Role of the LRRC8A-containing volume-regulated anion channel in intraischemic glutamate release and animal stroke outcomes

项目总结(30行) 在美国，缺血性中风是第五大死亡原因，也是导致长期中风的主要原因之一。 成年人的长期残疾。缺血性中风的特征之一是细胞肿胀，主要发生在 星形胶质细胞。为了调节它们的体积，细胞参与体积调节的阴离子通道(Vrac)，这是 氯离子通道可被包括谷氨酸在内的多种有机渗透物质渗透。过度激活 谷氨酸受体引起兴奋性毒性级联反应，最终导致钙介导的神经元损伤。这 肿胀激活的谷氨酸在缺血脑中释放的理论已经存在了几十年， Vrac的抑制剂(他莫昔芬和DCPIB)减少每搏输出量和缺血时谷氨酸的释放。 然而，对vrac的分子同一性和vrac抑制剂的特异性都缺乏了解。 阻碍了这一领域的进一步研究。最近发现vrac由来自LRRC8的蛋白质组成 具有LRRC8A-E亚基的家族已经为使用分子生物学工具进行新的研究打开了大门 特异性靶向vrac和必需的LRRC8A亚基。在以下工作中，我们计划建立LRRC8- 将vrac作为缺血内谷氨酸释放的主要贡献者，并提供了一种机制 非特异性vrac抑制剂的神经保护作用。在初步研究中，我们已经在体外表明 Vrac的异质性群体优先对谷氨酸具有渗透性，且vrac通道很可能 活跃在缺血半暗带，甚至可能是缺血核心。对于拟议的活体实验，我们 将使用转基因LRRC8A小鼠建立星形胶质细胞特异性LRRC8A基因敲除(KO)小鼠模型。 首先，我们将确定vrac抑制剂的神经保护特性是否源于它们对vrac的抑制。 通过测量星形细胞LRRC8A KO小鼠的每搏输出量和行为结果来评估活动 大脑中动脉远端永久性闭塞，这是一个中风的小鼠模型。我们还将测试 利用微透析技术，含LRRC8的vrac是脑缺血后谷氨酸释放的主要途径。 测量星形细胞LRRC8A KO动物的皮质谷氨酸。这项工作将有助于我们更多地了解 关于脑缺血损伤的复杂机制，并在某一天允许 更好的治疗缺血性中风的方法。通过未来两年的拟议工作和培训，我将 磨练我在疾病动物模型方面的知识和技能，并为自己成为一名成功的 医药研究领域的独立科学家。在Ruth L.Kirschstein National的帮助下 研究服务奖(NRSA)个人博士前奖学金，我将能够设计和执行研究 为缺血性卒中治疗建立一个新的靶点，并将非常适合未来的临床试验工作 神经退行性疾病患者的发展。

PROJECT SUMMARY (30 LINES) Ischemic stroke is the fifth leading cause of death in the United States, and one of the leading causes of long- term disability in adults. One of the hallmarks of ischemic stroke is cell swelling, which occurs primarily in astrocytes. In order to regulate their volume, cells engage volume regulated anion channels (VRAC), which are chloride channels that are permeable to a number of organic osmolytes, including glutamate. Overactivation of glutamate receptors causes an excitotoxic cascade that culminates in calcium-mediated neuronal damage. This theory of swelling-activated glutamate release in the ischemic brain has been around for several decades, as inhibitors of VRAC (tamoxifen and DCPIB) reduce stroke volumes and intraischemic glutamate release. However, the lack of both knowledge on the molecular identity of VRAC and specificity of VRAC inhibitors prevented further research in this field. The recent discovery that VRAC is composed of proteins from the LRRC8 family, with subunits LRRC8A-E, has opened the doors for new studies using molecular biology tools to specifically target VRAC and the essential LRRC8A subunit. In the following work, we plan to establish LRRC8- containing VRAC as a primary contributor to intraischemic glutamate release and provide a mechanism for the neuroprotective effects of nonspecific VRAC inhibitors. In preliminary studies, we have shown in vitro that heterogenous populations of VRAC are preferentially permeable to glutamate, and that VRAC channels are likely active in the ischemic penumbra and possibly even the ischemic core. For the proposed in vivo experiments, we will use transgenic floxed LRRC8A mice to develop an astrocyte-specific LRRC8A knockout (KO) mouse model. First, we will determine if the neuroprotective properties of VRAC inhibitors are due to their inhibition of VRAC activity by measuring stroke volumes and behavior outcomes in astrocytic LRRC8A KO mice following permanent distal occlusion of the middle cerebral artery, a mouse model of stroke. We will also test whether LRRC8-containing VRAC is a major pathway for intraischemic glutamate release, using microdialysis techniques to measure cortical glutamate from astrocytic LRRC8A KO animals. This work will help us to understand more about the complex mechanisms of damage in the ischemic brain, and someday allow for the development of better therapeutics for ischemic stroke. Through the proposed work and training over the next two years, I will hone my knowledge and skills in animal models of disease and prepare myself to become a successful, independent scientist in the field of pharmaceutical research. With the help of the Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship, I will be able to design and perform research establishing a new target for ischemic stroke treatment, and will be ideally suited for future work with clinical trial development for patients suffering from neurodegenerative diseases.