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.

项目概要 血脑屏障（BBB）的破坏是缺血性中风的一个标志性特征，会导致随后的 脑损伤。星形胶质细胞在 BBB 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 BBB 损伤之前，导致损伤进展。星形胶质细胞端足形式特化 与 BBB 密切相关的亚细胞区室。星形胶质细胞末足表达多种通道 离子转运蛋白，表明它们在维持离子和渗透稳态方面的特殊功能 和胶质血管信号传导。然而，星形胶质细胞末端离子转运机制的失调及其 对 BBB 损伤和神经退行性变的贡献尚未得到充分研究。生电钠 碳酸氢盐转运蛋白 1 (NBCe1/SLC4A4) 是表达的主要碳酸氢盐 (HCO3-) 转运蛋白 星形胶质细胞中含量丰富，在调节大脑 pH 稳态中发挥重要作用。研究表明 膜去极化、细胞外 HCO3- 和 Na+ 浓度升高，可以刺激转运 导致 HCO3- 和 Na+ 流入以及星形胶质细胞肿胀的活性。在大脑中，NBCe1 表达增加 在缺血性中风的亚急性期，在反应性星形胶质细胞中检测到了蛋白质，这与 随着神经元死亡和神经功能损害的增加。然而，NBCe1 蛋白是否存在于 血管周围反应性星形胶质细胞在中风后血脑屏障功能障碍中的作用仍不清楚。我们的初步 研究表明，在 Nbce1 cKO 小鼠的 GFAP+ 星形胶质细胞中靶向删除 Nbce1（Gfap-CreERT2+/- ；Nbce1fl/fl) 缺血后 1-7 天梗塞体积、脑肿胀和神经功能缺陷减少 中风。免疫细胞化学分析显示 BBB 完整性得到改善，AQP4 极化得以保留 星形胶质细胞，并减少 cKO 中风大脑中神经元细胞（NeuN+ 细胞）的损失。这些新颖的发现 表明 NBCe1 蛋白参与缺血性中风脑损伤，然而，潜在的分子 机制尚不清楚。我们假设 (1) NBCe1 活性的病理刺激有助于 中风引起的 BBB 损伤部分是由于星形胶质细胞 Na+i、pHi 和肿胀失调以及 AQP4 破坏所致 BBB 的丰富。 (2)选择性删除星形细胞Nbce1将保护BBB免受中风诱发的影响 通过保留 AQP4 极性和星形细胞稳态功能来减轻损伤。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中 NBCe1 表达增加是否与 AQP4 相关 中风大脑中的重新分布。选择性删除星形胶质细胞 Nbce1 对星形胶质细胞表型和功能的影响 将研究幼稚大脑和中风大脑的血脑屏障完整性。在目标 2 中，使用原位急性脑切片制剂 通过中风大脑和活细胞荧光成像，我们将确定 NBCe1 的激活及其对 [Na+]i [H+]i，以及 WT 和 Nbce1 cKO 星形胶质细胞的细胞体积变化。然后我们将使用邻近连接 定量 NBCe1-AQP4 相互作用并研究其对 AQP4 极化和 BBB 的影响 损害。这项研究的成功完成将为 NBCe1 蛋白如何发挥作用提供机制见解 在星形胶质细胞末端足部损伤中发挥作用，并导致血脑屏障功能障碍。