translational modifications, since actin is extensively modified [87]. C RHBDL4 expression in liver lysates of WT, APPtg, R4−/−, APPtg/R4−/+, and APPtg/R4−/− mice. β-tubulin as loading control. * probable non-specific cross-reactivity of the antibody. D Total (mutant human APP (hAPP), and endogenous mouse APP (mAPP)) APP and β-CTFs expression in brain lysates of WT, APPtg, R4−/−, APPtg/R4−/+, and APPtg/R4−/− mice. Expression was quantified and normalized to Ponceau-S. n = 8 per group, box and whisker plots represent minimum to maximum values with median center lines while blue “+” represents the mean; For APP: Two-tailed, unpaired t-test performed between WT and R4−/− group and one-way ANOVA (p < 0.0001) with Holm-Sidak multiple comparison test where APPtg is compared to all other groups. For β-CTFs: one-way ANOVA (p < 0.0001) with Holm-Sidak multiple comparison test where APPtg is compared to all other groups. Significant p values for t-test and post hoc analysis reported. E DEA soluble Aβ38, Aβ40, and Aβ42 concentration in APPtg, APPtg/R4−/+, and APPtg/R4−/− brains. n = 12–15 for mixed sexes, n = 6–9 for females (purple) and n = 5–7 for males (green). Box and whisker plots represent minimum to maximum values with median center lines while blue “+” represents the mean; For mixed sexes: two-way ANOVA (non-significant interaction, p < 0.0001 for Aβ species main effect and non-significant genotype main effect) with Tukey’s multiple comparison test. For females: two-way ANOVA (non-significant interaction, p < 0.0001 for Aβ species main effect and p = 0.0032 for the genotype main effect) with Tukey’s multiple comparison test. For males: two-way ANOVA (non-significant interaction, p = 0.0001 for Aβ species main effect and non-significant genotype main effect) with Tukey’s multiple comparison test. Significant p values for post hoc analysis reported. F Aβ42/Aβ40 or Aβ42/total Aβ38 + 40 + 42 ratios (DEA soluble) in APPtg, APPtg/R4−/+, and APPtg/R4−/− brains. n = 12–15 per group, mean ± SEM; non-significant Brown-Forsythe and Welch’s ANOVA (p = 0.557 for Aβ42/ Aβ40 ratio and p = 0.613 for Aβ42/total Aβ38 + 40 + 42 ratio). G Formic acid soluble Aβ42 concentration in APPtg, APPtg/R4−/+, and APPtg/R4−/− brains. n = 12–15 per group, mean ± SEM; non-significant Kruskal–Wallis test (p = 0.281). D–G Female data points are in purple and male in green. For all tests, assumptions of normality and variance were verified using Shapiro-Wilk test and Brown-Forsythe test, respectively.
