这项工作得到了由韩国政府 (MSIP) (NRF-2014K1A4A7A01074642) 和韩国国家研究基金会 (NRF) 个人科学家支持计划 (NRF-2013M3A9B5076486/NRF-2015R1D1A1A09057239) 资助的赠款。

1. 慢生产 注意: 根据国家卫生研究院 (NIH) 有关重组的研究指南, 对慢载体进行生产和操作。dna.用增强 YFP (SOD1WT-YFP 和 SOD1A4V-YFP) 标记的野生型和 A4V 突变体 SOD1 的质粒, 在金 et al 中描述。 11 两种基因融合产品均采用 PCR 引物对5和 #8242;-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3 和 #8242; 5 和 #8242;-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3 和 #8242; 插入 pTRIP 三角洲 U3 巨细胞病毒质粒 19 使用 XhoI 和 BsrGI 限制站点。在先前的实验中, HEK-293T 细胞的分裂率为1:4 至1:6 每两天。避免超过20通道和维持细胞在不到 60% 70-100 有助于确保良好的转染效率. 
<ol> <li> 在1天, 分裂 HEK-293T 细胞在 30-40% 汇合在 10 cm 直径组织培养板材 (3 x 10 6 细胞或盘) 在10毫升细胞培养基 (高 DMEM 与 10% FBS 和4毫米 l-谷氨酰胺) 为病毒生产. </li>
	<li> 保留10厘米直径组织培养板在 CO 2 孵化器 (37 和 #176; C, 5% CO 2 ) 过夜. </li>
	<li> 2 天, 准备一个含有10和 #181 的 DNA 转染溶液; g 慢载体 (pTRIP U3 CMV SOD1WT-YFP 或-SOD1A4V-YFP) 连同慢包装质粒 (5 和 #181; g pVSVg; 10 和 #181; g pCMV-dR8.71) ( 图 2A ) 20 . 添加125和 #181; 1 M CaCl 2 , 并将音量调整为500和 #181; 我使用蒸馏水。轻轻地加500和 #181; 0.05 米 HEPES 到混合物中, 室温下孵育10分钟. </li>
	<li> 用10毫升5ml 新鲜培养基取代 HEK-293T 细胞培养基与1毫升 DNA 转染溶液混合, 并在夜间孵育37和 #176; C, 5% CO 2 . </li>
	<li> 在3天, 用新鲜培养基取代细胞上清液. </li>
	<li> 在4天, 在15毫升的管中收获上清液, 并在 500 x g 离心5分钟, 以去除死细胞和碎片。通过0.45 和 #181 进一步净化上清液; m 过滤器使用大60毫升注射器。立即将一次性等分 (300 和 #181; L), 并存储在-80 和 #176; C。要保持最大的产品活动, 请避免冻结解冻周期. </li>
</ol> 2。慢转导 <ol> <li> 将单元格线拆分到60% 汇合处 (HEK-293 细胞和 SH-SY5Y; 5 x 10 5 单元格和 U2OS; 每井 1 x 10 个, 在 5-良好组织培养板块中6毫升的 DMEM 培养基中补充10% FBS. </li>
	<li> 将6个组织培养板保持在37和 #176; C 孵化器在 5% CO 2 过夜. </li>
	<li> 将冻结的慢从-80 和 #176 中取出; 在每次使用前将冰分冷冻并解冻; 不要冻结. </li>
	<li> 当病毒正在解冻时, 将含有血清的细胞培养基加热, 以符合细胞系的利益。一旦病毒完全解冻, 准备一个稀释 (1:3, 1:10, 一点半, 1:100) 在 DMEM 在一个新鲜的1.5 毫升离心管的范围. </li>
	<li> 将试管中的音量调到1毫升, 并降低血清介质. </li>
	<li> 添加2和 #181; 凝聚 (在4和 #181 的股票; g/和 #181; l) 到1毫升的病毒/媒体。移混合均匀, 并加入1毫升的混合物到细胞。用病毒孵育细胞 24 h. </li>
	<li> 删除病毒介质并替换为正常的 DMEM 介质, 并辅以 10% FBS。保持37和 #176 的单元格; C, 5% CO 2 . </li>
	<li> 监视单元格的增长并每两天更改一次区域性媒体。在汇合处, 将6井道扩展成直径10厘米的组织培养皿. </li>
	<li> 一旦单元格已得到充分扩展, 种子 1.5 x 10 4 每井50和 #181 的细胞; DMEM 培养基上的 384-试板, 通过显微镜检查 YFP 标记蛋白的表达水平。如果 YFP 标记的蛋白质细胞表达显示一个信噪比和 #8805; 3, 为相应的细胞线准备细胞储存. </li>
</ol> 3。蛋白酶体抑制剂的时间依赖性对蛋白质聚集的影响 注意: 使用自动显微镜执行图像采集 (请参阅 材料 )。
<ol> <li> 免除0.25 和 #181; 在 384-井平底板上, 亚砜或蛋白酶体抑制剂 L 的溶解在 100% ALLN, 2 毫米) 上. </li>
	<li> 手动播种 1.5 x 10 4 每个井在50和 #181 中的单元格; DMEM 介质在同一检测板上。蛋白酶体抑制剂 (ALLN) 最终浓度应为10和 #181; m. </li>
	<li> 将显微镜系统环境控制单元设置为37和 #176; C 和 5% CO 2 。实验设置的屏幕截图如 图 3 所示. </li>
	<li> 在广域荧光模式下操作显微镜, 使用以下设置的软件: 钻20X 目标, non-confocal 模式, 4 字段/好, 捕获间隔为一小时。每次运行结束时, 捕获的图像都会自动上载到服务器. </li>
</ol> 4。活细胞中 YFP 表达的时间推移成像和图像分析 (单通道) 注意: 以下步骤描述了软件的应用 ( 如 , 哥伦布)。
<ol> <li> 选择适当的算法来分割主要对象 (胞和聚合)。如果需要, 请调整背景阈值和对比度参数 ( 图 4 ). </li>
	<li> 使用和 #39 计数单元格; 查找单元格和 #39; YFP 强度通道的单个阈值为0.1。使用 and #39 确定聚合; 查找点和 #39; 具有相对 YFP 点强度和 #62 的算法; 0.2 和分裂系数为 1.0. </li>
</ol> 5。蛋白酶体抑制剂对活细胞蛋白质聚集的剂量响应效应 (两个通道) 注意: 使用自动显微镜进行图像采集。根据预期的 IC 50 值确定蛋白酶体抑制剂 (ALLN、Epoxomicin 和 MG132) 的浓度范围, 以确保最佳的曲线拟合. 
<ol> <li> 用标准的1:3 稀释系列准备 384-井储存聚丙烯板上的化合物的系列稀释。一定要把复合稀释好, 以确保化合物的浓度是准确的. </li>
	<li> 免除0.25 和 #181; 在空扁底黑384试板上的蛋白酶体抑制剂 L。我们使用的液体处理程序配备了384毛细管头能够转移体积. </li>
	<li> 种子 1.5 x 10 4 每个井在50和 #181 中的单元格; DMEM 介质在检测板上的 L。在37和 #176 上孵育检测板; C 和 5% CO 2 , 用于 24 h. </li>
	<li> 添加10和 #181; DMEM 介质 (5ml 37 和 #176; C) 与染色液 (赫斯特 33342, 10 毫克/毫升), 室温孵育10分钟. </li>
	<li> 启动百万分之一处理操作软件。实验设置的屏幕截图如 图 6 所示。选择 #34; 配置和 #34; 选项卡并选择20X 和 #160; 目标和正确的板型。确保和 #34; 衣领和 #34; 在目标上设置正确的值, 以便在不同的板型中进行适当的聚焦. </li>
	<li> 选择 #34; 显微镜和 #34; 标签. 定义曝光1作为 YFP (488 laser) 和曝光2作为赫斯特 (405 laser)。激活两个曝光的过滤器, 并将曝光1分配给照相机1和曝光2到照相机2。将曝光时间设为800毫秒, 曝光率为 1, 40 毫秒, 曝光率为 2. </li>
	<li> 选择曝光1。将焦点高度设置为0和 #181; m. 选择和 #34; 焦点和 #34;。一旦聚焦, 曝光摄像头1。调整焦点高度以优化曝光平面, 然后单击并 #34; 采取高度和 #34;。改变曝光时间和激光功率, 使最大像素强度为3000。保存曝光参数。重复曝光 2. </li>
	<li> 选择和 #34; 实验定义和 #34; 选项卡. 创建布局和 sublayout。拖放相关的布局、曝光、参考图像、skewcrop 文件和 sublayout。保存实验. </li>
	<li> 选择和 #34; 自动实验和 #34; tab 并获取图像。每次运行结束时, 捕获的图像都会自动上载到服务器. </li>
</ol> 6。两个通道图像的图像分析 <ol> <li> 选择软件算法来分割主对象 (核、胞和聚合) ( 图 7 )。
	<li> 通过对分段对象进行目视检查, 选择精确细分原子核的方法。赫斯特通道 1 (Ch1) 中的染色对象将用于确定单元格的数量。在通道 2 (Ch2) 的突变 SOD1-A4V 的 YFP 染色将用于确定总量. </li>
	<li> 使用 and #39 计数单元格; 查找核和 #39; 算法为赫斯特染色区域和 #62; 20 和 #181; m 2 , 其分割因子为 7.0, 单个阈值为 0.40, 对比度和 #62; 0.10. </li>
	<li> 创建数据表并确定 EC 50 使用 and #39 的每个化合物; 非线性回归和 #39; 绘图软件中的等式. </li>
</ol>

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作者没有什么可透露的。

有两种主要的方法来生成稳定的细胞系。第一次需要几个星期, 需要对基因组整合的质粒 DNA 载体进行瞬时转染和抗性选择。第二个需要几个小时的时间, 通过使用慢, 使这一协议的有效表达的目标蛋白在多个细胞系有限的努力。行程-CMV 矢量19是一个持续使用的载体, 具有保守的转导效率和稳定的转基因表达。令我们吃惊的是, 生成的三单元格线显示的是突变 SOD1 的可见聚合, 而不?...

蛋白质聚集是一种生物过程, 错误蛋白可以在神经退行性疾病 (淀粉样变性) 中起到致病剂的作用。在理解细胞功能障碍中聚合体的作用, 以及在促进发现影响病理发病的新因素方面, 蛋白质聚集性是必不可少的。活体细胞荧光标记蛋白的可视化是一种强有力的方法, 可以帮助开发适用于高含量筛选 (HCS)1、2、3、4的测试。
肌萎缩侧索硬化 (als) 被认为是一种 proteopathic 的疾病, 由存在的错误蛋白的倾向聚集和积累在运动神经元的家族 als (fALS) 和零星 als (销售)5,6.20% fALS 病例的一个子集与胞浆抗氧化酶铜锌超氧化物歧化因子 1 (SOD1)7,8的基因编码中的显性突变相关。提出了这一基因衍生性功能障碍的几个潜在原因, 包括 SOD1 变种的结构和功能的变化, 如异常稳定性, 增加的展开率, 和倾向于聚合9, 10。值得注意的是, 由 ALS 连接的 SOD1 变种和野生型 (重量) SOD1 所共享的唯一经过验证和潜在毒性的属性是形成条块分割的蛋白质聚合体或蛋白质夹杂物的倾向11,12.错误突变 SOD1, 是持续 polyubiquitinated 和退化的泛素-蛋白酶体系统。因此, 对蛋白酶体活性的抑制导致了突变体 SOD1 聚集13,14, 形成了由可溶性组分组成的无定形结构, 可与可溶性突变体 SOD1 交换。在胞15中。特别是, SOD1 突变 A4V (丙氨酸在密码子4改为缬氨酸) 是最常见的 ALS 引起的突变, 并导致快速神经的平均生存时间少于2年后疾病发作16。生化, SOD1 A4V 有增加的倾向 monomerize, 聚合, 并形成淀粉样孔隙;它的孔状聚合体类似于其他与疾病相关的突变体的淀粉样孔, 如α-核和β-淀粉样蛋白17。为了研究 SOD1-aggregate 积累的动力学规律, 对可溶性和不溶性 SOD1 聚合形式的监测方法有待发展。
我们以前已经显示, 使用活细胞成像和 HEK-293 细胞瞬时转染荧光蛋白标记 SOD1, ALS 相关的突变损害 SOD1 二和聚集11。虽然瞬态表达系统可以提供有关短期基因过度过度的生物学结果的有用信息, 但提供所需基因的稳定整合的方法可能更适合于检测的发展。因此, 慢载体提供了赋予哺乳动物细胞长期和调控基因表达的能力18。在这项研究中, 我们的重点是生成稳定细胞系转与重组慢轴承和突变 SOD1 标记的黄色荧光蛋白 (YFP)。使用活细胞成像显微镜和自动定量的 SOD1 聚集, 我们触发和量化 SOD1 聚集事件时抑制蛋白酶体。

<table><tbody><tr><td>ALLN (C&lt;sub&gt;20&lt;/sub&gt;H&lt;sub&gt;37&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;)</td><td>Millipore</td><td>208719</td><td></td></tr><tr><td>MG132 (C&lt;sub&gt;26&lt;/sub&gt;H&lt;sub&gt;41&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C2211</td><td></td></tr><tr><td>Epoxomicin (C&lt;sub&gt;28&lt;/sub&gt;H&lt;sub&gt;50&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>E3652</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H-3570</td><td></td></tr><tr><td>Opera</td><td>Perkin Elmer</td><td>OP-QEHS-01</td><td></td></tr><tr><td>Opera EvoShell software</td><td>Perkin Elmer</td><td>Ver 1.8.1</td><td></td></tr><tr><td>Operetta</td><td>Perkin Elmer</td><td>OPRT1288</td><td></td></tr><tr><td>Harmony Imaging software</td><td>Perkin Elmer</td><td>Ver 3.0.0</td><td></td></tr><tr><td>Columbus Image analysis software</td><td>Perkin Elmer</td><td>Ver 2.3.2</td><td></td></tr><tr><td>CyBi Hummingwell liquid handling</td><td>CyBio AG</td><td>OL 3387 3 0110</td><td></td></tr></tbody></table>

assay development for high content quantification of sod1 mutant protein aggregate formation in living cells

肌萎缩侧索硬化症 (ALS) 是一种致命的神经退行性疾病, 可导致遗传突变的基因编码铜锌超氧化物歧化酶 1 (SOD1)。SOD1 的结构不稳定性和家族性 als 患者的 SOD1-positive 包裹体的检测支持错误和/或聚合 SOD1 在 als 病理中的潜在因果作用。在这项研究中, 我们描述了一个细胞分析的发展, 旨在量化的动态 SOD1 聚集在活体细胞的高含量的筛选方法。利用慢向量, 我们生成了表达野生型和突变 A4V SOD1 标记的稳定细胞系, 黄色荧光蛋白, 发现这两种蛋白质都表达了胞没有任何聚集的迹象。有趣的是, 只有 SOD1 A4V 稳定表达在 HEK-293, 但不是在 U2OS 或 SH-SY5Y 细胞线, 形成了聚集后, 蛋白酶体抑制剂治疗。我们表明, 这是可能的量化聚合的基础上, 剂量反应分析的各种蛋白酶体抑制剂, 并跟踪集料形成动力学的延时显微镜。我们的方法引入了定量的可能性, ALS 突变的作用, SOD1 在聚合形成和筛选的小分子, 防止 SOD1 A4V 聚集。

使用慢生成稳定的单元格线:在图 1中说明了监测 SOD1 蛋白聚合体的总体策略。在第一步, 我们生成了一个慢表达载体, SOD1 稳定基因传递到细胞系 (步骤 1)。制备了两个慢矢量编码 YFP 标签 SOD1 和 SOD1 A4V (SOD1WT-YFP 和 SOD1A4V-YFP 分别) 与包装和包络质粒 (图 2A)。在慢转导 (步骤 2) 测试的细胞线 (HEK-293, U2OS 和 SH-SY5Y) 保持健?...

Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

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

我们描述一种方法来量化错误蛋白质的聚集。我们的协议细节慢诱导稳定细胞线生成, 自动共焦成像, 和图像分析的蛋白质集料。作为一个说明性应用, 我们研究了小分子在促进 SOD1 聚集的时间和剂量依赖性的方式的影响。

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 ...

assay-development-for-high-content-quantification-sod1-mutant-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.1K 次观看.  Institut Pasteur Korea. 我们描述一种方法来量化错误蛋白质的聚集。我们的协议细节慢诱导稳定细胞线生成, 自动共焦成像, 和图像分析的蛋白质集料。作为一个说明性应用, 我们研究了小分子在促进 SOD1 聚集的时间和剂量依赖性的方式的影响。

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

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

科研

神经科学

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.

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.

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 ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

微粒相关α synuclein 聚合体对原代神经元的外源性管理--纤维形成的强大细胞模型

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

生物学

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

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.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

使用荧光相关光谱检测蛋白质聚合

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

一种基于人源酵母模型研究α-突触核蛋白毒性和聚集的方法

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Source: Lee, H. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/56425">Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.

蛋白质聚集体形成测定:一种在蛋白酶体抑制剂诱导下检测和定量培养细胞中蛋白质聚集的方法

Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)
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