Name: quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms
Uploaded: 2018-01-25T12:30:00
Description: Watch this Scientific Journal Video about Quantitative Measurement of Î³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

The authors thank the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, for technical support. This study was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2320-B-001-016-MY3 to Y.-F. L.), the Program for Translational Innovation of Biopharmaceutical Development &#8211; Technology Supporting Platform Axis Scheme (NP7 to Y.-F.L.), and Academia Sinica (to Y.-F.L.).

1. Measurement of Luciferase Reporter Signals, which Correspond to &#947;-Secretase Cleavage of APP-C99 or N&#916;E
NOTE: Please refer to the previous publications for detailed descriptions of the generation of CG and NG cells20,21.
<ol>
	<li>Seed NG or CG cells (20, 000 cells/well) onto 96-well microplates at a final volume of 200 &#956;L/well in growth medium that is composed of Dulbecco&#39;s Modified Eagle Medium (DMEM) p...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha,+interleukin-1beta,+and+interferon-gamma+stimulate+gamma-secretase-mediated+cleavage+of+amyloid+precursor+protein+through+a+JNK-dependent+MAPK+pathway.">Tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma stimulate gamma-secretase-mediated cleavage of amyloid precursor protein through a JNK-dependent MAPK pathway.</a> J Biol Chem. 279 (47), 49523-49532 (2004).</li><li>Wang, B. 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The promising outcomes from a phase 1b trial of aducanumab immunotherapy for AD have firmly established the critical role of A&#946; in the pathogenesis of AD22 and suggest that anti-A&#946; approaches are still a viable strategy for the development of anti-AD drugs. Here, we describe cell-based assays for quantifying &#947;-secretase-catalyzed proteolysis of APP-C99 and N&#916;E using firefly luciferase reporter systems. APP-C99 and N&#916;E are the two most well-studied &#947;-secretase substrat...

Oligomeric forms of amyloid-&#946; (A&#946;) are believed to be the primary cause of neurodegeneration in the brains of patients suffering from Alzheimer&#39;s disease (AD)1. A&#946; peptides are produced by the stepwise cleavage of amyloid precursor protein (APP), first by &#946;-secretase and then by &#947;-secretase2. In the past decade, therapeutic approaches toward treatment of AD have focused on the prevention of A&#946; production3. The majority of studies have focused on either the augmentation of &#945;-secretase activity, which can preclude &#947;-secretase cleavage of APP and thereby decrease the production of A&#946;, or the inhibition of &#946;-and/or &#947;-secretase activities4. Unfortunately, non-selective inhibition of &#946;- or &#947;-secretases results in unavoidable side effects that are due to interference with the functioning of other physiological substrates of &#946;- and &#947;-secretase5,6. With regard to &#947;-secretase inhibitors, recent studies have reported the discovery of a number of chemical and genetic modulators that can regulate A&#946; production while exerting negligible effects on the essential &#947;-secretase-mediated processing of Notch7,8,9,10,11,12, 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 &#947;-secretase-mediated APP processing.
&#947;-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 &#947;-secretase-catalyzed APP processing in the absence of affecting Notch processing will be essential for minimizing Notch-related side effects of &#947;-secretase inhibitors, and this selectivity is thought to be a primary factor that will dictate the biological efficacy of potential &#947;-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 &#947;-secretase14,15. A previous study has demonstrated that the differential inhibition of &#947;-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 &#947;-secretase toward APP and Notch17,18. Using protocols similar to those described herein, we have recently discovered a novel class of (D)-leucinamides that potently modulate &#947;-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 &#947;-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 &#947;-secretase toward two distinct substrates, the 99-amino acid C-terminal fragment of APP (APP-C99) and the extracellular domain-deleted Notch peptide (N&#916;E). Both APP-C99 and N&#916;E have been widely used as direct substrates of &#947;-secretase in various assays. To generate homogeneous and consistent luciferase reporter gene assays for the &#947;-secretase cleavage of either APP-C99 or N&#916;E, 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 N&#916;E (N&#916;E-GV). Using these novel substrate-specific &#947;-secretase assays, we now provide a method to quantify and distinguish the catalytic activity of &#947;-secretase toward distinct substrates (APP-C99 in CG cells, or N&#916;E in NG cells). Moreover, the substrate-specific &#947;-secretase assays were designed to be conducive to high-content screening platforms. These newly generated &#947;-secretase assays could pave the way for the discovery of novel &#947;-secretase modulators that could improve cognitive function by suppressing A&#946; production without eliciting undesirable Notch inhibition for the next-generation treatment of AD.

<table>
	<tbody>
		<tr>
			<td>VictorLight luminescence plate reader</td>
			<td>PerkinElmer</td>
			<td>2030-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type 2 Biological Safety Cabinet</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>CL-387,785</td>
			<td>EMD Calbiochem</td>
			<td>233100-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Merck</td>
			<td>565770</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher</td>
			<td>12100046</td>
			<td>main compnent of growth medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Thermo Fisher</td>
			<td>A1113903</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher</td>
			<td>R25005</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Gibco</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytomer</td>
			<td>Sigma-Aldrich</td>
			<td>BR717805 Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Nunc</td>
			<td>156545</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Steady-Glo luciferase assay reagent</td>
			<td>Promega</td>
			<td>E2510</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Revco</td>
			<td>Ultima II</td>
			<td></td>
		</tr>
		<tr>
			<td>T-REx293 cell line</td>
			<td>Invitrogen</td>
			<td>R71007</td>
			<td></td>
		</tr>
		<tr>
			<td>Wallac 1420 software version 3.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>instrument control software of the luminescence microplate reader</td>
		</tr>
	</tbody>
</table>

quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-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-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-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.

Inhibition of ErbB2 by CL-387,785 can differentially promote the processing of APP-C99 by &#947;-secretase without affecting Notch cleavage 
To establish a cell-based assay that can quantitatively measure the proteolytic cleavage of a particular &#947;-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...

Watch this Scientific Journal Video about Quantitative Measurement of Î³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

The overall goal of this quantitative cell-based reporter gene assay for gamma-secretase is to examine the differential effect of putative gamma-secretase modulators on gamma-secretase-mediated cleavage. This method can help to answer the key questions in the Alzheimer disease field, such as identification of novel gamma-secretase modulators for the treatment of Alzheimer disease. The advantage of this technique is that it is a highly efficient assay system, and it can easily identify gamma-secretase modulators.The implication of this technique extend toward therapy of Alzheimer disease through the potential discovery of anti-AD drugs with minimal side effects. This method can provide insight to the substrate selectivity of gamma-secretase. It also can be applied to the studies of other diseases, such as neuroblastoma.Individual new to this method may struggle with careful removal and addition of cell culture media to 96 microplate, as too much disturbance may complicate the final results. Begin this procedure with preparation of NG and CG cells, as referenced in the text protocol. Count NG or CG cells with a hemocytometer.Then, seed the cells at 20, 000 cells per well onto 96-well microplates at a final volume of 200 microliters per well in growth medium. Transfer the microplates to a humidified carbon dioxide incubator, and incubate at 37 degrees Celsius overnight. Following overnight incubation, remove 100 microliters of growth medium from each well.Add 100 microliters per well of growth medium containing two micrograms per milliliter of tetracycline with either 0.2%dimethyl sulfoxide, two micromolar CL-387, 785, two micromolar DAPT, or other related ErbB1/ErbB2 inhibitors. Incubate the treated cells and microplates at 37 degrees Celsius for 24 hours. Following incubation, remove 150 microliters per well of growth medium from the microplates.Then, add 50 microliters per well of luciferase assay reagent. Next, transfer the microplates to a luminescence microplate reader, and maintain them at room temperature for five minutes. Determine the firefly luciferase-emitted luminescence using a predefined program stored in the luminescence microplate reader.First, open the instrument control software. Then, from the Tool menu, select Luminometry. Under the Parameters setting, choose No Filter for emission filter, tick Normal for emission aperture, enter one for counting time, and click on OK to set up the luminescence reading.Under the Instrument control tab, scroll through the list of active protocols, and select the pre-stored protocol for reading luminescence. Under the Live display tab, define the scale from the pull-down menu, and select Logarithm or Linear for the type. Under the Temperature tab, select Off for plate heating.Finally, click the Start button to perform the luminescence reading. Export the results into a spreadsheet format using the Explorer embedded within the control software. Estimate background luminescence from CG or NG cells treated with growth medium that does not include tetracycline.Define a luminescence signal emitted by CG or NG cells treated with 0.1%DMSO in growth medium containing one microgram per milliliter tetracycline alone as 100%relative gamma-secretase activity. Finally, normalize luciferase signals from CG or NG cells that are cultured with growth medium containing tetracycline and either CL-387, 785 or DAPT to the luciferase signal emitted by DMSO-treated CG or NG cells. Shown here, are representative results demonstrating the quantitative measurements of gamma-secretase cleavage of APP-C99 in the CG cells.Cells were treated with one micromolar of CL-387, 785 or DAPT in the presence of one microgram per milliliter tetracycline at 37 degrees Celsius for 24 hours. The luminescence derived from gamma-secretase-mediated proteolysis of C99-GV was quantified after the addition of 100 microliters per well of luciferase assay reagent. The luciferase signal emitted by cells treated with vehicle alone was set as 100%relative luminescence.Representative results demonstrating the quantitative measurements of gamma-secretase cleavage of N-delta-E in the NG cells are shown here. Similarly, cells were treated with one micromolar of CL-387, 785 or DAPT in the presence of one microgram per milliliter tetracycline at 37 degrees Celsius for 24 hours. The luminescence derived from gamma-secretase-mediated proteolysis of N-delta-E-GV was quantified after the addition of 100 microliters per well of luciferase assay reagent.The luciferase signal emitted by cells treated with vehicle alone was set as 100%relative luminescence. Once mastered, this technique only requires a hands-on time of one to two hours, pending the number of compounds to exam. After its development, this technique paved the way for the researcher in the field of Alzheimer disease to explore genetic and chemical modifier of gamma-secretase in pursuit of novel therapeutics for Alzheimer disease.

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Neuroscience

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...