Imaging Calpain/Bioenergetic Interactions in Neuronal Excitotoxicity

描述（由申请人提供）：每年有70万美国人患有中风，目前有150万美国人患有帕金森病。这些只是涉及谷氨酸"兴奋性毒性"的几种衰弱性病理中的两种，谷氨酸"兴奋性毒性"是由神经递质谷氨酸的过度释放和随后的钙渗透性NMDA型谷氨酸受体的过度激活引起的神经元损伤机制。阻断这些受体的药物在临床试验中基本上不成功，并且具有严重的副作用，这强调了对替代干预靶点的需求。在NMDA受体活化下游发生的两个主要事件是线粒体功能障碍和钙依赖性钙蛋白酶蛋白酶的活化，线粒体是细胞的关键钙缓冲和能量产生（"生物能"）细胞器。尽管钙蛋白酶抑制剂在小动物疾病模型中显示出早期的前景，但对钙蛋白酶蛋白酶的靶点以及钙蛋白酶是否有助于线粒体功能障碍知之甚少。为了实现设计合理的神经疾病治疗方法的最终目标，关键是要了解这些蛋白酶在神经退行性过程中何时以及如何发挥作用。这项研究将测试的核心假设，钙蛋白酶在线粒体功能障碍和钙稳态失调，神经元兴奋性毒性的特点发挥了致病作用。本研究的目的一是开发一种灵敏、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干扰方法将严格建立这种新技术的特异性。本研究目的2中的实验将评估谷氨酸激发神经元中钙蛋白酶激活的时间过程、程度和因果作用，以及细胞内钙的变化和线粒体功能的改变。将开发两项新技术，广泛推进生物医学研究的竞技场，以了解和治疗神经疾病的病理后果。他们将允许调查人员：1。结合关键生理参数动态测量完整神经元中破坏性蛋白酶的活性;以及2.可靠地区分损伤神经元中线粒体和质膜电位的变化。这项研究将为详细研究神经退行性变的细胞机制提供坚实的基础和有力的工具。

DESCRIPTION (provided by applicant): 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.