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

Mol Brain. 2008 Nov 4;1:15. doi: 10.1186/1756-6606-1-15.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amyloid Precursor Protein Secretases / antagonists & inhibitors*
  • Amyloid Precursor Protein Secretases / metabolism
  • Amyloid beta-Protein Precursor / metabolism*
  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism
  • Dipeptides / pharmacology
  • Embryo, Nonmammalian / drug effects
  • Enzyme-Linked Immunosorbent Assay
  • Gene Expression Regulation / drug effects
  • Mutation / genetics
  • Phenotype
  • Protease Inhibitors / pharmacology*
  • Protein Structure, Tertiary
  • Receptors, Notch / antagonists & inhibitors
  • Receptors, Notch / metabolism*
  • Recombinant Proteins / metabolism
  • Signal Transduction / drug effects
  • Substrate Specificity / drug effects
  • Zebrafish / embryology
  • Zebrafish / genetics
  • Zebrafish Proteins / genetics
  • Zebrafish Proteins / metabolism

Substances

  • Amyloid beta-Protein Precursor
  • Basic Helix-Loop-Helix Transcription Factors
  • Dipeptides
  • Her6 protein, zebrafish
  • N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester
  • Protease Inhibitors
  • Receptors, Notch
  • Recombinant Proteins
  • Zebrafish Proteins
  • Amyloid Precursor Protein Secretases