Imaging Calpain/Bioenergetic Interactions in Neuronal Excitotoxicity

描述(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氏症。这只是涉及谷氨酸“兴奋性毒性”的几种衰弱病理中的两种，兴奋性毒性是一种由神经递质谷氨酸过度释放导致神经元损伤的机制，从而导致钙渗透型NMDA型谷氨酸受体过度激活。阻断这些受体的药物在临床试验中基本上没有成功，而且有严重的副作用，这突显了寻找替代干预目标的必要性。NMDA受体激活下游发生的两个主要事件是线粒体功能障碍，线粒体是细胞中关键的钙缓冲和能量产生(生物能)细胞器，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希望，但对钙蛋白酶的靶标以及钙蛋白酶是否导致线粒体功能障碍知之甚少。为了实现设计合理的神经疾病疗法的最终目标，了解这些蛋白水解酶何时以及如何在神经退行性变过程中发挥作用是至关重要的。这项研究将检验一个中心假设，即钙激活蛋白在神经元兴奋性毒性所特有的线粒体功能障碍和钙稳态失调中起致病作用。这项研究的目标1中的实验将开发一种灵敏而特异的荧光指示剂，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项新技术的特异性。本研究目标2中的实验将评估谷氨酸刺激的神经元中钙蛋白酶激活的时间进程、程度和因果作用，涉及细胞内钙变化和线粒体功能的变化。将开发两项新技术，这两项技术将广泛推进生物医学研究领域，了解和治疗神经疾病的病理后果。它们将使研究人员能够：1.结合关键生理参数，动态测量完整神经元中破坏性蛋白水解酶的活性；2.可靠地区分受损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.