Human Aβ1-42 peptides inhibit proteolysis of multiple γ-secretase substrates.
(A) The western blot presents de novo generated NICDs in detergent-based γ-secretase activity assays, using NOTCH1-3xFLAG at 0.4 μM and 1.2 μM as a substrate, supplemented with human Aβ1-42 peptides at concentrations ranging from 0.5 to 10 μM. The graphs present the quantification of the western blot bands for NICDs. The pink and green lines correspond to 0.4 μM and 1.2 μM substrate concentrations, respectively. The data are normalized to the NICD levels generated in the DMSO conditions, considered as 100% and presented as mean ± SEM, N=3-5.
(B) Analysis of the de novo ICD generation in cell-free detergent-based γ-secretase activity assays is shown. The graph presents the quantification of the western blots. The data are shown as mean ± SEM, N=3-18. The statistics were calculated using one-way ANOVA and multiple comparison of predefined columns, with Šidák correction test, with respective DMSO supplemented reactions set as a reference, **** p<0.0001.
(C) PanCad-FL and PanCad-CTF levels in ReNcell VM cells treated for 24h with human Aβ1-42 peptides at 1 μM or GSI (InhX) at 2 μM concentration were quantified by western blotting. The PanCad-CTF/FL ratio was calculated from the integrated density of the corresponding bands. The data are presented as mean ± SEM, N=6-8. The statistics were calculated using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. *p<0.05.
(D) PC12 wild type or PC12 deficient for TrkA (PC12nnr5) were incubated with human Aβ1-42 or p3 17-42 peptides, GSI (compound E) or vehicle (DMSO) for 72h. The images present immunocytochemical analyses of cleaved caspase 3, and the graph corresponding quantification of the percentage of cleaved caspase 3 positive cells. The statistics were calculated within PC12 and PC12nnr5 groups using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. The data are presented as mean ± SEM, N=3. **p<0.01,****p<0.0001.
(E) Representative western blot demonstrates the accumulation of p75-CTFs in the cells treated with human Aβ1-42 peptide or GSI. The data are presented as mean ± SEM, N=3. The statistics were calculated within PC12 and PC12nnr5 groups using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. *p<0.05, **p<0.01.
(F) Mouse primary neurons were treated with human Aβ1-42 (1 μM), p3 17-42 (1 μM), GSI (compound E, 10 μM) or vehicle control, in the absence or presence of K252α inhibitor at 0.5 μM. Level of apoptosis in basal frontal cholinergic neurons (BFCNs) was analyzed by immunostaining for choline acetyltransferase (ChAT) and cleaved caspase 3. Representative images are shown. The graph presents the quantification of the percentage of cleaved caspase-3 positive cells among ChAT positive cells. The data are presented as mean ± SEM, N=3. The statistics were calculated within -K252α and +K252α groups using one-way ANOVA and multiple comparison Dunnett’s test, with DMSO set as a reference. **p<0.01, ***p<0.001.