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

• 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
• 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

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，水通道蛋白-这两种分子途径。 4 (AQP4) 和 SUR1-TRPM4 与水肿有关，通过 AQP4 的水输送主要受水肿影响。 质膜中存在的 AQP4 通道的限速数量，以及跨膜 最近有报道称，在培养细胞中，AQP4 的表面定位受渗透梯度的调节。 Ca2+/钙调蛋白 (CAM)，CAM 结合导致 AQP4 羧基末端构象变化 尽管这一开创性的发现暗示了一种潜在的重要途径。 操纵AQP4影响脑肿胀，动态调节机制是一大知识空白 我们在缺血后血管周围星形胶质细胞中捕获了 AQP4 在体内缺血中的表面定位。 endfeet，Na+通过SUR1-TRPM4流入将Na+/Ca2+交换器1（NCX1）转变为“Ca2+进入模式”（CaEnt-NCX1）， 它挤出 Na+ 并导致 Ca2+ 流入，CaEnt-NCX1 介导的 Ca2+ 流入激活 CAM 驱动 AQP4 在足底表面定位，促进足底肿胀、血脑屏障 (BBB) 功能障碍 我们理论的核心是新的发现和新的初步数据：在小鼠脑组织中。 大脑中动脉闭塞 (MCAo)，SUR1-TRPM4（ATP 耗尽激活的“Na+ 通道”）和 在 MCAo 后脑切片中，Ca2+ 的初步数据显示，NCX1 在血管周围星形胶质细胞终脚中表达上调。 血管周围末足成像显示，SUR1-TRPM4 的激活导致末足 Ca2+ 增加， 被 SUR1 抑制剂和 CaEnt-NCX1 抑制剂阻断。我们最近的理论也很重要。 开发了一种测量脑切片中 AQP4 表面定位的方法，这是一项重要进展 这为研究 MCAo 后脑切片中的缺血后脑肿胀提供了一种新的工具。 表明 AQP4 的表面定位因缺血而增加，并且这种增加被预处理所阻断 在这个项目中，我们将通过追求确认和扩展这些初步数据。 具体目标如下： 在具体目标 1 中，利用我们开发的新型小鼠模型 （Slc8a1fl/fl；+GFAP-cre/ERT2 小鼠），我们将描述星形胶质细胞 NCX1 在缺血后 BBB 中的作用 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 表面中的作用 在特定目标 3 中，使用具有转基因表达的 PC::G5-tdT 小鼠（GCaMP5G 和 tdTomato）。 由 pGFAP-cre/ERT2 调节，我们将描述星形胶质细胞 NCX1 在 MCAo 后血管周围的作用 该项目专注于星形胶质细胞末端 NCX1 和 AQP4， 提出了一种新理论，通过整合 SUR1-TRPM4 和 AQP4 水肿途径来统一 中间元素——NCX1——在脑水肿的一种新的分子机制中，我们预计科学。 该项目的产生将产生巨大影响，并具有巨大的转化潜力。

项目成果

Brain Swelling versus Infarct Size: A Problematizing Review.

脑肿胀与梗死面积：有问题的回顾。

• 发表时间: 2024-02-28
• 影响因子: 3.3
• 作者: Simard, J Marc;Wilhelmy, Bradley;Tsymbalyuk, Natalya;Shim, Bosung;Stokum, Jesse A;Evans, Madison;Gaur, Anandita;Tosun, Cigdem;Keledjian, Kaspar;Ciryam, Prajwal;Serra, Riccardo;Gerzanich, Volodymyr
• 通讯作者: Gerzanich, Volodymyr

Canagliflozin, an Inhibitor of the Na+-Coupled D-Glucose Cotransporter, SGLT2, Inhibits Astrocyte Swelling and Brain Swelling in Cerebral Ischemia.

Canagliflozin 是 Na 偶联 D-葡萄糖协同转运蛋白 SGLT2 的抑制剂，可抑制脑缺血时的星形胶质细胞肿胀和脑肿胀。

• 发表时间: 2023-09-06
• 影响因子: 6
• 作者: Shim, Bosung;Stokum, Jesse A;Moyer, Mitchell;Tsymbalyuk, Natalya;Tsymbalyuk, Orest;Keledjian, Kaspar;Ivanova, Svetlana;Tosun, Cigdem;Gerzanich, Volodymyr;Simard, J Marc
• 通讯作者: Simard, J Marc