Fig 1: T2DM affects ?-secretase activity differently in WT and hAPP NL/F mice.(A) Plot showing the comparison of the Aß42 peptide content in the homogenization buffer (HB)-soluble fraction extracted from cerebral cortices of WT and hAPP NL/F knockin (NL/F) mice with T2DM or control condition (CTL), (WT-CTL n = 7; WT-T2DM n = 9; NL/F-CTL n = 10; NL/F-T2DM n = 10). Statistical analysis was performed by Kruskal–Wallis test (P < 0.0001), followed by Dunn’s multiple comparisons test (*P < 0.05, ***P < 0.001, ****P < 0.0001). Mann–Whitney test also revealed a significant effect of T2DM in NL/F mice (###P = 0.0005). Aß42 content was normalized to the total protein present in this fraction and determined by BCA assay. (B) Plot showing the comparison of the Aß42 peptide content in the guanidine–hydrochloride-soluble fraction (Insoluble Aß) extracted from cerebral cortices of WT and NL/F mice, CTL and T2DM (WT-CTL n = 7; WT-T2DM n = 8; NL/F-CTL n = 9; NL/F-T2DM n = 8). Statistical analysis was performed by Kruskal–Wallis test (P < 0.0001), followed by Dunn’s multiple comparisons test (*P < 0.05, ***P < 0.001, ****P < 0.0001). Mann–Whitney test also revealed a significant effect of T2DM in NL/F mice (###P = 0.0006). (C) Representative immunoblots of PS1-CTF, APP, APP-CTF, and NRG1-CTF in total lysates of brain cortical samples from CTL or T2DM WT and NL/F mice. (C, D) Graph showing relative PS1-CTF protein levels (quantified from immunoblot experiments as the one shown in panel (C)) normalized to the WT control group (WT-CTL n = 11; WT-T2DM n = 12; NL/F-CTL n = 13; NL/F-T2DM n = 10). Statistical analysis was performed by one-way ANOVA (P = 0.0471), followed by Tukey’s multiple comparisons test (*P < 0.05). Unpaired t test also revealed a significant effect of T2DM in NL/F mice (#P = 0.0163, data normalized to the NL/F control group). Actin was used as loading control. (C, E) Graph showing the ratio of APP-CTF/APP protein levels (quantified from immunoblot experiments as the one shown in panel (C)) normalized to the WT control group (WT-CTL n = 10; WT-T2DM n = 12; NL/F-CTL n = 13; NL/F-T2DM n = 12). Statistical analysis was performed by one-way ANOVA (P < 0.0001), followed by Tukey’s multiple comparisons test (**P < 0.01, ****P < 0.0001). Unpaired t test also revealed a significant effect of T2DM in WT mice (#P = 0.0081). Actin was used as loading control. (C, F) Graph showing relative NRG1-CTF protein levels (quantified from immunoblot experiments as the one shown in panel (C)) normalized to the WT control group (WT-CTL n = 11; WT-T2DM n = 13; NL/F-CTL n = 13; NL/F-T2DM n = 12). One-way ANOVA revealed no significant differences among experimental conditions. Unpaired t test revealed a significant decrease mediated by T2DM in WT mice (#P = 0.0229), and a significant increase in NL/F mice (#P = 0.0273, data normalized to the NL/F control group). Calnexin was used as loading control. All plots show mean values ± SEM.