Name: modeling age-associated neurodegenerative diseases in caenorhabditis elegans
Uploaded: 2020-08-15T14:00:00
Description: Watch this Scientific Journal Video about Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans at JoVE.com

我们感谢查尼奥塔基斯 M. 和库纳基斯 K. 的录像录制和编辑。K.P. 由希腊研究和创新基金会（HFRI）和研究与技术总秘书处（GSRT）提供赠款。N.T. 的资金来自欧洲研究理事会 （ERC – GA695190 – MANNA）、欧盟委员会框架方案和希腊教育部的赠款。

1. 由超活性离子通道引起的坏死细胞死亡
注：在退化素的基因家族，包括mec-4 和 deg-3 等，功能增益突变导致产生多活性离子通道，触发蠕虫3中机械化所需的六个接触受体神经元的坏死细胞死亡。退化素的异常刺激引起的坏死与哺乳动物的兴奋毒性有若干机械和形态相似性。维持能量代谢和钙平衡对坏死11期间神经元的生存起着至关...

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在这里，我们介绍和描述广泛访问的方法，增长，同步和微观检查一些多功能的C.elegans模型研究年龄依赖性神经退化。特别是，我们使用高活性离子通道诱发坏死和蛋白质聚集诱导,神经毒性,,,,1、2、3、4、5、7、9、10、11,2来评估和剖析与年龄相关的神经元崩溃的7,</s...

在过去的二十年里，C.elegans被广泛用作研究坏死细胞死亡的分子机制的模型有机体。C. elegans提供特别良好的特征和映射的神经系统、透明的身体结构和多种遗传和成像方法，以监测体内细胞功能和整个衰老过程中的生存。因此，已经开发了几个C.elegans神经退化的遗传模型来评估神经元的生存能力。特别是，描述良好的线虫模型包括多活性离子通道诱发坏死1,1，2，3,3和细胞死亡触发增加蛋白质聚集4，5，6，7，8，9，105,6,7,8,9,10和中暑411，12，等等。11,12
短期暴露在亚致命温度下，对坏死细胞死亡产生抵抗力，由线虫和哺乳动物神经元11的后续热应激触发。有趣的是，线虫在温和的高温下每天预置，可防止神经退化，而神经退化是由多种刺激引起的，例如离子不平衡（例如，mec-4（u231）和/或deg-3（u662）和蛋白质聚集（例如，α-合成素和聚Q40）11，13。11,13
在这里，我们描述了使用 C.elegans来 监测和评估人类疾病成熟模型中依赖年龄的神经退化的通用方法，如兴奋性触发细胞死亡、帕金森氏症和亨廷顿舞蹈症。此外，我们强调热预置在几种神经退化模型中的神经保护作用。这些技术与遗传和/或药理学筛选相结合，将识别和鉴定新型细胞死亡调节剂，具有潜在的治疗兴趣。

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym</td>
			<td>8,40,004</td>
			<td></td>
		</tr>
		<tr>
			<td>AM101: rmsIs110[prgef-1Q40::YFP]</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate (CaCl2&#8729;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>SERVA Electrophoresis</td>
			<td>17101.01</td>
			<td></td>
		</tr>
		<tr>
			<td>deg-3(u662)V or deg-3(d)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Maintain animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>DIC microscope (Nomarsky)</td>
			<td>Zeiss</td>
			<td></td>
			<td>Axio Vert A1</td>
		</tr>
		<tr>
			<td>Dissecting stereomicroscope</td>
			<td>Nikon Corporation</td>
			<td></td>
			<td>SMZ645</td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>EVOS Cell Imaging Systems</td>
		</tr>
		<tr>
			<td>Escherichia coli OP50 strain</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner Petri dishes (60 mm x 15 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>P5237</td>
			<td></td>
		</tr>
		<tr>
			<td>image analysis software</td>
			<td>Fiji</td>
			<td></td>
			<td>https://fiji.sc</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>1,37,010</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>EMD Millipore</td>
			<td>1,04,873</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>mec-4(u231)X or mec-4(d)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td>Maintain animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>Microscope slides (75 mm x 25 mm x 1 mm)</td>
			<td>Marienfeld, Lauda-Koenigshofen</td>
			<td>10 006 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass (18 mm x 18 mm)</td>
			<td>Marienfeld, Lauda-Koenigshofen</td>
			<td>01 010 30</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2011 Excel software package</td>
			<td>Microsoft Corporation, Redmond, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>1,06,586</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode growth medium (NGM) agar plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin stock solution</td>
			<td>Sigma-Aldrich</td>
			<td>N3503</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>BD, Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>EMD Millipore</td>
			<td>1,06,40,41,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard equipment for preparing agar plates (autoclave, Petri dishes, etc.)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard equipment for maintaining worms (platinum wire pick, incubators, etc.)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>statistical analysis software</td>
			<td>GraphPad Software Inc., San Diego, USA</td>
			<td></td>
			<td>GraphPad Prism software package</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>L9756</td>
			<td></td>
		</tr>
		<tr>
			<td>UA44: Is[baIn1; pdat-1&#945;-syn, pdat-1GFP]</td>
			<td></td>
			<td></td>
			<td>Upon request: G. Caldwell (University of Alabama, Tuscaloosa AL)</td>
		</tr>
	</tbody>
</table>

modeling age-associated neurodegenerative diseases in caenorhabditis elegans

对抗人类神经退行性疾病并管理其普遍的社会经济影响正成为全球优先事项。尽管它们对人的生命质量和保健系统有有害影响，但大多数人类神经退行性疾病仍然无法治愈和不可预防。因此，针对此类疾病开发新的治疗干预措施正成为当务之急。神经元回路和功能的年龄相关恶化在像低蠕虫卡诺哈布迪炎和人类等不同生物体中进化地得到Caenorhabditis elegans保存，这表示在底层细胞和分子机制上具有相似性。C. elegans是一种高度可塑性的基因模型，它提供了良好的神经系统、身体透明度以及多种遗传和成像技术，用于评估衰老期间的神经元活动和质量控制。在这里，我们介绍和描述利用一些多功能线虫模型的方法，包括高活性离子通道诱导坏死（例如，deg-3（d）和mec-4（d）和蛋白质聚合（例如，α-syunclein和多聚谷氨酸）诱导神经毒性，以监测和解剖与年龄相关的神经元分解的细胞和分子基础。这些动物神经退化模型，加上细胞死亡调节剂的遗传和药理学屏幕，将导致对神经元功能与年龄相关的分解的前所未有的理解，并提供与人类健康和生活质量具有广泛相关性的关键见解。

过度活跃的离子通道引起的坏死细胞死亡 使用此处介绍的程序，mec-4（u231）和deg-3u662）突变胚胎要么在34°C下孵育25分钟，要么保持在20°C的标准温度下。孵化后，在两组动物的L1幼虫阶段确定神经元细胞尸体的数量。从热冲击预置卵子中孵化的线虫中，坏死细胞死亡减少（图1A-1B）。
蛋白质聚合诱导神经退...

Watch this Scientific Journal Video about Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans at JoVE.com

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

在这里，我们介绍和描述广泛的方法，利用一些多功能线虫模型，包括高活性离子通道诱导坏死和蛋白质聚合诱导神经毒性，以监测和解剖年龄相关的神经退行性疾病的细胞和分子基础。

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

在过去的几十年里，由于生活水平的提高和医学科学的进步，人类的寿命延长了。年龄是许多威胁生命的疾病的最大危险因素，包括神经退行性疾病，如阿尔茨海默氏症、帕金森氏症和亨廷顿舞蹈症。在这里，我们演示了利用一些通用新方法模型的方法，包括高活性离子通道诱发坏死和蛋白质聚合诱导神经毒性，以监测和解剖神经退化的细胞分子机制。峰值健康幼虫的制作四个，使三个牛顿线虫到线虫生长介质板，与E.Coli使用解剖立体显微镜。放置10和4个线虫，寄生酶板，并在20摄氏度的标准温度下生长。五天后，用1 mL和9缓冲液清洗盘子，用1.5mL管收集动物。在30，000g下离心30秒，取出上一代。添加 0.5 mL 电镀溶液。电镀溶液储存在室温下。漩涡和定期监测，直到它们都溶解。避免电镀时间超过五分钟。在10，000g下离心30秒，取出上一代。用 1 mL 和 9 个缓冲器清洗调色板的两倍。在 10000 g 下离心 30 秒，然后取出上一液。洗涤后，将鸡蛋重新用200微升M9缓冲液中重新冷冻，并在34摄氏度的水浴中孵育25分钟。在20摄氏度下保持一组单独的鸡蛋。移液 100 微升含有控制或热冲击处理鸡蛋，并把它们放在非种子 10 GN 板。每个盘子至少包含100到200个鸡蛋。在20摄氏度下孵育卵子，直到孵化。使用和M9缓冲液清洗板，收集L1寿命和线虫在1.5毫升管。在10，000g下离心30秒。卸下上一点并保留调色板。加入100微升20毫摩尔M9利文缓冲液，麻醉线虫。带10微升的M9悬浮液缓冲器，包含L1表，并安装在2%agar-ls点轻轻地放在样品顶部的盖玻片上。使用 DIC 显微镜观察手表。通过计算具有线虫特征虚拟辅助外观的细胞，扫描630个受体神经元的退化。通过选择并转移15至20个L4幼虫，首先在细菌C和GM板上转移线虫。孵育，让线虫在20摄氏度的标准温度下生长。在34摄氏度的培养箱中移动板，每天进行30分钟的预置。然后在20摄氏度的标准温度下返回预置线虫。在 agar-ls 点的中心添加 10 微升 20 毫摩尔 M9 利文格维缓冲液滴。挑选各自的转基因线虫，并在M9利方滴中转移。每滴放置 20 到 30 个线虫。轻轻地将盖板滑放在样品顶部。用指甲油密封盖滑，在整个成像过程中保持湿度。使用荧光显微镜与相机相结合，以 20 倍的放大率检测和捕获头部区域的研究图像。监测七天的转基因线虫多巴胺神经细胞死亡。测量四天大转基因动物头部区域的神经元政治聚集体，表达与活人融合的240种。坏死细胞死亡诱发超活性离子通道是线虫中最主要的，以前有热休克预体蛋。热休克预置，促进对7天大成人耳塞细胞死亡的预测，减少4天大成人头部区域Q40蛋白质聚集量。衰老是细胞和组织平衡的逐渐退化造成的损害的结果。因此，理解与年龄相关的神经元分解是重中之重。提出的技术与遗传学和药理学屏幕相结合，可能导致正常细胞死亡调节剂与潜在的治疗兴趣的通知。

Research

JoVE Journal

Biology

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Interview: Protein Folding and Studies of Neurodegenerative Diseases

专访：蛋白质折叠与神经退行性疾病的研究

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

科研

生物学

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

建模年龄相关的神经退行性疾病在卡诺哈布迪蒂斯埃利根