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

脑水肿和脑肿胀是缺血性脑卒中的主要并发症。两种分子途径，水通道蛋白- 4（AQP 4）和SUR 1-TRPM 4与水肿有关。通过AQP 4的水运输主要由以下因素控制： 存在于质膜中的AQP 4通道的限速数量，以及通过跨膜 渗透梯度最近报道，在培养的细胞中，AQP 4的表面定位受 Ca 2 +/钙调蛋白（CAM），CAM结合引起AQP 4羧基末端构象变化 驱动细胞表面定位。尽管这一开创性的发现暗示了一种潜在的重要方法， 操纵AQP 4影响脑肿胀，一个主要的知识空白是动态调节机制 AQP 4在体内缺血时的表面定位。我们假设在缺血后的血管周围星形胶质细胞中 通过SUR 1-TRPM 4的Na+流入使Na+/Ca 2+交换器1（NCX 1）转变为“Ca 2+进入模式”（CaEnt-NCX 1）， CaEnt-NCX 1介导的Ca 2+内流激活CAM， AQP 4在末端足中的表面定位促进末端足肿胀、血脑屏障（BBB）功能障碍 和脑肿胀我们理论的核心是新的发现和新的初步数据： 大脑中动脉闭塞（MCAo），SUR 1-TRPM 4（ATP耗竭激活的“Na+通道”）和 NCX 1在血管周围星形胶质细胞终足中上调。在MCAo后的脑切片中，Ca 2+的初步数据 血管周围终足的成像显示，SUR 1-TRPM 4的激活引起终足中Ca 2+的增加， 被SUR 1抑制剂和CaEnt-NCX 1抑制剂阻断。我们的理论的另一个核心是， 开发了一种方法来测量脑切片中AQP 4的表面定位，这是一个重要的发展， 这为研究缺血后脑肿胀提供了一种新的工具。在大脑中动脉闭塞后的脑切片中，初步数据显示 表明AQP 4表面定位因缺血而增加，且这种增加可被预处理阻断 CaEnt-NCX 1抑制。在本项目中，我们将通过以下方式确认并扩展这些初步数据： 以下具体目标：在具体目标1中，利用我们开发的新型小鼠模型， （Slc 8a 1fl/fl;+GFAP-cre/ERT 2小鼠），我们将表征星形胶质细胞NCX 1在缺血后BBB中的作用 功能障碍和脑肿胀在具体目标2中，利用我们独特的方法来量化表面 定位AQP 4在脑切片中，我们将表征星形胶质细胞NCX 1在后MCAo AQP 4表面的作用 本地化在特定目标3中，使用具有转基因表达的PC：：G5-tdT小鼠（GCaMP 5G和tdTomato） 通过pGFAP-cre/ERT 2的调节，我们将描述星形胶质细胞NCX 1在MCAo后血管周围的作用。 足端肿胀和Ca 2+信号传导。该项目主要针对星形胶质细胞末端NCX 1和AQP 4， 提出了一种新的理论，该理论通过将 中间元件-NCX 1-脑水肿新分子机制。我们预计， 从这个项目中产生的将是非常有影响力的，并将具有巨大的翻译潜力。

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.