Elevated Zinc in Ischemia and Reperfusion

描述(申请人提供)：中风是美国第三大致死原因，也是成人致残的主要原因。随着人口老龄化，美国中风患者的数量可能会增加。目前描述兴奋性毒性损伤的模型，如缺血再灌注(I/R)损伤，主要集中在钙离子积聚导致细胞死亡的概念上。由于氧自由基(ROS)的存在和钙离子与ROS之间的相互作用造成的氧化损伤是缺血/再灌注分子生物学中的重要步骤。然而，针对钙稳态和ROS产生的实验性治疗(如钙通道阻滞剂和ROS清除剂)在减少神经元损伤的体积或严重程度方面并不是非常成功。越来越多的证据表明，另一种二价离子锌也参与了脑外伤、癫痫、脑缺血和再灌流后兴奋性毒性神经元的死亡。这项研究背后的总体假设是，I/R的急性神经损伤与细胞内锌离子或锌离子超载增加有关，这可能是细胞死亡或随后的脑变性的先兆。这项建议的具体目标1将检验以下假设：锌离子升高通过促进ROS的产生在神经元氧化损伤中发挥主要作用，以及ROS介导的模拟缺血-葡萄糖剥夺(OGD)和再灌流引起的神经元损伤在很大程度上是锌依赖的。我们将评估钙离子和锌离子在OGD和再灌流过程中对ROS产生的特殊贡献，并表征细胞内锌离子增加与相应的细胞死亡发展的关系。这一目的的意义在于破译和区分锌离子引起的和钙离子引起的ROS产生过程，以便设计针对相关离子的特定治疗干预措施。在特定的目标2中，我们将检验锌离子介导的神经元损伤依赖于其与ROS的相互作用的假设。具体地说，在OGD过程中，功能障碍的线粒体触发ROS的产生，随后加剧了锌离子的积累；然后，升高的锌离子通过激活NADPH氧化酶，在再灌流中放大了ROS的产生。探讨缺氧性脑损伤/再灌流过程中锌蓄积与ROS生成的关系及其机制。在具体目标3中，我们将确定急性脑损伤时锌离子的蓄积来源，并研究线粒体、内质网、高尔基体和溶酶体等细胞内存储的锌离子释放。从贮藏库中释放出的锌离子可能参与了早期锌离子的积累和随后的线粒体功能障碍。该项目的长期目标是阐明神经元损伤的新调节机制，并确定新的治疗方式或康复策略，以预防或减轻急性脑损伤后的神经退行性疾病。

DESCRIPTION (provided by applicant): Stroke is the third leading cause of death in the United States and the leading cause of adult disability. With the aging of the population, the number of stroke patients in the US is likely to grow. Current models describing excitotoxic injury, such as during ischemia and reperfusion (I/R) injury, center on the concept of calcium (Ca2+) ion accumulation leading to cell death. Oxidative damage due to the presence of radical oxygen species (ROS) and cross-talk between Ca2+ and ROS is the essential step in the molecular biology of ischemia/reperfusion. However, experimental treatments targeting Ca2+ homeostasis and ROS generation (e.g. Ca2+ channel blockers and ROS scavengers) have not been very successful in reducing the volume or severity of neuronal damage. Accumulating evidence suggests that another divalent ion zinc (Zn2+) is also involved in excitotoxic neuronal death after head trauma, epilepsy, cerebral ischemia and reperfusion. The Overall Hypothesis behind the proposed research is that acute neural injury in I/R is associated with an increase in intracellular Zn2+ or Zn2+ overload that could be the precursor for cell death or subsequent brain degeneration. The specific aim 1 of this proposal will test the hypothesis that Zn2+ elevation plays a major role in oxidative damage of neuron by facilitating ROS generation, and that ROS mediated neuronal damages in simulated ischemia by oxygen-glucose deprivation (OGD) and reperfusion are largely Zn2+-dependent. We will evaluate the specific contributions made by Ca2+ and Zn2+ towards ROS generation during OGD and reperfusion, and to characterize the relationship between increases of intracellular Zn2+ and corresponding development of cell death. The significance of this aim is to decipher and differentiate Zn2+-caused and Ca2+-caused events in ROS generation, so that specific therapeutic interventions can be designed to target the relevant ion. In specific aim 2 we will test the hypothesis that Zn2+-mediated neuronal injury depends on its interaction with ROS. Specifically, during the OGD, the dysfunctional mitochondria triggers ROS production and subsequently aggravate Zn2+ accumulation; the elevated Zn2+ then amplifies ROS production, by activating NADPH oxidase, in reperfusion. The mechanisms and time course of Zn2+ accumulation in relation to ROS generation during the course of OGD/reperfusion will be explored. In specific aim 3, we will determine the source of Zn2+ accumulation upon acute brain injury, and to study the Zn2+ release from intracellular storages such as mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes. The release of Zn2+ from the storages may contribute to early Zn2+ accumulation and subsequent mitochondrial dysfunction. The long-term goal of the project is to elucidate novel regulatory mechanisms for neuronal injury and to identify new therapeutic modalities or rehabilitation strategies to prevent or attenuate neurodegenerative disorders after acute brain injury.