Cell Swelling-Activated Chloride Channel in Ischemic Stroke

项目总结 中风是第三大致死原因，也是导致老年人长期严重残疾的首要原因。 美国。大约87%的中风被归类为缺血性中风，这是由于 血管内的阻塞，为大脑某一区域提供富氧血液。尽管有疗效， 溶栓(通过组织型纤溶酶原激活剂，t-PA)和血栓摘除术(通过手术)的安全性，大多数 由于时间窗口狭窄，患者没有资格接受治疗。因此，缺血性中风是一个巨大的未得到满足的疾病 医疗需要；确定新的中风治疗靶点是当务之急。星形胶质细胞和神经元肿胀 是许多神经疾病的病理标志，包括缺血性中风和创伤性脑 受伤。它在发病机制中的作用尚不清楚。细胞肿胀激活体积- 调节阴离子通道(Vrac)，它通常通过中介促进调节量的降低 外流氯化物和有机渗透分子，随后释放渗透压所需的水。然而， 大脑中持续的vrac激活被认为是有害的。例如，vrac已被 被认为是肿胀的星形胶质细胞过度释放谷氨酸的主要途径，这种过度- 刺激神经元NMDA受体并引起兴奋性毒性。尽管在30年中进行了密集的研究， Vrac的分子身份是一个长期存在的谜。由于这一差距，以前的证据 支持vrac的病理作用主要是基于非特异性的药物抑制剂。我们 开发了一种创新的高通量分析方法，并通过全基因组RNAi筛选， 成功地鉴定出一个新的膜蛋白SWELL1(LRRC8A)是唯一必需的vrac亚基。 我们的初步数据显示，星形胶质细胞中依赖Swell1的vrac直接释放谷氨酸， 这增强了神经元的兴奋性。重要的是，Swell1星形胶质细胞特异性KO小鼠 在短暂性大脑中动脉闭塞(TMCAO)卒中模型中保护脑损伤。这 拟议的研究计划将结合创新方法，包括细胞和急性脑片 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，以及体内实验性中风模型，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。建议的研究完成后， 建立Swell1通道作为卒中的关键病理介质并提供新的“可用药”离子 不仅是中风，还有其他与异常细胞相关的神经系统疾病的经络靶点 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.