Oxidative stress-mediated β-secretase activity and sAPPβ levels are reduced by γ-secretase inhibitors. (A) The lipid peroxidation product HNE enhances β- and γ-secretase activities. SH-SY5Y cells were pre-incubated with the β-secretase inhibitor BI4 or the γ-secretase inhibitors DAPT and L685,458 for 1 h before treatment with HNE. Cells were collected after 24 h of incubation with 5 μM or 10 μM HNE. The cell lysates were tested for β- and γ-secretase activities by addition of secretase-specific peptide substrate conjugated to the reporter molecules EDANS and DABCYL. (B) HNE treatment increased the amounts of secreted Aβ42. After treatment of SH-SY5Y cells stably expressing mutant (Swedish) APP for 24 h, medium was harvested and analyzed by a sandwich ELISA for specific quantification of secreted Aβ42. (C) γ-secretase inhibitors suppress HNE-induced sAPPβ levels. After treatment of SH-SY5Y cells stably expressing wild type APP for 24 h, cells were harvested and analyzed by Western blots for APP, APP-CTFs and actin. Culture media were also analyzed by Western blot for sAPPβ.

γ-secretase proteins regulate BACE1 promoter activity. (A) Quantitative real time PCR analysis was performed on RNA samples from rat cortical neurons that had been exposed for 3 hours to the indicated treatments. γ-secretase inhibitors (compound E, 10 nM; L685,458, 2 μM) were added for 2 hours before HNE exposure. Values are the means and SD of 3 independent experiments. (B) HNE increases human BACE1 promoter activity, an effect suppressed by γ-secretase inhibitors. SH-SY5Y cell were transfected with BACE1 promoter constructs pB1P-A or pGL3-Basic (negative control) in association with renilla luciferase vector pCMT-Rluc to verify transfection efficiency. Twenty four hours after transfection, cells were treated for 3 hours with HNE in the presence of the indicated γ-secretase inhibitors (compound E, 10 nM; L685,458, 2 μM). BACE1 transcriptional activity was measured using a dual-luciferase assay. Values are the mean and SD of three independent experiments. *P < 0.01 compared to control, ^#P < 0.01 and ^##P < 0.001 compared to the 10 μM HNE treated samples. (C) BACE1 promoter activity is increased by PS1, FAD PS1 mutations and AICD in PS-deficient cells. PS^−/− fibroblasts were transiently co-transfected with pB1P-A and PS1, PS1-M146L, PS1-ΔE9, PS1-D257A, PS1-D385A, or AICD. After 24 hr incubation, BACE1 promoter activity was analyzed. Results were obtained from four independent experiments, each performed in triplicate (mean and SD). *P < 0.01 compared to vector transfected control. (D) BACE1 protein levels are increased by AICD expression. PS^−/− fibroblasts were transiently transfected with increasing amounts of AICD cDNA, and then BACE1 and β-actin levels were measured by immunoblot analysis.

Evidence that γ-secretase induces an increase in β-secretase levels in AD. (A) Linear regression analysis of β-secretase and γ-secretase activities in inferior parietal lobule and cerebellum from clinically diagnosed and neuropathologically confirmed AD (open rectangle; □) and non-demented control subjects (filled rectangle; ■). (B) Wild-type and BACE ^−/− MEFs were exposed to HNE (15 μM) for 2 hours. BACE1 levels were examined by immunoblot analysis. (C, D) Effects of γ-secretase inhibitors (GSI) on HNE-mediated induction of BACE1. Primary cultured rat cortical (C) and hippocampal (D) neurons were pretreated with the indicated inhibitors (compound E, 10 nM; L685,458, 2 μM; WPE-III-31C, 2 μM; pepstatin A, 2 μM; β-secretase Inhibitor IV, 2 μM) for 2 hours, and then cells were exposed to HNE for 16 hours. BACE1, BACE2, PS1, ADAM10, TACE, and β-actin levels were evaluated by immunoblot analysis.

Oxidative stress-induced BACE1 expression is reduced in presenilin-deficient cells. (A, B) Wild type, PS1^−/− (A) and PS1^−/−/PS2^−/− (PS^−/−) MEFs (B) were treated with HNE (5, 10, and 15 μM) or H₂O₂ (10, 20, and 50 μM) for 3 hours. Some wild-type cells were also co-treated with GSI (2 μM L685,458). BACE1, BACE2, PS1, and β-actin protein levels were measured by immunoblot analysis. (C) Oxidative stress-induced BACE1 expression is partially reduced in PS2 deficient cells. Wild type, PS1^−/− and PS1^−/−/PS2^−/− MEFs were treated with HNE (5, 10, and 15 μM) or H₂O₂ (10, 20, and 50 μM) for 3 hours. BACE1, BACE2, PS1, and β-actin expression were evaluated by immunoblot analysis. (D, E) SH-SY5Y cells were transfected with BACE1 promoter construct pB1P-A, in combination with PS1 or Nicastrin siRNAs. At 36 hours, cells were harvested and analyzed for BACE1 promoter activity by dual-luciferase assay (D) or were used to examine levels of PS1, Nicastrin, BACE1, and β-actin by immunoblotting (E). Values are the mean and SD of three independent experiments. *P < 0.01 compared to control.

γ-secretase inhibitor reduces BACE1 expression in the brains of AD model mice. PS1KI and 3xTgAD mice were injected with γ-secretase inhibitor (GSI) or vehicle every day for 7 days. (A) Cerebral cortex tissue was homogenized and BACE1 and β-actin levels were examined by immunoblots. (B) Quantitative analysis showed increased levels of BACE1 in PS1KI and 3xTgAD mice compared to wild type mice, and a significant reduction following γ-secretase inhibitor treatment. Values are the means and SD (5 mice/group). *P < 0.001 and **P < 0.05 compared to wild-type. ^#P < 0.01.