Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

Summary

We have successfully generated two substrate-specific γ-secretase assays. Both cell-based assays presented here are designed to quantify γ-secretase enzymatic activities via the output of firefly luciferase reporters.

Abstract

We have developed a pair of cell-based reporter gene assays to quantitatively measure γ-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor γ-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-ΔE (NΔE; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress γ-secretase cleavage of APP-C99 in CG cells, but not NΔE in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for γ-secretase modulators, and these responses are in stark contrast to the pan-inhibition of γ-secretase induced by DAPT. Our studies provide direct evidence that γ-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

Introduction

Oligomeric forms of amyloid-β (Aβ) are believed to be the primary cause of neurodegeneration in the brains of patients suffering from Alzheimer's disease (AD)¹. Aβ peptides are produced by the stepwise cleavage of amyloid precursor protein (APP), first by β-secretase and then by γ-secretase². In the past decade, therapeutic approaches toward treatment of AD have focused on the prevention of Aβ production³. The majority of studies have focused on either the augmentation of α-secretase activity, which can preclude γ-secretase cleavage of APP and thereby de....

Protocol

1. Measurement of Luciferase Reporter Signals, which Correspond to γ-Secretase Cleavage of APP-C99 or NΔE

NOTE: Please refer to the previous publications for detailed descriptions of the generation of CG and NG cells^20,21.

1. Seed NG or CG cells (20, 000 cells/well) onto 96-well microplates at a final volume of 200 μL/well in growth medium that is composed of Dulbecco's Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS), 5 μg/mL blasticidin, 250 μg/mL antibiotic (e.g., zeocin), and 200 μg/mL hygromycin B.

Results

Inhibition of ErbB2 by CL-387,785 can differentially promote the processing of APP-C99 by γ-secretase without affecting Notch cleavage
To establish a cell-based assay that can quantitatively measure the proteolytic cleavage of a particular γ-secretase substrate, we generated the HEK293-derived CG cell line by stable co-transfection of a tetracycline-inducible C-terminally Gal4/VP16-tagged APP-C99 and a Gal4 promoter-driven firefly luc.......

Discussion

The promising outcomes from a phase 1b trial of aducanumab immunotherapy for AD have firmly established the critical role of Aβ in the pathogenesis of AD²² and suggest that anti-Aβ approaches are still a viable strategy for the development of anti-AD drugs. Here, we describe cell-based assays for quantifying γ-secretase-catalyzed proteolysis of APP-C99 and NΔE using firefly luciferase reporter systems. APP-C99 and NΔE are the two most well-studied γ-secretase substrat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
VictorLight luminescence plate reader	PerkinElmer	2030-0010
Class II, Type 2 Biological Safety Cabinet	Thermo Scientific	1300 Series A2
CL-387,785	EMD Calbiochem	233100-1MG
DAPT	Merck	565770
Dulbecco’s Modified Eagle Medium (DMEM)	Thermo Fisher	12100046	main compnent of growth medium
Fetal bovine serum (FBS)	Thermo Fisher	16000044
Blasticidin	Thermo Fisher	A1113903
Zeocin	Thermo Fisher	R25005
Hygromycin B	Gibco	10687010
Hemocytomer	Sigma-Aldrich	BR717805 Aldrich
96-well microplate	Nunc	156545
Tetracycline	Sigma	T7660
Dimethyl sulfoxide (DMSO)	Sigma	D2650
Steady-Glo luciferase assay reagent	Promega	E2510
Humidified CO2 incubator	Revco	Ultima II
T-REx293 cell line	Invitrogen	R71007
Wallac 1420 software version 3.0	PerkinElmer		instrument control software of the luminescence microplate reader