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


CONCLUSIONS
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.
Yang
Ting
T
Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard University, Boston, MA, USA. tyang3@partners.org
Arslanova
Dilyara
D
Gu
Yongli
Y
Augelli-Szafran
Corinne
C
Xia
Weiming
W
D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004625:Embryo, Nonmammalian / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011480:Protease Inhibitors / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D015027:Zebrafish / Q000196:embryology / Q000235:genetics; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor; D051792:Basic Helix-Loop-Helix Proteins; D004151:Dipeptides; D011480:Protease Inhibitors; D051880:Receptors, Notch; D011994:Recombinant Proteins; D029961:Zebrafish Proteins; C426854:Her6 protein, zebrafish; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester