Cell Swelling-Activated Chloride Channel in Ischemic Stroke

项目摘要 中风是第三大死亡原因，也是导致严重长期残疾的主要原因， 美国的大约87%的中风被归类为缺血性中风，其发生是由于缺血性中风。 向大脑某个区域供应富氧血液的血管内的阻塞。尽管疗效 溶栓（通过组织纤溶酶原激活剂，t-PA）和血栓切除术（通过手术）的安全性， 由于时间窗狭窄，患者不适合接受治疗。因此，缺血性中风是一个巨大的未满足的 医疗需求;确定新的中风治疗靶点势在必行。星形胶质细胞和神经元肿胀 是许多神经系统疾病的病理标志，包括缺血性中风和创伤性脑 损伤它如何导致发病机制尚不清楚。细胞膨胀激活了体积- 调节阴离子通道（VRAC），通常通过介导 氯离子和有机渗透物的流出，随后释放出有毒的水。然而，在这方面， 大脑中持续的VRAC激活被认为是有害的。例如，VRAC已经 被认为是肿胀的星形胶质细胞释放过量谷氨酸的主要途径， 刺激神经元NMDA受体并引起兴奋性毒性。尽管30年来进行了大量的研究， VRAC的分子身份长期以来是个谜由于这一差距，以前的证据 支持VRAC的病理作用主要基于非特异性药理学抑制剂。我们 开发了一种创新的高通量检测方法，并通过全基因组RNAi筛选， 成功鉴定了一种新的膜蛋白SWELL 1（LRRC 8A）作为唯一的VRAC必需亚基。 我们的初步数据显示，星形胶质细胞中的Swell 1依赖性VRAC直接释放谷氨酸， 从而增强神经元的兴奋性。重要的是，Swell 1星形胶质细胞特异性KO小鼠显著 在短暂性大脑中动脉闭塞（tMCAO）中风模型中免受脑损伤。这 拟议的研究计划将结合联合收割机创新的方法，包括细胞和急性脑切片 电生理学，活细胞成像，高通量化学筛选，细胞类型特异性KO小鼠 模型和体内实验中风模型，以阐明细胞肿胀的重要作用- 激活氯通道在缺血性卒中发病机制中的作用完成拟议的研究将 确立Swell 1通道为中风关键病理介质并提供新的“可药用”离子 通道靶点不仅用于中风，还用于与异常细胞相关的其他神经系统疾病 肿胀.

PROJECT SUMMARY Stroke is the third leading cause of death and also the leading cause of serious long-term disability in the United States. About 87% of all strokes are classified as ischemic, which occurs as a result of an obstruction within a blood vessel supplying oxygen-rich blood to an area of the brain. Despite the efficacy and safety of thrombolysis (by tissue plasminogen activator, t-PA) and thrombectomy (by surgery), most patients are ineligible for treatment due to the narrow time-window. Thus, ischemic stroke is a huge unmet medical need; it's imperative to identify new stroke therapeutic targets. Swelling of astrocytes and neurons is a pathological hallmark of many neurological diseases, including ischemic stroke and traumatic brain injury. How it contributes to the pathogenesis remains unclear. Cell swelling activates the Volume- Regulated Anion Channel (VRAC), which typically facilitates regulatory volume decrease by mediating efflux of chloride and organic osmolytes, followed by release of osmotically obligated water. However, persistent VRAC activation in the brain is thought to be detrimental. For example, VRAC has been proposed to be a major pathway for the excessive glutamate release from swollen astrocytes, which over- stimulates neuronal NMDA receptors and causes excitotoxicity. Despite intense research in 3 decades, the molecular identity of VRAC was a longstanding mystery. Due to this gap, the previous evidence supporting VRAC's pathological role was mainly based on nonspecific pharmacological inhibitors. We developed an innovative high-throughput assay and through a genome-wide RNAi screen, have successfully identified a novel membrane protein SWELL1 (LRRC8A) as the only essential VRAC subunit. Our preliminary data showed that Swell1-dependent VRAC in astrocytes directly releases glutamate, which enhances neuronal excitability. Importantly, Swell1 astrocyte-specific KO mice were significantly protected from brain damage in transient middle cerebral artery occlusion (tMCAO) stroke model. This proposed research program will combine innovative approaches including cell and acute brain slice electrophysiology, live cell imaging, high-throughput chemical screening, cell-type specific KO mouse models, and in vivo experimental stroke models to elucidate the important role of the cell swelling- activated chloride channel in the pathogenesis of ischemic stroke. Completion of the proposed study will establish Swell1 channel as a key pathological mediator in stroke and provide a new “druggable” ion channel target for not only stroke, but also other neurological disease associated with abnormal cell swelling.