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, zeocin) 和200微克/毫升霉 B。
	<ol>
<li>用例计数单元格。</li>
		<li>将板转移到湿润 CO2孵化器, 并在一夜之间在37° c 孵育。</li>
	</ol>
</li>
	<li>从每个井中取出100μ l 的生长培养基。</li>
	<li>添加100μ l/好的生长培养基含有2微克/毫升四环素与0.2% 二甲基亚砜 (亚砜), 2 μ m CL-387,785, 2 μ m n-[n-(35-difluorophenacetyl-丙)]-s-氨 t butylester (DAPT), 或其他相关的 ErbB1/ErbB2 抑制剂。</li>
	<li>在板37° c 的24小时内孵育经治疗的 CG 或 NG 细胞。</li>
	<li>从板中取出150μ l/井生长培养基。</li>
	<li>添加50μ l/井荧光测定试剂 (请参阅材料表)。</li>
	<li>
将板转换为发光微读取器。
	<ol>
<li>在室温下保持板5分钟的温和搅拌。</li>
	</ol>
</li>
	<li>
使用存储在发光微读卡器中的预定义程序确定萤火虫荧光 (FL) 发出的发光。
	<ol>
<li>打开仪器控制软件 (请参阅仪器详细信息的材料表)。</li>
		<li>从 "工具" 菜单中选择 "Luminometry"。<ol>
<li>在 "参数" 设置下, 选择 "无过滤器" 进行发射过滤, 对发射孔径进行 "正常" 刻度, 输入 "1" 计算时间, 点击 "OK" 设置发光读数。</li>
		</ol>
</li>
		<li>在 "工具控件" 选项卡中, 滚动活动协议列表, 并选择预存协议以读取发光。</li>
		<li>在 "实时显示" 选项卡下, 从下拉列表中定义刻度, 并为类型选择 "对数" 或 "线性"。</li>
		<li>在 "温度" 选项卡下, 选择 "关闭" 为板加热。</li>
		<li>单击 "开始" 按钮执行发光读数。</li>
		<li>根据需要, 使用内嵌在控制软件中的 "资源管理器" 将结果导出为电子表格格式。</li>
	</ol>
</li>
</ol>2. γ-分泌活性的定量
<ol><li>
定义一个由 CG 或 NG 细胞所发射的发光信号, 在含有1微克/毫升四环素的生长培养基中处理0.1% 甲基亚砜, 作为100% 相对γ-分泌活性。
	<ol>
<li>通过对不含四环素的生长培养基进行细胞处理, 来估计 CG 或 NG 细胞的背景发光。</li>
	</ol>
</li>
	<li>通过含有1微克/毫升四环素的生长培养基和1μ m CL-387,785 或1μ m 的 DAPT 与由亚砜治疗的 cg 或 ng 细胞 (100% 相对γ-分泌活动) 发出的荧光信号, 使荧光信号从 cg 或 ng 细胞正常化。定义于 2.1)。</li>
</ol>

<ol>
	<li>Selkoe, D. J., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease+at+25+years.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+at+a+glance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+cascade+hypothesis+for+Alzheimer's+disease:+an+appraisal+for+the+development+of+therapeutics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+strategies+for+disease+modification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-secretase+enzyme+BACE+in+health+and+Alzheimer's+disease:+regulation,+cell+biology,+function,+and+therapeutic+potential.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin+diversifies+its+portfolio.">Presenilin diversifies its portfolio.</a> Trends Genet. 23 (3), 140-150 (2007).</li><li>Kukar, T. L., 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=Substrate-targeting+gamma-secretase+modulators.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+gamma-secretase+reduces+beta-amyloid+deposition+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orphan+G+protein-coupled+receptor+3+modulates+amyloid-beta+peptide+generation+in+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Alzheimer's+disease+amyloid-beta+formation+by+casein+kinase+I.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gleevec+increases+levels+of+the+amyloid+precursor+protein+intracellular+domain+and+of+the+amyloid-beta+degrading+enzyme+neprilysin.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-secretase+activating+protein+is+a+therapeutic+target+for+Alzheimer's+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+substrates+of+presenilin/gamma-secretase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+Evaluation+of+BMS-708163,+a+Potent,+Selective+and+Orally+Bioavailable+gamma-Secretase+Inhibitor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACS+chemical+neuroscience+molecule+spotlight+on+Begacestat+(GSI-953).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gamma-secretase+modulation+differentiates+inhibitor+compound+selectivity+between+two+substrates+Notch+and+amyloid+precursor+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cell-based+method+for+determining+the+gamma-secretase+enzyme+activity+against+both+Notch+and+APP+substrates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+selective+gamma-secretase+inhibitors+for+treatment+of+Alzheimer's+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+small+molecular+(D)-leucinamides+as+potent,+Notch-sparing+gamma-secretase+modulators.">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/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. 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. A., Miele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Secretase+inhibitors+and+modulators.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Prodromal+Alzheimer+Disease+With+Avagacestat:+A+Randomized+Clinical+Trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+between+gamma-secretase+inhibitors+and+modulators+on+cognitive+function+in+amyloid+precursor+protein-transgenic+and+nontransgenic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Agents+Targeting+gamma-Secretase+Increase+Risk+of+Cancer+and+Cognitive+Decline+in+Alzheimer's+Disease+Patients:+A+Systematic+Review+and+Meta-Analysis.">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>

作者没有什么可透露的。

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

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。

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

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

6.7K 次观看.  Academia Sinica. 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。

视频: Quantitative Measurement of γ-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

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

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

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-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

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 fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

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

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal 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

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 research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

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

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

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

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

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

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

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

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

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

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

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, ...

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 superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

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.

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

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine 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 ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

在体外陶氏蛋白聚集与药物筛选研究方法

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...