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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.

项目总结