Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein

Abstract

Background: Deposition of amyloid-β protein (Aβ) is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from γ-secretase cleavage of amyloid precursor protein (APP). In addition to APP, γ-secretase also cleaves other type I integral membrane proteins, including the Notch receptor, a key molecule involved in embryonic development.

Results: To explore selective γ-secretase inhibitors, a combination of five methods was used to systematically determine these inhibitors' profiles on the γ-secretase cleavage of APP and Notch. When two potent γ-secretase inhibitors, compound E (cpd E) and DAPT, were used in a conventional in vitro γ-secretase activity assay, cpd E completely blocked Aβ generation from the cleavage of substrate APP C100, but only had a minor effect on Notch cleavage and NICD generation. Next, cpd E and DAPT were applied to HEK293 cells expressing a truncated Notch substrate NotchDeltaE. Both cpd E and DAPT were more potent in blocking Aβ generation than NICD generation. Third, a reporter construct was created that carried the NICD targeting promoter with three Su(H) binding sequences followed by the luciferase gene. We found that the inhibition of NICD generation by cpd E and DAPT was consistent with the reduced expression of luciferase gene driven by this Notch targeting promoter. Fourth, levels of "Notch-Aβ-like" (Nβ*) peptide derived from two previously reported chimeric APP with its transmembrane domain or the juxtamembrane portion replaced by the Notch sequence were quantified. Measurement of Nβ* peptides by ELISA confirmed that EC₅₀'s of cpd E were much higher for Nβ* than Aβ. Finally, the expression levels of Notch target gene her6 in cpd E or DAPT-treated zebrafish were correlated with the degree of tail curvature due to defective somitogenesis, a well characterized Notch phenotype in zebrafish.

Conclusion: Our ELISA-based quantification of Aβ and Nβ* in combination with the test in zebrafish provides a novel approach for efficient cell-based screening and in vivo validation of APP selective γ-secretase inhibitors.

Figure 1

Generation of NICD and Aβ from purified APP and Notch substrate C100 and N100 in an in vitro γ-secretase activity assay. The E. coli generated APP- and Notch-based, 100-residue γ-secretase substrates C100-Flag and N100-Flag were mixed with the membrane vesicles solubilized in CHAPSO after DMSO or compounds were added. The mixture was incubated at 37°C for 4 hours. A. A dose-dependent inhibition of NICD generation by DAPT. Generation of NICD was detected by Western blot (WB) with antibody 1744 specifically recognizing N-terminus of NICD. B. Generation of NICD was inhibited in the presence of 100 nM of cpd E. C. Levels of NICD determined by WB were quantified by densitometry (dotted line). Levels of Aβ generated from the γ-secretase cleavage of C100 in the presence of DAPT were determined by ELISA (solid line). Comparison of NICD and Aβ generation in the presence of DAPT suggests that high concentrations of DAPT were more potent in blocking Aβ than NICD generation. D. NICD (dotted line) and Aβ (solid line) generation in the presence of cpd E were compared. Cpd E inhibited Aβ generation with an IC₅₀ of 1 nM and is more potent in inhibiting Aβ than NICD.

Figure 2

Generation of NICD and Aβ from NotchΔE and APP expressing cells. A. Twenty four hrs after the construct carrying NotchΔE was transfected into APP expressing HEK293 cells, cells were treated with DAPT or cpd E for 8 hr and lysed for WB with antibody 1744 to specifically detect the N-terminus of NICD. Bottom panel, the antibody against α-tubulin was applied to normalize the amounts of lysates used for WB. B. Levels of NICD determined by WB were quantified by densitometry (dotted line). Levels of Aβ generated from the γ-secretase cleavage of APP in the presence of DAPT were determined by ELISA. Comparison of NICD and Aβ generation in the presence of DAPT suggests that high concentrations of DAPT had a greater inhibition of Aβ than NICD. C. NICD (dotted line) and Aβ (solid line) production from cpd E-treated cells were compared. Cpd E inhibited Aβ generation with an IC₅₀ of ~8 nM, and it shows a greater inhibition of Aβ than NICD. D. A luciferase reporter construct driven by HES1 promoter was transfected into HEK293 cells followed by treatment with cpd E or DAPT. Both γ-secretase inhibitors blocked transcriptional activation of NICD dependent luciferase activity.

Figure 3

Generation of Aβ and Nβ* from chimeric APP-Notch expressing cells. A. The juxtamembrane portion of the APP ectodomain (the α-secretase cleavage region) was replaced by the corresponding sequence in Notch (α-NOTCH). Levels of Nβ* in the media from cells expressing APP-α-Notch (dotted line) and levels of Aβ from APP expressing cells (solid line) were determined by ELISA. B. A chimeric cDNA constructs express APP with its transmembrane domain (TMD) replaced by the Notch TMD (APP-m-NOTCH). Levels of Aβ (solid line) and Nβ* (dotted line) were determined by ELISA.

Figure 4

Treatment of zebrafish embryos with DAPT causes curved tails. A. A stock of DAPT or cpd E in DMSO was diluted in embryo medium, and increasing concentrations of DAPT or cpd E were applied to de-chorionated zebrafish embryos incubated at 28°C from 24 hpf to 48 hpf. Control embryos were mock-treated with embryo medium containing the same concentration of DMSO. Treatment of zebrafish embryos with 50 μM DAPT caused curved trunk and tails. B. DAPT- or cpd E-treated embryos were kept until 4 dpf, and images were acquired at 40 × magnification.

Figure 5

Expression levels of Notch target gene her6 are consistent with the curvature phenotype. Increasing concentrations of DAPT or cpd E were applied to de-chorionated zebrafish embryos from 24 hpf until 48 hpf, and in situ hybridization of compound-treated embryos was carried out at 48 hpf using the her6 probe. At least 10 to 20 embryos were examined for each experiment. Images were taken at the 64× magnification for stained embryos.