Imaging Calpain/Bioenergetic Interactions in Neuronal Excitotoxicity

每年有70万美国人患有中风，目前有150万美国人患有中风 帕金森氏症。这些只是涉及谷氨酸的几种衰弱病理中的两种 “兴奋性毒性”，神经递质过度释放导致神经元损伤的一种机制 谷氨酸和随之而来的钙通透性NMDA型谷氨酸受体的过度激活。毒品，即 阻断这些受体在临床试验中很大程度上是不成功的，并有严重的副作用， 强调需要有替代的干预目标。下游发生的两个主要事件 NMDA受体激活是线粒体的功能障碍，是钙缓冲和能量的关键 细胞细胞器的生成(“生物能量”)，以及钙依赖的钙蛋白酶的激活。 尽管钙蛋白酶抑制剂在小动物疾病模型中显示出了早期的前景，但人们对此知之甚少。 关于钙蛋白酶的靶标，以及钙蛋白酶是否导致线粒体功能障碍。至 要达到为神经疾病设计合理疗法的最终目标，关键是要理解 这些蛋白水解酶何时以及如何在神经退化过程中发挥作用。这项研究将考验中央 关于钙调蛋白在线粒体功能障碍和钙调节失调中的作用的假说 动态平衡是神经元兴奋性毒性的特征。本研究目标1中的实验将 开发一种灵敏和特异的荧光指示剂，用于细胞内钙蛋白酶活性的活细胞成像。 基因缺失和RNA干扰方法将严格确立这一新的特异性 技术。研究目标2中的实验将评估时间进程、程度和因果作用 谷氨酸刺激的神经元中钙激活与细胞内钙和钙离子的变化 线粒体功能的改变。将开发两项新技术，广泛推进 了解和治疗神经疾病的病理后果的生物医学研究领域。 它们将使研究人员能够：1.动态测量完整神经元中破坏性蛋白酶的活性 结合关键的生理参数；以及2.可靠地区分线粒体和 受损神经元的质膜电位。本文的研究将为今后的研究提供坚实的基础和有力的支持 用于详细研究神经变性的细胞机制的工具。

Every year 700,000 Americans are afflicted by stroke and 1.5 million Americans currently suffer from Parkinson's Disease. These are just two of several debilitating pathologies involving glutamate "excitotoxicity", a mechanism of neuronal injury caused by excessive release of the neurotransmitter glutamate and consequent overactivation of calcium-permeable NMDA-type glutamate receptors. Drugs that block these receptors have been largely unsuccessful in clinical trials and have serious side effects, underscoring the need for alternative targets of intervention. Two prime events that occur downstream of NMDA receptor activation are dysfunction of mitochondria, the pivotal calcium buffering and energy generating ("bioenergetic") organelle of the cell, and activation of calcium-dependent calpain proteases. Although calpain inhibitors have shown early promise in small animal disease models, very little is known about the targets of calpain proteases and whether calpains contribute to mitochondrial dysfunction. To achieve the eventual goal of designing rational therapeutics for neurodisease, it is critical to understand when and how these proteases function in the neurodegenerative process. This study will test the central hypothesis that calpains play a causative role in the mitochondrial dysfunction and deregulation of calcium homeostasis that are characteristic of neuronal excitotoxicity. The experiments in aim 1 of this study will develop a sensitive and specific fluorescent indicator for live-cell imaging of intracellular calpain activity. Genetic deletion and RNA interference approaches will rigorously establish the specificity of this novel technique. The experiments in aim 2 of the study will assess the time course, extent, and causal role of calpain activation in glutamate-challenged neurons with respect to changes in intracellular calcium and alterations in mitochondrial function. Two new technologies will be developed that will broadly advance the arena of biomedical research in understanding and treating the pathological consequences of neurodisease. They will allow investigators to: 1. Kinetically measure the activity of destructive proteases in intact neurons in conjunction with key physiological parameters; and 2. Reliably distinguish changes in mitochondria and plasma membrane potentials in injured neurons. This study will provide a strong foundation and powerful tools for the detailed investigation of cellular mechanisms underlying neurodegeneration.