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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要血脑屏障的破坏（BBB）是缺血性中风的标志性特征，随后导致脑损伤。星形胶质细胞在BBB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损害，导致损伤进展。星形胶质细胞终身制形式专业与BBB密切关联的亚细胞室。星形胶质细胞表达几个渠道，离子转运蛋白表明其在维持离子和渗透稳态方面的专业功能和神经血管信号。但是，星形胶质细胞端膜离子运输机制的失调及其了解对BBB损伤和神经退行性的贡献。电源钠碳酸氢盐转运蛋白1（NBCE1/SLC4A4）是表达的主要碳酸氢盐（HCO3-）转运蛋白绝对在星形胶质细胞中在调节脑pH稳态的调节中起着重要作用。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+影响的活性以及星形胶质细胞肿胀。在大脑中，NBCE1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蛋白质，该蛋白质相关随着神经元死亡和神经功能障碍的增加。但是，NBCE1蛋白是否在血管周围反应性星形胶质细胞在中风后在BBB功能障碍中起作用。我们的初步研究表明，NBCE1 CKO小鼠的GFAP+星形胶质细胞中NBCE1的靶向缺失（GFAP-CREERT2 +/-- ; nbce1fl/fl）减少梗塞体积，脑肿胀和神经功能在缺血后的1-7天定义中风。免疫细胞化学分析显示BBB完整性提高，并保留AQP4极化在星形胶质细胞和CKO中风大脑中神经元细胞（Neun+细胞）的损失中。这些新颖的发现表明NBCE1蛋白参与缺血性中风脑损伤，但是，潜在的分子机制是未知的。我们假设（1）NBCE1活性的病理刺激有助于中风引起的BBB损害部分通过使星形胶质细胞NA+I，PHI和肿胀和破坏AQP4而损害 BBB的富集。（2）星形胶质细胞NBCE1的选择性删除将保护BBB免受中风诱导的通过保留AQP4极性和星形胶质稳态功能来损害。为了研究这一点，在AIM 1中，我们将调查反应性星形胶质细胞终端中NBCE1的表达是否与AQP4有关中风大脑的重新分布。选择性删除星形胶质细胞NBCE1对星形胶质细胞表型和幼稚和中风大脑中的BBB完整性将研究。在AIM 2中，使用原位急性脑切片制剂从中风大脑和活细胞荧光成像中，我们将确定NBCE1激活及其对其对 [Na+] I [H+] I，WT和NBCE1 CKO星形胶质细胞的细胞体积变化。然后，我们将使用接近连接测定量化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提供有关NBCE1蛋白如何如何的机械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。