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 decrease the production of Aβ, or the inhibition of β-and/or γ-secretase activities⁴. Unfortunately, non-selective inhibition of β- or γ-secretases results in unavoidable side effects that are due to interference with the functioning of other physiological substrates of β- and γ-secretase^5,6. With regard to γ-secretase inhibitors, recent studies have reported the discovery of a number of chemical and genetic modulators that can regulate Aβ production while exerting negligible effects on the essential γ-secretase-mediated processing of Notch^{7,8,9,10,11,12}, however, successful therapeutics have not yet been developed. Thus, further systematic screens are warranted to discover novel genetic and chemical modifiers that may selectively modulate γ-secretase-mediated APP processing.

γ-Secretase is known to cleave more than 90 different membrane-anchored proteins. Among these substrates is Notch, whose activity is critical for cell fate determination and differentiation during development¹³. Selectively modulating γ-secretase-catalyzed APP processing in the absence of affecting Notch processing will be essential for minimizing Notch-related side effects of γ-secretase inhibitors, and this selectivity is thought to be a primary factor that will dictate the biological efficacy of potential γ-secretase modulators for the chronic treatment of AD. There have been a number of arylsulfonamide derivatives, such as GSI-953 (begacestat) and BMS-708163 (avagacestat), that have been found to exhibit potent and selective inhibition of γ-secretase^14,15. A previous study has demonstrated that the differential inhibition of γ-secretase cleavage of APP and Notch can be observed using a quantitative ELISA-based assay in combination with in vivo validation using zebrafish¹⁶. Furthermore, cell-based assay paradigms have been established to address the potential substrate selectivity of γ-secretase toward APP and Notch^17,18. Using protocols similar to those described herein, we have recently discovered a novel class of (D)-leucinamides that potently modulate γ-secretase cleavage of APP with Notch-sparing selectivity¹⁹. Together, this collection of studies provides a proof-of-concept supporting the notion that the substrate selectivity/availability of γ-secretase can be subject to chemical modulation and experimental verification. However, these assay platforms often rely on relatively low-throughput readouts and labor-intensive approaches that might not meet industrial standards for drug discovery programs.

We have recently generated cell-based luciferase reporter gene assays that can quantitatively determine the catalytic activity of γ-secretase toward two distinct substrates, the 99-amino acid C-terminal fragment of APP (APP-C99) and the extracellular domain-deleted Notch peptide (NΔE). Both APP-C99 and NΔE have been widely used as direct substrates of γ-secretase in various assays. To generate homogeneous and consistent luciferase reporter gene assays for the γ-secretase cleavage of either APP-C99 or NΔE, we generated one HEK-derived stable cell line (CG) that constitutively expresses a Gal4 promoter-driven firefly luciferase reporter (Gal4-Luc) and Gal4/VP16-tagged APP-C99 fusion protein (C99-GV). In addition, we generated another HEK-derived stable cell line (NG) that constitutively expresses Gal4-Luc and Gal4/VP16-tagged NΔE (NΔE-GV). Using these novel substrate-specific γ-secretase assays, we now provide a method to quantify and distinguish the catalytic activity of γ-secretase toward distinct substrates (APP-C99 in CG cells, or NΔE in NG cells). Moreover, the substrate-specific γ-secretase assays were designed to be conducive to high-content screening platforms. These newly generated γ-secretase assays could pave the way for the discovery of novel γ-secretase modulators that could improve cognitive function by suppressing Aβ production without eliciting undesirable Notch inhibition for the next-generation treatment of AD.

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.
   1. Count cells with a hemocytometer.
   2. Transfer the microplates to a humidified CO₂ incubator and incubate at 37 °C overnight.
2. Remove 100 μL of growth medium from each well.
3. Add 100 μL/well of growth medium containing 2 μg/mL tetracycline with either 0.2% dimethyl sulfoxide (DMSO), 2 μM CL-387,785, 2 μM N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), or other related ErbB1/ErbB2 inhibitors.
4. Incubate treated CG or NG cells in microplates at 37 °C for 24 h.
5. Remove 150 μL/well growth medium from microplates.
6. Add 50 μL/well luciferase assay reagent (please see Table of Materials).
7. Transfer the microplates to a luminescence microplate reader.
   1. Maintain the microplates at room temperature for 5 min with gentle agitation.
8. Determine the Firefly luciferase (FL)-emitted luminescence using a pre-defined program stored in the luminescence microplate reader.
   1. Open the instrument control software (see Table of Materials for instrument details).
   2. From the 'Tool' menu, select 'Luminometry'.
      1. Under the 'Parameters' settings, choose 'No filter' for Emission Filter, tick 'Normal' for Emission Aperture, enter '1' for Counting Time, and click on 'OK' to set up the luminescence reading.
   3. Under the 'Instrument control' tab, scroll through the list of active protocols, and select the pre-stored protocol for reading luminescence.
   4. Under the 'Live display' tab, define the Scale from the pull-down list, and select 'Logarithm' or 'Linear' for the Type.
   5. Under the 'Temperature' tab, select 'Off' for Plate heating.
   6. Click the 'Start' button to perform the luminescence reading.
   7. As needed, export results into a spreadsheet format using the 'Explorer' embedded within the control software.

2. Quantification of γ-Secretase Activity

1. Define a luminescence signal emitted by CG or NG cells treated with 0.1% DMSO in growth medium containing 1 μg/mL tetracycline alone as 100% relative γ-secretase activity.
   1. Estimate background luminescence from CG or NG cells by treating cells with growth medium that does not include tetracycline.
2. Normalize luciferase signals from CG or NG cells that are cultured with growth medium containing 1 μg/mL tetracycline and either 1 μM CL-387,785 or 1 μM of DAPT with the luciferase signal emitted by DMSO-treated CG or NG cells (100% relative γ-secretase activity, as defined in 2.1).

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...

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