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 ÃŽÂ³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

作者感谢中央研究院细胞与有机体生物学研究所的核心设施, 以提供技术支持。这项研究得到了台湾科学技术部的支持 (最多 103-2320-001-016-MY3 至 Y。L.), 生物制药发展的转化创新方案–技术支持平台轴方案 (NP7 至 y.---), 中央研究院 (对 y---)。

1. 荧光报告器信号的测量, 对应于 APP-C99 或 NΔE 的γ-分泌劈裂
注: 有关 CG 和 NG 单元格生成的详细说明, 请参阅以前的出版物20,21。
<ol><li>
种子 NG 或 CG 细胞 (20, 000 细胞/井) 到96井板在最后的体积200μ l/井在生长培养基, 由 Dulbecco 氏改良的鹰培养基 (DMEM) 加上10% 胎牛血清 (FBS), 5 微克/毫升 blasticidin, 250 微克/毫升抗生素 (例如</...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2+regulates+autophagic+flux+to+modulate+the+proteostasis+of+APP-CTFs+in+Alzheimer's+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibody+aducanumab+reduces+Abeta+plaques+in+Alzheimer's+disease.">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. 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在 ad 的 aducanumab 免疫治疗的1b 期试验中, 有希望的结果已经牢固确立了 a 在 ad22发病机制中的关键作用, 并认为反 a 方法仍然是开发抗 ad 药物的可行策略。在这里, 我们描述了以细胞为基础的定量分析γ-分泌催化水解 APP-C99 和 NΔE 使用萤火虫荧光报告系统。APP-C99 和 NΔE 是两个最有研究的γ-分泌基底, 同时代表γ-分泌13的致病和生理活动。我们的发现, 下调 ErbB2 的...

聚的淀粉样β (a) 的形式被认为是导致阿尔茨海默氏病 (AD)1患者大脑中神经的主要原因。a 肽是由淀粉样前体蛋白 (APP) 的逐步裂解产生的, 首先是β-分泌, 然后是γ-分泌2。在过去的十年中, 治疗 AD 的治疗方法侧重于预防 a 的生产3。大多数研究都集中在α-分泌活性的增强上, 这可以排除应用的γ-分泌分裂, 从而减少 a 的产生, 或抑制β和/或γ-分泌活动4。不幸的是, 非选择性抑制β或γ-secretases 导致不可避免的副作用, 这是由于干扰功能的其他生理基质的β和γ-分泌5,6。关于γ-分泌抑制剂, 最近的研究报告发现了一些化学和遗传调节剂, 可以调节 a 的生产, 而对必要的γ-分泌介导的加工缺口的影响微不足道的7 ,8,9,10,11,12, 但是, 成功的治疗方法尚未开发。因此, 有必要进一步系统的屏幕, 以发现新的遗传和化学修饰, 可能选择性地调节γ-分泌介导的应用程序处理。
γ-分泌是已知的切割超过90种不同的膜锚定蛋白。在这些基体之中是切口, 它的活动是关键的细胞命运决心和分化在发展期间13。在没有影响缺口处理的情况下, 选择性地调节γ-分泌-催化的应用处理, 对于最小化γ-分泌抑制剂的缺口相关副作用至关重要, 而这种选择性被认为是一个主要因素, 将规定了潜在的γ-分泌调节剂对 AD 慢性治疗的生物学功效。有许多 arylsulfonamide 衍生物, 例如 GSI-953 (begacestat) 和 BMS-708163 (avagacestat), 被发现表现出强有力的和有选择性的抑制γ-分泌14,15。先前的一项研究表明, 使用定量 ELISA 法结合使用斑马鱼16的活体验证, 可以观察应用和切口的γ-分泌裂解的差异抑制。此外, 建立了基于细胞的检测范式, 以解决γ-分泌对 APP 和凹槽1718的潜在基底选择性。使用类似于本文所述的协议, 我们最近发现了一种新的类 (D)-leucinamides, 能调制的γ-分泌分裂的应用程序与缺口备用选择性19。这一集合的研究提供了一个证明的概念, 支持的概念, 基板的选择性/可用性γ-分泌可以受到化学调制和实验验证。然而, 这些化验平台往往依赖相对较低的吞吐量读数和劳动密集型的方法, 可能不符合工业标准的药物发现计划。
我们最近生成了基于细胞的荧光报告基因检测, 可以定量地确定γ-分泌对两个不同基底的催化活性, 即应用 (APP-C99) 的 99-氨基甲酸 C 末端片段和胞外域删除的缺口肽 (NΔE)。APP-C99 和 NΔE 在各种测定方法中被广泛用作γ-分泌的直接基质。为了生成均匀和一致的荧光报告基因检测的γ-分泌分裂的 APP-C99 或 NΔE, 我们产生了一个 HEK 衍生稳定细胞线 (CG) 组成表达 Gal4 启动驱动萤火虫荧光记者 (Gal4-Luc) 和 Gal4/VP16-tagged APP-C99 融合蛋白 (C99-GV)。此外, 我们还生成了另一个 HEK 的稳定细胞系 (NG), 组成表达 Gal4-Luc 和 Gal4/VP16-tagged NΔE (NΔE)。利用这些新的基质特异γ-分泌测定方法, 我们现在提供了一种定量和区分γ-分泌对不同基底 (APP-C99 在 CG 细胞中, 或 NΔE 在 NG 细胞中) 的催化活性。此外, 基板特定的γ-分泌检测的目的是有利于高含量的筛选平台。这些新生成的γ-分泌检测方法可以为发现新的γ-分泌调节剂铺平道路, 通过抑制 a 的产生, 而不引起对下一代治疗的不良抑制, 从而提高认知功能。的广告。

<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

我们已经开发了一对细胞为基础的记者基因检测, 定量测量γ-分泌分裂的不同基质。本手稿描述的程序, 可用于监测γ-分泌介导的 APP-C99 或缺口, 使用 Gal4 启动器驱动的萤火虫荧光记者系统的裂解。染 HEK293 细胞与 Gal4-driven 荧光报告基因和 Gal4/VP16-tagged C 终端片段 (APP-C99) 建立了稳定的 co-非晶胞蛋白的测定。CG 细胞), 或 Gal4/VP16-tagged 缺口-δ (NΔE;NG 细胞)。利用这些记者并行分析, 我们已经证明, ErbB2 抑制剂, CL-387,785, 可以优先抑制γ分泌分裂的 APP-C99 在 CG 细胞, 但不是 NΔE 在 NG 细胞。CL-387,785 治疗时, CG 和 NG 细胞所表现的差异反应是γ-分泌调节剂的首选特性, 这些反应与 DAPT 诱导的γ-分泌的泛抑制形成了鲜明的对比。我们的研究提供了直接的证据证明γ-分泌活动向不同的基底可以被区分在一个细胞的背景。因此, 这些新的化验方法可能是药物发现的有用工具, 用于改进 AD 疗法。

通过CL-387,785 抑制 ErbB2 可差异促进 APP-C99 的处理γ-分泌不影响缺口解理 为了建立一种能定量测量特定γ - 分泌基质的蛋白水解的细胞分析方法 , 我们通过稳定的联合转染四环素诱导的 c - 终末期 Gal4 / VP16 - tagged , 生成了 HEK293 - derived 的 CG 细胞系。APP-C99 和一个 Gal4 启动驱动萤火虫荧光记者基因。还建立了一个平行 NG 细胞线, 以检测 N...

Watch this Scientific Journal Video about Quantitative Measurement of ÃŽÂ³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

Quantitative Measurement of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

基于细胞荧光检测平台的γ-分泌介导的淀粉样前体蛋白和缺口裂解的定量测定

我们已经成功地生成了两个基片特定的γ-分泌检测。这两种基于细胞的检测方法都被设计成通过萤火虫荧光记者的输出来量化γ-分泌酶的活性。

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

Research

JoVE Journal

Neuroscience

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

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

Selection of Aptamers for Amyloid ÃƒÅ½Ã‚Â²-Protein, the Causative Agent of Alzheimer's Disease

淀粉样β-蛋白，阿尔茨海默氏病的病原体适配的选择

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

科研

神经科学

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

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

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

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

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

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

受体激活的测量通过荧光素酶活性：哺乳动物的嗅觉受体的高通量分析

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

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

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

低聚淀粉样蛋白-β的立体灌注到小鼠海马

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

使用F9稀土LACZ基于Cell的报告基因检测在E8.5胚胎和神经球文化相对维甲酸水平的定量测量

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

Modeling Amyloid-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

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

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

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

定量自动放射法测定维沃脑蛋白合成区域速率

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