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

含有 LRRC8A 的容量调节阴离子通道在缺血内谷氨酸释放和动物中风结果中的作用

• 批准号: 10056171
• 负责人: Corinne Sage Wilson
• 金额: $ 4.55万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-11 至 2021-09-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10056171
• 关键词: Adult; Alteplase; Animal Disease Models; Animal Model; Animals; Anions; Astrocytes; Back; Behavioral; Blood Vessels; Blood flow; Brain; Brain Injuries; Calcium; Cause of Death; Cells; Chloride Channels; Clinical; Clinical Trials; Complex; Data; Development; Distal; Exclusion Criteria; FDA approved; Family; Fellowship; Future; Glial Fibrillary Acidic Protein; Glutamate Receptor; Glutamates; Glycolysis; Goals; Hour; Human; Immunohistochemistry; Impairment; In Vitro; Individual National Research Service Award; Injury; Ischemic Brain Injury; Ischemic Penumbra; Ischemic Stroke; Knock-out; Knockout Mice; Knowledge; Learning; Leucine-Rich Repeat; Link; LoxP-flanked allele; Measures; Mediating; Messenger RNA; Metabolic; Microdialysis; Middle Cerebral Artery Occlusion; Modeling; Modernization; Molecular; Molecular Biology; Motor; Mus; Neurodegenerative Disorders; Neuroglia; Neurons; Neurotransmitters; Pathologic; Pathway interactions; Patients; Permeability; Pharmacologic Substance; Pharmacology; Pharmacotherapy; Play; Population; Process; Property; Proteins; Publishing; Reporting; Research; Rodent Model; Role; Route; Scientist; Sensory; Specificity; Stains; Stroke; Stroke Volume; Swelling; Tamoxifen; Techniques; Testing; Therapeutic; Time; Tissues; Training; Transgenic Organisms; United States; Western Blotting; Work; acute stroke; behavioral outcome; channel blockers; clinical practice; design; disability; excitotoxicity; experimental study; glutamatergic signaling; improved outcome; in vivo; inhibitor/antagonist; knockout animal; leucine-rich repeat protein; member; middle cerebral artery; mouse model; new therapeutic target; post stroke; pre-doctoral; prevent; sex; side effect; skills; stroke model; stroke outcome; stroke patient; stroke therapy; theories; tool; translational scientist; treatment strategy

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.

项目概要（30行） 缺血性中风是美国第五大死亡原因，也是长期死亡的主要原因之一。 成年人的长期残疾。缺血性中风的特征之一是细胞肿胀，其主要发生在 星形胶质细胞。为了调节它们的体积，细胞参与体积调节阴离子通道（VRAC），其是 氯离子通道可渗透许多有机渗透剂，包括谷氨酸盐。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 缺血性脑中肿胀激活谷氨酸释放的理论已经存在了几十年， VRAC抑制剂（他莫昔芬和DCPIB）减少每搏输出量和缺血内谷氨酸释放。 然而，缺乏对VRAC分子特性和VRAC抑制剂特异性的了解， 阻止了这一领域的进一步研究。最近发现VRAC由来自LRRC 8的蛋白质组成， 具有亚基LRRC 8A-E的家族，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LRRC 8A亚基。在接下来的工作中，我们计划建立LRRC 8- 含有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，我们已经在体外证明， VRAC的异质群体优先渗透谷氨酸，且VRAC通道可能 在缺血半暗带甚至可能在缺血核心中具有活性。对于提议的体内实验，我们 将使用转基因floxed LRRC 8A小鼠来开发星形胶质细胞特异性LRRC 8A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是由于其抑制VRAC 通过测量星形胶质细胞LRRC 8A KO小鼠的每搏输出量和行为结果， 大脑中动脉永久远端闭塞，中风小鼠模型。我们还将测试 利用微透析技术，含LRRC 8的VRAC是缺血内谷氨酸释放的主要途径 以测量来自星形胶质细胞LRRC 8A KO动物的皮质谷氨酸。这项工作将有助于我们了解更多 关于缺血性脑损伤的复杂机制，并有一天允许发展 更好的治疗缺血性中风的方法。通过未来两年的工作和培训，我将 磨练我在疾病动物模型方面的知识和技能， 独立的科学家在制药研究领域。在Ruth L. Kirschstein国家 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 建立了缺血性卒中治疗的新靶点，非常适合未来的临床试验工作 为患有神经退行性疾病的患者开发。