Fig. 2
L41 treatment prevents DYRK1A proteolysis in APP/PS1 mice hippocampus. a, b Western blot of hippocampus from APP/PS1 mice or littermates treated with vehicle or L41, showing lower levels of DYRK1A (90 kDa) immunoblotting with the α-DYRK1A-Cter antibody in vehicle-treated APP/PS1 mice (n = 6) compared to littermates (n = 6) (One-way ANOVA, p < 0.005). DYRK1A protein levels in L41-treated APP/PS1 mice (n = 7) were higher than in vehicle-treated APP/PS1 mice (One-way ANOVA, p < 0.005) and similar to what observed in littermates (One-way ANOVA, ns). c Calpain activity assessed by a fluorescent method, showing higher calpain activity in hippocampus from both vehicle-treated (n = 6) and L41-treated APP/PS1 mice (n = 7) compared to littermates (n = 6) (One-way ANOVA, p < 0.05 for both). There was no significant difference between L41-treated and vehicle-treated APP/PS1 mice (One-way ANOVA, ns). d DYRK1A protein levels did not correlate with calpain activity (r2 = 0.43; ns). e HPLC assay for total endogenous DYRK1A activity showing no differences between hippocampus from littermates (n = 9) and vehicle- or L41-treated APP/PS1 mice (n = 9 and 8, respectively) (One-way ANOVA, ns). f Representative images from immunohistochemical staining using the α-DYRK1A-Cter antibody, of hippocampal slices from littermates, vehicle- and L41-treated APP/PS1 mice, showing neuronal staining in the CA1 and Stratum Radiatum (StrR) regions (see enlargement at the bottom). g Representative images from immunohistochemical staining, using the α-DYRK1A-Nter antibody, of hippocampal slices, showing neuronal staining for L41-treated APP/PS1 mice and littermates in the CA1 and Stratum Radiatum (StrR) regions. Neuronal staining was observed in both the CA1 and StrR regions, whereas additional astrocyte staining was mostly observed in the Stratum Radiatum (StrR) region of vehicle-treated APP/PS1 mice. h Laser confocal microscopy showing double staining using α-DYRK1A-Cter (red) and anti-GFAP (green) antibodies. There were no differences in α-DYRK1A-Cter staining in GFAP positive cells between littermates (n = 6, astrocytes = 73), vehicle- (n = 6, astrocytes = 90), and L41-treated APP/PS1 mice (n = 6, astrocytes = 85) (One-way ANOVA, ns). i Laser confocal microscopy showing double staining using α-DYRK1A-Nter antibody (red) and anti-GFAP (green). α-DYRK1A-Nter staining was higher in the GFAP positive area in vehicle-treated APP/PS1 mice (n = 6, astrocytes = 105) compared to littermates (n = 6, astrocytes = 50) (One-way ANOVA, p < 0.0005). α-DYRK1A-Nter immunoreactivity was lower in the GFAP positive area of L41-treated (n = 6, astrocytes = 83) than vehicle-treated APP/PS1 mice (One-way ANOVA, p < 0.0005). Data represent the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Significant differences between littermates and vehicle-treated APP/PS1 mice are indicated by *p < 0.05, **p < 0.005 and ***p < 0.0005. Significant differences between vehicle- and L41-treated APP/PS1 mice are indicated by #p < 0.05, ##p < 0.005 and ###p < 0.0005