Aquaporin-4 regulation by NCX1 in post-ischemic brain swelling

NCX1 对缺血后脑肿胀中水通道蛋白 4 的调节

基本信息

批准号：
10650854
负责人：
J. Marc Simard
金额：
$ 38.63万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10650854
关键词：
Astrocytes Binding Blood Vessels Blood brain barrier dysfunction Brain Brain Edema Brain Ischemia Calmodulin Cause of Death Cell surface Clinical Trials Cultured Cells Data Development Diazoxide Edema Elements Evans blue stain Female Genetic Techniques Glial Fibrillary Acidic Protein Glyburide Hour Image Ischemia Ischemic Stroke Knowledge Laboratories Link Measures Mediating Membrane Methods Middle Cerebral Artery Occlusion Modeling Molecular Molecular Conformation Mus Neurological outcome Osmosis Pathway interactions Play Public Health Regulation Reperfusion Therapy Reporting Role Science Seminal Signal Transduction Slice Surface Swelling Water Work aquaporin 4 brain tissue experimental study in vivo inhibitor male mouse model new therapeutic target novel pharmacologic pre-clinical public health relevance receptor sulfonylurea receptor theories tool transgene expression translational potential

项目摘要

Brain edema and brain swelling are major complications of ischemic stroke. Two molecular pathways, aquaporin- 4 (AQP4), and SUR1-TRPM4, have been linked to edema. Water transport via AQP4 is governed principally by the rate-limiting number of AQP4 channels present in the plasmalemmal membrane, and by the transmembrane osmotic gradient. It was recently reported that in cultured cells, surface localization of AQP4 is regulated by Ca2+/calmodulin (CAM), with CAM binding causing a conformational change in the carboxyl terminus of AQP4 that drives cell-surface localization. Although this seminal discovery hints at a potentially important way to manipulate AQP4 to influence brain swelling, a major gap in knowledge is the mechanism of dynamic regulation of surface localization of AQP4 in ischemia in vivo. We hypothesize that in post-ischemic perivascular astrocyte endfeet, Na+ influx via SUR1-TRPM4 shifts Na+/Ca2+ exchanger 1 (NCX1) into “Ca2+ entry mode” (CaEnt-NCX1), which extrudes Na+ and causes Ca2+ influx, and that CaEnt-NCX1-mediated Ca2+ influx activates CAM to drive surface localization of AQP4 in endfeet that promotes endfoot swelling, blood-brain barrier (BBB) dysfunction and brain swelling. Central to our theory are new findings and new preliminary data: In mouse brain tissues after middle cerebral artery occlusion (MCAo), both SUR1-TRPM4 (a “Na+ channel” activated by ATP depletion) and NCX1 are upregulated in perivascular astrocyte endfeet. In post-MCAo brain slices, preliminary data with Ca2+ imaging of perivascular endfeet show that activation of SUR1-TRPM4 causes an increase in Ca2+ in endfeet that is blocked by inhibitors of SUR1 and by inhibitors of CaEnt-NCX1. Also central to our theory is our recent development of a method to measure the surface localization of AQP4 in brain slices, an important development that provides a novel tool for studying post-ischemic brain swelling. In post-MCAo brain slices, preliminary data indicate that surface localization of AQP4 is increased by ischemia, and the increase is blocked by pretreatment with CaEnt-NCX1 inhibition. In this project, we will confirm and expand on these preliminary data by pursuing the following specific aims: In Specific Aim 1, making use of a novel mouse model we developed (Slc8a1fl/fl;+GFAP-cre/ERT2 mouse), we will characterize the role of astrocyte NCX1 in post-ischemic BBB dysfunction and brain swelling. In Specific Aim 2, making use of our unique method to quantify surface localization of AQP4 in brain slices, we will characterize the role of astrocyte NCX1 in post-MCAo AQP4 surface localization. In Specific Aim 3, using PC::G5-tdT mice with transgene expression (GCaMP5G and tdTomato) regulated by pGFAP-cre/ERT2, we will characterize the role of astrocyte NCX1 in post-MCAo perivascular endfoot swelling and Ca2+ signaling. This project, which is focused on astrocyte endfoot NCX1 and AQP4, advances a novel theory that unifies the SUR1-TRPM4 and AQP4 edema pathways by incorporating an intermediate element – NCX1 – in a novel molecular mechanism of brain edema. We anticipate that the science emerging from this project will be highly impactful, and will have great translational potential.

脑水肿和脑肿胀是缺血性中风的主要并发症。两种分子途径，水通道蛋白 4（AQP4）和SUR1-TRPM4已与水肿相关。通过AQP4的水运输主要由静脉内膜中存在的AQP4通道的速率限速数，并通过跨膜渗透梯度。最近有报道说，在培养的细胞中，AQP4的表面定位受 Ca2+/钙调蛋白（CAM），带有CAM结合，导致AQP4羧基末端的构象变化这驱动细胞表面定位。尽管第二个发现暗示了一种潜在的重要方法操纵AQP4影响脑肿胀，知识的主要差距是动态调节的机制 AQP4在体内缺血中的表面定位。我们假设在缺血后的周围星形胶质细胞中 Endfeet，Na+通过SUR1-TRPM4的影响将Na+/Ca2+交换器1（NCX1）转移到“ Ca2+进入模式”（CAENT-NCX1），挤压Na+并引起Ca2+影响，并且Caent-NCX1介导的Ca2+影响激活CAM驱动凸轮 AQP4的表面定位在Endfeet中促进终脚吞咽，血脑屏障（BBB）功能障碍和大脑肿胀。我们理论的核心是新发现和新的初步数据：在小鼠脑组织中大脑中动脉闭塞（MCAO），均为SUR1-TRPM4（ATP耗竭激活的“ Na+通道”）和 NCX1在血管周围的星形胶质细胞终端中上调。在mcao后大脑切片中，具有CA2+的初步数据血管周末末端的成像表明，SUR1-TRPM4的激活在Endfeet中导致Ca2+的增加被SUR1的抑制剂和CAENT-NCX1的抑制剂阻塞。我们理论的核心也是我们最近的开发一种测量AQP4在脑切片中的表面定位的方法，这是一个重要的发展这为研究后脑肿胀提供了一种新颖的工具。在mcao后大脑切片中，初步数据表明缺血增加了AQP4的表面定位，并且通过预处理阻止了增加 caent-ncx1抑制。在这个项目中，我们将通过追求这些初步数据确认并扩展以下特定目的：在特定目标1中，利用我们开发的新型鼠标模型（SLC8A1FL/FL;+GFAP-CRE/ERT2小鼠），我们将表征星形胶质细胞NCX1在缺血后BBB中的作用功能障碍和脑肿胀。在特定目标2中，利用我们独特的方法来量化表面 AQP4在脑切片中的定位，我们将表征星形胶质细胞NCX1在MCAO后AQP4表面的作用本土化。在特定的目标3中，使用具有转换表达式的PC :: G5-TDT小鼠（GCAMP5G和TDTOMATO）由PGFAP-CRE/ERT2调节，我们将表征星形胶质细胞NCX1在MCAO后血管周围的作用终脚肿胀和Ca2+信号传导。该项目的重点是星形胶质细胞端脚NCX1和AQP4，通过编码一个新的理论，该理论统一了SUR1-TRPM4和AQP4水肿途径中间元素 - NCX1 - 在一种新型的脑水肿分子机制中。我们预计科学该项目的出现将具有很高的影响力，并且将具有巨大的翻译潜力。