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




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Molecular Brain

, 1:15

First Online: 04 November 2008Received: 29 September 2008Accepted: 04 November 2008

Abstract

BackgroundDeposition 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.

ResultsTo 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 NotchΔE. 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 SuH 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.

ConclusionOur 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.

AbbreviationsADAlzheimer-s disease

Aβamyloid β protein

APPamyloid precursor protein

AblAbelson leukemia

cpd Ecompound E

dpfdays post fertilization

ECeffective concentration

HEKhuman embryonic kidney

hpfhours post fertilization

Nβ*Notch-Aβ-like

NICDNotch intracellular domain

PSPresenilin

TMDtransmembrane domain

WBWestern blot.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-6606-1-15 contains supplementary material, which is available to authorized users.

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Autor: Ting Yang - Dilyara Arslanova - Yongli Gu - Corinne Augelli-Szafran - Weiming Xia

Fuente: https://link.springer.com/







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