Smith Graham S T, Voyer-Grant Janine A M, Harauz George
Department of Molecular and Cellular Biology, University of Guelph, Berlgium.
J Vis Exp. 2012 Jan 13(59):3422. doi: 10.3791/3422.
The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis. In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience), and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation. We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein). Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.
中枢神经系统会经历多种应激和神经损伤，这些损伤会产生许多不良影响，最终导致神经元数量和功能减少。受损的轴突会向细胞外基质释放兴奋性分子，包括钾离子或谷氨酸，这反过来又会对包括星形胶质细胞和少突胶质细胞在内的支持性神经胶质细胞造成进一步的损伤。如果损伤持续存在，细胞将经历程序性细胞死亡（凋亡），这由许多成熟的信号转导级联反应调节和激活。创伤性脑损伤、脑缺血和癫痫发作后可发生凋亡和组织坏死。凋亡调节的一个经典例子是半胱氨酸依赖性天冬氨酸定向蛋白酶家族，即胱天蛋白酶。包括胱天蛋白酶在内的活化蛋白酶也与包括阿尔茨海默病、亨廷顿病和多发性硬化症在内的慢性神经退行性疾病中的细胞死亡有关。在本实验方案中，我们描述了使用NucView 488胱天蛋白酶-3底物来测量永生化N19-少突胶质细胞（OLG）细胞培养物中胱天蛋白酶-3介导的凋亡率，该细胞培养物在暴露于不同的细胞外应激，如高浓度的钾离子或谷氨酸后。条件永生化N19-OLG细胞系（代表O2A祖细胞）由安东尼·坎帕尼奥尼博士（加州大学洛杉矶分校塞梅尔神经科学研究所）提供，此前已用于研究髓鞘基因表达和导致OLG分化的信号转导的分子机制。我们发现该细胞系在用与RFP或GFP（红色或绿色荧光蛋白）融合的外源性髓鞘碱性蛋白（MBP）构建体转染时表现稳定。在这里，用80 mM氯化钾或100 mM谷氨酸钠处理N19-OLG细胞培养物，以模拟轴突渗漏到细胞外基质中诱导凋亡。我们使用了一种双功能胱天蛋白酶-3底物，它含有一个DEVD（天冬氨酸-谷氨酸-缬氨酸-天冬氨酸）胱天蛋白酶-3识别亚基和一种DNA结合染料。该底物迅速进入细胞质，在那里被细胞内的胱天蛋白酶-3切割。染料NucView 488被释放并进入细胞核，在那里它与DNA结合并在488 nm处发出绿色荧光，表明细胞发生凋亡。使用NucView 488胱天蛋白酶-3底物可进行实时活细胞成像。在本视频中，我们还描述了永生化N19-OLG细胞的培养和转染，以及活细胞成像技术。
