Astrocytic NBCe1 in regulation of blood brain barrier integrity

项目摘要 血脑屏障（BBB）的破坏是缺血性卒中的标志性特征，其导致随后的脑缺血性卒中。 脑损伤星形胶质细胞在血脑屏障调节中起重要作用，研究表明星形胶质细胞功能障碍 通常先于BBB损伤，其导致损伤进展。星形胶质细胞终足形成专门的 与BBB密切相关的亚细胞区室。星形胶质细胞终足表达几个通道， 离子转运蛋白，表明它们在维持离子和渗透稳态中的特殊功能 和神经胶质血管信号。然而，星形胶质细胞终足离子转运机制的失调及其对细胞增殖的抑制作用可能与星形胶质细胞终足的功能有关。 对BBB损伤和神经退行性变的贡献尚未得到充分研究。产电钠 碳酸氢盐转运蛋白1（NBCe 1/SLC 4A 4）是主要的碳酸氢盐（HCO 3-）转运蛋白， 大量存在于星形胶质细胞中，在调节脑pH稳态中起重要作用。研究表明 膜去极化、细胞外HCO 3-和Na+浓度升高可刺激转运 活性导致HCO 3-和Na+内流，以及星形胶质细胞肿胀。在大脑中，NBCe 1的表达增加 在缺血性卒中的亚急性期，在反应性星形胶质细胞中检测到一种蛋白， 神经元死亡和神经功能损害增加。然而，NBCe 1蛋白是否在 血管周围反应性星形胶质细胞在卒中后BBB功能障碍中的作用仍不清楚。我们的初步 研究显示，在Nbce 1 cKO小鼠中，GFAP+星形胶质细胞中Nbce 1的靶向缺失（Gfap-CreERT 2 +/- 在缺血后1-7天，NBCE 1 fl/fl）可减少梗死体积、脑肿胀和神经功能缺损 中风免疫细胞化学分析显示改善的BBB完整性，和AQP 4极化的保存 在星形胶质细胞中，减少cKO中风脑中神经元细胞（NeuN+细胞）的损失。这些新发现 提示NBCe 1蛋白参与缺血性脑卒中脑损伤，然而， 机制不明。我们假设（1）NBCe 1活性的病理性刺激有助于 脑卒中诱导的血脑屏障损伤部分是由于星形胶质细胞Na+i、pHi和肿胀失调以及AQP 4破坏所致 在BBB富集。(2)选择性缺失星形胶质细胞Nbce 1将保护BBB免受脑卒中诱导的 保护AQP 4极性和星形胶质细胞稳态功能。为了研究这一点，在目标1中，我们将 研究反应性星形胶质细胞终足中NBCe 1表达增加是否与AQP 4相关 中风脑内的再分布选择性缺失星形胶质细胞Nbce 1对星形胶质细胞表型的影响 将研究初治和卒中脑中的BBB完整性。在目标2中，使用原位急性脑切片制备 从中风脑和活细胞荧光成像，我们将确定NBCe 1激活及其对脑缺血的影响。 [Na+]i [H+]i和WT和Nbce 1 cKO星形胶质细胞中的细胞体积变化。然后我们将使用邻位连接 定量NBCe 1-AQP 4相互作用并研究其对AQP 4极化和BBB的影响的测定 损害这项研究的成功完成将为NBCe 1蛋白如何在体内发挥作用提供机制上的见解。 在星形胶质细胞末端损伤中起作用，并导致BBB功能障碍。

PROJECT SUMMARY Disruption of blood brain barrier (BBB) is a hallmark feature of ischemic stroke that leads to subsequent brain damage. Astrocytes play important roles in BBB regulation and studies show that astrocyte dysfunction often precedes BBB damage that contributes to injury progression. The astrocyte endfeet form specialized subcellular compartments that closely associates with BBB. Astrocytic endfeet express several channels and ion transporters, indicating their specialized functions in the maintenance of ionic and osmotic homeostasis and gliovascular signaling. However, dysregulation of astrocytic endfeet ion transport mechanisms and their contribution to the BBB damage and neurodegeneration are understudied. The electrogenic sodium bicarbonate transporter 1, (NBCe1/SLC4A4), is the major bicarbonate (HCO3-) transporter expressed abundantly in astrocytes that play important roles in regulation of brain pH homeostasis. Studies indicate that membrane depolarization, elevated extracellular HCO3-, and Na+ concentrations, can stimulate transport activity leading to HCO3- and Na+ influx, and astrocyte cell swelling. In brain, increased expression of NBCe1 protein has been detected in reactive astrocytes during sub-acute phase of ischemic stroke, which correlates with increased neuronal death and neurological functional impairment. However, whether NBCe1 protein in perivascular reactive astrocytes plays a role in BBB dysfunction after stroke remains unknown. Our preliminary studies revealed that targeted deletion of Nbce1 in GFAP+ astrocytes in Nbce1 cKO mice (Gfap-CreERT2+/- ;Nbce1fl/fl) reduced infarct volume, brain swelling and neurological function deficits at 1-7 days after ischemic stroke. Immunocytochemical analysis showed improved BBB integrity, and preservation of AQP4 polarization in astrocytes and reduced loss of neuronal cells (NeuN+ cells) in the cKO stroke brains. These novel findings suggest that NBCe1 protein is involved in ischemic stroke brain injury, however, the underlying molecular mechanisms are unknown. We hypothesize that (1) pathological stimulation of NBCe1 activity contributes to stroke-induced BBB damage in part by dysregulating astrocyte Na+i, pHi and swelling and disrupting AQP4 enrichment at the BBB. (2) selective deletion of astrocytic Nbce1 will protect BBB against the stroke-induced damage by preserving AQP4 polarity and astrocytic homeostatic functions. To study this, in Aim 1, we will investigate whether increased NBCe1 expression in reactive astrocyte endfeet is associated with AQP4 redistribution in stroke brain. The impact of selective deletion of astrocytic Nbce1 on astrocyte phenotypes and BBB integrity in naïve and stroke brains will be studied. In Aim 2, using in situ acute brain slice preparations from stroke brains and live cell fluorescence imaging, we will determine the NBCe1 activation and its effect on [Na+]i [H+]i, and cell volume changes in WT and Nbce1 cKO astrocytes. We will then use proximity ligation assay to quantify the NBCe1-AQP4 interactions and study its effect on AQP4 polarization and the BBB damage. Successful completion of this study will provide mechanistic insights into how NBCe1 protein could play a role in astrocyte end feet damage and contributes to BBB dysfunction.