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Updated: Jun 28, 2026
Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms
Published on: January 25, 2018
Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates
Ting Yang1, Dilyara Arslanova, Yongli Gu
1Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard University, Boston, MA, USA. tyang3@partners.org
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.
