Notch1 and Amyloid Precursor Protein Are Competitive Substrates for Presenilin1-dependent γ-Secretase Cleavage

Oksana Berezovska,Christine Jack,Amy Deng,Nicole Gastineau,G William Rebeck,Bradley T Hyman

doi:10.1074/jbc.m008268200

Oksana Berezovska, Christine Jack + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m008268200

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Abstract

Proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases results in the production of a highly amyloidogenic Abeta peptide, which deposits in the brains of Alzheimer's disease patients. Similar gamma-secretase processing occurs in another transmembrane protein, Notch1, releasing a potent signaling molecule, the Notch C-terminal domain. It has been shown that both events are dependent on a presenilin-dependent protease. We now test the hypothesis that activated Notch1 and APP are competitive substrates for the same proteolytic activity in neurons. Treatment of neurons with the native Notch ligand, Delta, induces endogenous Notch1 intramembraneous cleavage and diminishes Abeta production in a dose-dependent manner. Complementary experiments showed that the converse was also true. Overexpressing human APP (APP(695Sw)) in neurons leads to a decrease in endogenous Notch1 signal transduction, as assessed by a CBF1 luciferase transcription assay, by Notch C-terminal domain nuclear translocation in vitro and by analysis of Notch C-terminal domain generation and Notch1 staining in vivo. In summary, two complementary approaches suggest that APP and Notch1 are physiologically relevant competitive substrates for gamma-secretase activity.

Highlights

Amyloid peptide (A␤)1 is the major component of senile plaques in the brains of Alzheimer’s disease (AD) patients
amyloid precursor protein (APP) Competes with Notch1 for ␥-Secretase—To test the hypothesis that APP competes with Notch1 for ␥-secretase processing, we activated endogenous Notch1 signaling in primary neurons by treatment with different concentrations of the Notch ligand, Dl-Fc, for 2 days and measured the amount of A␤ in conditioned medium
Correlative decreases of total A␤ after treatment with Dl-Fc in a dose-dependent manner, with the highest dose leading to a decrease of about 45% (p Ͻ 0.001) in A␤ production (Fig. 1B)

Summary

The abbreviations used are

A␤, amyloid peptide; PS1, presenilin; APP, amyloid precursor protein; AD, Alzheimer’s disease; NICD, Notch C-terminal domain; CBF1, C-promoter binding factor 1; Dl-Fc, Fcconjugated Delta; CM, conditioned medium; CTF, C-terminal fragment; ELISA, enzyme-linked immunosorbent assay. Tain; photoaffinity labeling of PS1 by potent ␥-secretase inhibitors indicates that PS1 may contain the active site of ␥-secretase [7, 8] Another single pass transmembrane receptor, Notch, undergoes proteolytic processing upon ligand binding by an intramembraneous protease, resulting in the release and nuclear translocation of the signaling C-terminal domain of the Notch molecule, NICD (9 –11). We examined Notch cellular localization in the hippocampal formation of adult APP695Sw-overexpressing mice and found a statistically significant decrease in nuclear localization of Notch, suggesting that Notch activation is less efficient in the presence of increased APP expression in vivo Taken together, these data support the idea that A␤ generation and Notch activation are competing biological processes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION