Astrocytic NBCe1 in regulation of blood brain barrier integrity

星形胶质细胞 NBCe1 对血脑屏障完整性的调节

• 批准号: 10810958
• 负责人: Gulnaz Begum
• 金额: $ 42.84万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-11 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10810958
• 关键词: Acute; Astrocytes; Basement membrane; Bicarbonates; Biochemical; Biological Assay; Blood - brain barrier anatomy; Blood Vessels; Blood brain barrier dysfunction; Brain; Brain Edema; Brain Injuries; Cause of Death; Cell Volumes; Cells; Central Nervous System; Endothelial Cells; Equilibrium; Functional disorder; Glial Fibrillary Acidic Protein; Goals; Homeostasis; Immunofluorescence Immunologic; Impairment; In Situ; Infarction; Injury; Ion Transport; Ions; Ischemic Stroke; Knockout Mice; Ligation; Link; Maintenance; Membrane; Membrane Potentials; Molecular; Mus; Nerve Degeneration; Nervous System Physiology; Neurologic; Osmosis; Pathologic; Pericytes; Phase; Phenotype; Physiological; Play; Preparation; Proteins; Regulation; Rest; Role; Signal Transduction; Slice; Sodium Bicarbonate; Stains; Stroke; Structure; Swelling; Technology; Testing; Tissues; Transmission Electron Microscopy; aquaporin 4; blood-brain barrier disruption; conditional knockout; disability; extracellular; fluorescence imaging; foot; functional disability; improved; insight; neural; neuron loss; novel; pH Homeostasis; post stroke; preservation; response; stroke model

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.

项目总结 血脑屏障(BBB)的破坏是缺血性卒中的一个显著特征，它会导致随后的 脑部受损。星形胶质细胞在血脑屏障调节中发挥重要作用，研究表明星形胶质细胞功能障碍 通常发生在导致损伤进展的血脑屏障损伤之前。星形胶质细胞末端足形成特化 与血脑屏障密切相关的亚细胞室。星形细胞末端脚表达几种渠道和 离子转运体，表明其在维持离子和渗透平衡方面的特殊功能 和神经胶质血管信号。然而，星形细胞端足离子转运机制的调节失调及其 对血脑屏障损伤和神经退行性变的作用尚未得到充分研究。生电钠 碳酸氢盐转运蛋白1(NBCe1/SLC4A4)是表达碳酸氢盐(HCO3-)的主要转运蛋白 大量存在于星形胶质细胞中，在调节大脑pH动态平衡方面发挥重要作用。研究表明， 膜去极化，细胞外HCO3-和Na+浓度升高，可以刺激运输 活性导致HCO3-和Na+内流，星形胶质细胞肿胀。在脑内，NBCe1的表达增加 在缺血性中风的亚急性期，已在反应性星形胶质细胞中检测到蛋白质，这与 神经元死亡和神经功能损害增加。然而，NBCe1蛋白是否存在于 血管周围反应性星形胶质细胞在卒中后血脑屏障功能障碍中的作用尚不清楚。我们的预赛 研究表明，在Nbce1 CKO小鼠GFAP+星形胶质细胞中靶向缺失Nbce1(GFAP-CreERT2+/- (Nbce1fl/fl)减少脑缺血后1-7天的脑梗塞体积、脑肿胀和神经功能缺失 卒中。免疫细胞化学分析显示血脑屏障完整性得到改善，AQP4极化得以保留 CKO卒中大鼠脑内星形胶质细胞减少，神经细胞(Neun+细胞)减少。这些新发现 提示NBCe1蛋白参与了缺血性卒中脑损伤，然而，潜在的分子 机制尚不清楚。我们假设(1)NBCe1活性的病理性刺激有助于 卒中致血脑屏障损伤部分通过调节星形胶质细胞Na+i、Phi和肿胀及破坏AQP4 在BBB进行浓缩。(2)选择性缺失星形细胞Nbce1基因将保护血脑屏障免受卒中的影响 通过保留AQP4极性和星形细胞内稳功能造成的损伤。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NBCe1表达增加是否与AQP4相关 卒中后脑组织的再分布。星形胶质细胞Nbce1选择性缺失对星形胶质细胞表型和功能的影响 幼稚和中风大脑中的血脑屏障完整性将被研究。在目标2中，使用原位急性脑片制备 从卒中脑和活细胞荧光成像中，我们将确定NBCe1的激活及其对 [Na+]i[H+]i和细胞体积的变化。然后我们将使用近距离结扎术 NBCe1-AQP4相互作用的定量测定及其对AQP4极化和血脑屏障的影响 损坏。这项研究的成功完成将为NBCe1蛋白如何 在星形胶质细胞末端足部损伤中起作用，并导致血脑屏障功能障碍。