Name: Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms (Video) | JoVE Journal
Uploaded: 2018-01-25T12:30:00.000+00:00
Duration: 6 min 40 s
Description: Learn quantitative γ-secretase assays using cell-based luciferase reporter platforms to identify modulators for Alzheimer disease research. This method enables efficient screening of therapeutic candidates with minimal side effects.

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5 minutes

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) plus 10% fetal bovine serum (FBS), 5 &#956;g/mL blasticidin, 250 &#956;g/mL antibiotic (e.g., zeocin), and 200 &#956;g/mL hygromycin B.
	<ol>
		<li>Count cells with a hemocytometer.</li>
		<li>Transfer the microplates to a humidified CO2 incubator and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Remove 100 &#956;L of growth medium from each well.</li>
	<li>Add 100 &#956;L/well of growth medium containing 2 &#956;g/mL tetracycline with either 0.2% dimethyl sulfoxide (DMSO), 2 &#956;M CL-387,785, 2 &#956;M N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), or other related ErbB1/ErbB2 inhibitors.</li>
	<li>Incubate treated CG or NG cells in microplates at 37 &#176;C for 24 h.</li>
	<li>Remove 150 &#956;L/well growth medium from microplates.</li>
	<li>Add 50 &#956;L/well luciferase assay reagent (please see Table of Materials).</li>
	<li>Transfer the microplates to a luminescence microplate reader.
	<ol>
		<li>Maintain the microplates at room temperature for 5 min with gentle agitation.</li>
	</ol>
	</li>
	<li>Determine the Firefly luciferase (FL)-emitted luminescence using a pre-defined program stored in the luminescence microplate reader.
	<ol>
		<li>Open the instrument control software (see Table of Materials for instrument details).</li>
		<li>From the &#39;Tool&#39; menu, select &#39;Luminometry&#39;.
		<ol>
			<li>Under the &#39;Parameters&#39; settings, choose &#39;No filter&#39; for Emission Filter, tick &#39;Normal&#39; for Emission Aperture, enter &#39;1&#39; for Counting Time, and click on &#39;OK&#39; to set up the luminescence reading.</li>
		</ol>
		</li>
		<li>Under the &#39;Instrument control&#39; tab, scroll through the list of active protocols, and select the pre-stored protocol for reading luminescence.</li>
		<li>Under the &#39;Live display&#39; tab, define the Scale from the pull-down list, and select &#39;Logarithm&#39; or &#39;Linear&#39; for the Type.</li>
		<li>Under the &#39;Temperature&#39; tab, select &#39;Off&#39; for Plate heating.</li>
		<li>Click the &#39;Start&#39; button to perform the luminescence reading.</li>
		<li>As needed, export results into a spreadsheet format using the &#39;Explorer&#39; embedded within the control software.</li>
	</ol>
	</li>
</ol>
2. Quantification of &#947;-Secretase Activity
<ol>
	<li>Define a luminescence signal emitted by CG or NG cells treated with 0.1% DMSO in growth medium containing 1 &#956;g/mL tetracycline alone as 100% relative &#947;-secretase activity.
	<ol>
		<li>Estimate background luminescence from CG or NG cells by treating cells with growth medium that does not include tetracycline.</li>
	</ol>
	</li>
	<li>Normalize luciferase signals from CG or NG cells that are cultured with growth medium containing 1 &#956;g/mL tetracycline and either 1 &#956;M CL-387,785 or 1 &#956;M of DAPT with the luciferase signal emitted by DMSO-treated CG or NG cells (100% relative &#947;-secretase activity, as defined in 2.1).</li>
</ol>

<ol><li>Selkoe, D. J., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+amyloid+hypothesis+of+Alzheimer%27s+disease+at+25+years%5BTitle%5D)+AND+%22EMBO+Mol+Med%22%5BJournal%5D)">The amyloid hypothesis of Alzheimer's disease at 25 years.</a> EMBO Mol Med. 8 (6), 595-608 (2016).</li>
<li>Wolfe, M. S., Guenette, S. Y. <a target="_blank" href="http://jcs.biologists.org/content/120/18/3157">APP at a glance.</a> J Cell Sci. 120 (Pt 18), 3157-3161 (2007).</li>
<li>Karran, E., Mercken, M., De Strooper, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+amyloid+cascade+hypothesis+for+Alzheimer%27s+disease%3A+an+appraisal+for+the+development+of+therapeutics%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics.</a> Nat Rev Drug Discov. 10 (9), 698-712 (2011).</li>
<li>Citron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alzheimer%27s+disease%3A+strategies+for+disease+modification%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Alzheimer's disease: strategies for disease modification.</a> Nat Rev Drug Discov. 9 (5), 387-398 (2010).</li>
<li>Vassar, R., Kovacs, D. M., Yan, R., Wong, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+beta-secretase+enzyme+BACE+in+health+and+Alzheimer%27s+disease%3A+regulation%2C+cell+biology%2C+function%2C+and+therapeutic+potential%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">The beta-secretase enzyme BACE in health and Alzheimer's disease: regulation, cell biology, function, and therapeutic potential.</a> J Neurosci. 29 (41), 12787-12794 (2009).</li>
<li>Parks, A. L., Curtis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presenilin+diversifies+its+portfolio%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">Presenilin diversifies its portfolio.</a> Trends Genet. 23 (3), 140-150 (2007).</li>
<li>Kukar, T. L., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/18548070">Substrate-targeting gamma-secretase modulators.</a> Nature. 453 (7197), 925-929 (2008).</li>
<li>Kounnas, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+gamma-secretase+reduces+beta-amyloid+deposition+in+a+transgenic+mouse+model+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Modulation of gamma-secretase reduces beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease.</a> Neuron. 67 (5), 769-780 (2010).</li>
<li>Thathiah, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+orphan+G+protein-coupled+receptor+3+modulates+amyloid-beta+peptide+generation+in+neurons%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons.</a> Science. 323 (5916), 946-951 (2009).</li>
<li>Flajolet, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+Alzheimer%27s+disease+amyloid-beta+formation+by+casein+kinase+I%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Regulation of Alzheimer's disease amyloid-beta formation by casein kinase I.</a> Proc Natl Acad Sci U S A. 104 (10), 4159-4164 (2007).</li>
<li>Eisele, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gleevec+increases+levels+of+the+amyloid+precursor+protein+intracellular+domain+and+of+the+amyloid-beta+degrading+enzyme+neprilysin%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Gleevec increases levels of the amyloid precursor protein intracellular domain and of the amyloid-beta degrading enzyme neprilysin.</a> Mol Biol Cell. 18 (9), 3591-3600 (2007).</li>
<li>He, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gamma-secretase+activating+protein+is+a+therapeutic+target+for+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease.</a> Nature. 467 (7311), 95-98 (2010).</li>
<li>Haapasalo, A., Kovacs, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+many+substrates+of+presenilin%2Fgamma-secretase%5BTitle%5D)+AND+%22J+Alzheimers+Dis%22%5BJournal%5D)">The many substrates of presenilin/gamma-secretase.</a> J Alzheimers Dis. 25 (1), 3-28 (2011).</li>
<li>Gillman, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery+and+Evaluation+of+BMS-708163%2C+a+Potent%2C+Selective+and+Orally+Bioavailable+gamma-Secretase+Inhibitor%5BTitle%5D)+AND+%22ACS+Med+Chem+Lett%22%5BJournal%5D)">Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable gamma-Secretase Inhibitor.</a> ACS Med Chem Lett. 1 (3), 120-124 (2010).</li>
<li>Hopkins, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ACS+chemical+neuroscience+molecule+spotlight+on+Begacestat+%28GSI-953%29%5BTitle%5D)+AND+%22ACS+Chem+Neurosci%22%5BJournal%5D)">ACS chemical neuroscience molecule spotlight on Begacestat (GSI-953).</a> ACS Chem Neurosci. 3 (1), 3-4 (2012).</li>
<li>Yang, T., Arslanova, D., Gu, Y., Augelli-Szafran, C., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+gamma-secretase+modulation+differentiates+inhibitor+compound+selectivity+between+two+substrates+Notch+and+amyloid+precursor+protein%5BTitle%5D)+AND+%22Mol+Brain%22%5BJournal%5D)">Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein.</a> Mol Brain. 1, 15(2008).</li>
<li>McKee, T. D., Loureiro, R. M., Dumin, J. A., Zarayskiy, V., Tate, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+cell-based+method+for+determining+the+gamma-secretase+enzyme+activity+against+both+Notch+and+APP+substrates%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">An improved cell-based method for determining the gamma-secretase enzyme activity against both Notch and APP substrates.</a> J Neurosci Methods. 213 (1), 14-21 (2013).</li>
<li>Basi, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyloid+precursor+protein+selective+gamma-secretase+inhibitors+for+treatment+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Alzheimers+Res+Ther%22%5BJournal%5D)">Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease.</a> Alzheimers Res Ther. 2 (6), 36(2010).</li>
<li>Liao, Y. F., Tang, Y. C., Chang, M. Y., Wang, B. J., Hu, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery+of+small+molecular+%28D%29-leucinamides+as+potent%2C+Notch-sparing+gamma-secretase+modulators%5BTitle%5D)+AND+%22Eur+J+Med+Chem%22%5BJournal%5D)">Discovery of small molecular (D)-leucinamides as potent, Notch-sparing gamma-secretase modulators.</a> Eur J Med Chem. 79, 143-151 (2014).</li>
<li>Liao, Y. F., Wang, B. J., Cheng, H. T., Kuo, L. H., Wolfe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor+necrosis+factor-alpha%2C+interleukin-1beta%2C+and+interferon-gamma+stimulate+gamma-secretase-mediated+cleavage+of+amyloid+precursor+protein+through+a+JNK-dependent+MAPK+pathway%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ErbB2+regulates+autophagic+flux+to+modulate+the+proteostasis+of+APP-CTFs+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 114 (15), E3129-E3138 (2017).</li>
<li>Sevigny, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+antibody+aducanumab+reduces+Abeta+plaques+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The antibody aducanumab reduces Abeta plaques in Alzheimer's disease.</a> Nature. 537 (7618), 50-56 (2016).</li>
<li>Golde, T. E., Koo, E. H., Felsenstein, K. M., Osborne, B. A., Miele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((gamma-Secretase+inhibitors+and+modulators%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">gamma-Secretase inhibitors and modulators.</a> Biochim Biophys Acta. 1828 (12), 2898-2907 (2013).</li>
<li>Coric, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+Prodromal+Alzheimer+Disease+With+Avagacestat%3A+A+Randomized+Clinical+Trial%5BTitle%5D)+AND+%22JAMA+Neurol%22%5BJournal%5D)">Targeting Prodromal Alzheimer Disease With Avagacestat: A Randomized Clinical Trial.</a> JAMA Neurol. 72 (11), 1324-1333 (2015).</li>
<li>Mitani, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+effects+between+gamma-secretase+inhibitors+and+modulators+on+cognitive+function+in+amyloid+precursor+protein-transgenic+and+nontransgenic+mice%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Differential effects between gamma-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice.</a> J Neurosci. 32 (6), 2037-2050 (2012).</li>
<li>Penninkilampi, R., Brothers, H. M., Eslick, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+Agents+Targeting+gamma-Secretase+Increase+Risk+of+Cancer+and+Cognitive+Decline+in+Alzheimer%27s+Disease+Patients%3A+A+Systematic+Review+and+Meta-Analysis%5BTitle%5D)+AND+%22J+Alzheimers+Dis%22%5BJournal%5D)">Pharmacological Agents Targeting gamma-Secretase Increase Risk of Cancer and Cognitive Decline in Alzheimer's Disease Patients: A Systematic Review and Meta-Analysis.</a> J Alzheimers Dis. 53 (4), 1395-1404 (2016).</li>
</ol>

The authors have nothing to disclose.

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&amp;rsquo;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 CO&lt;sub&gt;2&lt;/sub&gt; 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

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 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...

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 ...

quantitative-measurement-secretase-mediated-amyloid-precursor-protein

Research

JoVE Journal

Neuroscience

This video demonstrates the procedure of measuring γ-secretase activity in genetically modified human cells using a tetracycline-inducible system. The process involves inducing APP gene expression with tetracycline, generating amyloid-β peptides, and activating luciferase production. The bioluminescence resulting from the luciferase-substrate reaction is then measured, reflecting γ-secretase activity.

Luciferase Assay for Measuring &#947;-Secretase Activity in Human Cells

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Assays for the Degradation of Misfolded Proteins in Cells

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

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

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid β protein on brain tissues of Alzheimer's disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Here, we describe a yeast system reconstituting human γ-secretase. This system allows for the identification and study of mutations affecting activity and for screening of γ-secretase inhibitors (GSIs). Utilizing the loss of function properties of familial Alzheimer's disease mutants, it is possible to screen for γ-secretase modulators (GSMs).

Saccharomyces cerevisiae Models of Alzheimer's Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by &#947;-Secretase

Learn quantitative γ-secretase assays using cell-based luciferase reporter platforms to identify modulators for Alzheimer disease research. This method enables efficient screening of therapeutic candidates with minimal side effects.

Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms (Video) | JoVE Journal

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

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