SLC4A11 Mitochondrial Uncoupling and ROS Production in Corneal Endothelium

SLC4A11 角膜内皮线粒体解偶联和 ROS 产生

• 批准号: 10393579
• 负责人: Joseph Aurelio Bonanno
• 金额: $ 41.2万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10393579
• 关键词: Address; Aging; Amino Acids; Ammonia; Antioxidants; Apical; Apoptosis; Autophagocytosis; Back; Bicarbonates; Blindness; Buffers; Carcinoma; Catabolism; Cell Density; Cell Line; Cell membrane; Cells; Citric Acid Cycle; Colon Carcinoma; Consumption; Cornea; Corneal Endothelium; Corneal dystrophy; Corneal edema; Data; Defect; Down-Regulation; Electron Transport; Endothelial Cells; Endothelium; Feeds; Fuchs' Endothelial Dystrophy; Functional disorder; Genes; Glutamates; Glutamine; Goals; Human; Hydration status; In Vitro; Inflammation; Inherited; Knock-out; Knockout Mice; Laboratories; Lactate Transporter; Lead; Malignant Epithelial Cell; Measurement; Measures; Medical; Membrane Potentials; Membrane Proteins; Membrane Transport Proteins; Metabolism; Mitochondria; Mitochondrial Membrane Protein; Modeling; Mus; Mutation; NADP; Na(+)-K(+)-Exchanging ATPase; Oryctolagus cuniculus; Pathology; Phenotype; Physiology; Production; Property; Proteins; Pump; Reactive Oxygen Species; Reduced Glutathione; Role; Source; Stimulus; Taurine; Testing; Tissues; Transfection; Transplantation; Trauma; UCP2 protein; Variant; Vision; Work; basolateral membrane; carbonate dehydratase; cell type; in vivo; insight; loss of function; mitochondrial dysfunction; mitochondrial membrane; mouse model; novel; prevent; therapy development; transcriptome sequencing

“SLC4A11 Mitochondrial Uncoupling and ROS Production in Corneal Endothelium” ABSTRACT Defects in the gene SLC4A11 cause Congenital Hereditary Endothelial Dystrophy and some forms of Fuchs Dystrophy. The goal of this study is to understand the role of this membrane transporter in normal corneal endothelial metabolism and how SLC4A11 deficiency leads to Corneal Dystrophy. The corneal endothelial “pump” maintains corneal hydration and transparency. When the “pump” fails due to trauma, inflammation, ageing, or dystrophy, corneal edema ensues, transparency is lost, and vision is significantly degraded. The usual therapy is transplantation, which is not without significant compromises and complications. A hallmark of Corneal Endothelial Dystrophies is mitochondrial dysfunction. Our laboratory has shown that SLC4A11 is an NH3 dependent electrogenic H+ transporter. We have found that glutamine is actively metabolized by the endothelium producing NH3 and enhancing ATP formation. Slc4a11 knock out shows significant corneal edema, lactate accumulation, altered mitochondrial physiology, and ROS. These data have led to the overarching hypothesis that corneal endothelium actively metabolize glutamine and that the absence of SLC4A11 alters glutamine metabolism, leading to mitochondrial dysfunction, ROS, and eventual apoptosis. Preliminary data indicate that SLC4A11 is both a plasma membrane and a mitochondrial membrane protein, leading to the novel hypothesis that SLC4A11 is a mitochondrial uncoupler. Using multiple in vitro & in vivo complementary approaches these hypotheses will be tested in three aims. Aim 1 will determine how glutamine metabolism is facilitated in Corneal Endothelial mitochondria. The hypothesis is that Slc4a11 is an NH3 sensitive mitochondrial uncoupler that works in conjunction with Uncoupling Protein-2 and the potential mitochondrial buffer taurine to facilitate Glutamine catabolism. Aim 2 will examine the source of ROS and ROS as a stimulus to Apoptosis in Slc4a11 KO. Our hypothesis is that apoptosis is accelerated by ROS, which is generated by the interaction of NH3 with an energized electron transport chain and reduced by SLC4A11 uncoupling. Aim 3 will identify the cause of corneal edema in Slc4a11 KO Mice. We will test the hypothesis that loss of Slc4a11 secondarily induces downregulation of key proteins that facilitate lactate transport. Completion of this study will establish the role of SLC4A11 and glutamine in endothelial metabolism; provide new insight for mechanisms that facilitate glutamine metabolism yet alleviate NH3 induced ROS production that will be transferable to a wide array of glutamine metabolizing tissues; and provide insight for development of therapies for endothelial dystrophies.

“SLC 4A 11线粒体解偶联和角膜内皮细胞中的ROS产生” 摘要 基因SLC 4A 11的缺陷导致先天性遗传性内皮营养不良和某些形式的 富克斯营养不良本研究的目的是了解这种膜转运蛋白在正常角膜中的作用。 内皮代谢以及SLC 4A 11缺陷如何导致角膜营养不良。角膜内皮 “泵”保持角膜水合和透明度。当“泵”因创伤、炎症而失效时， 老化或营养不良，角膜水肿，透明度丧失，视力显著下降。通常的 治疗是移植，这不是没有重大的妥协和并发症。角膜的标志 内皮营养不良是线粒体功能障碍。我们的实验室已经表明，SLC 4A 11是一种NH3 依赖性生电H+转运蛋白。我们已经发现谷氨酰胺被内皮细胞积极代谢 产生NH3并增强ATP形成。Slc 4a 11敲除显示显著的角膜水肿，乳酸 积累，改变线粒体生理学和ROS。这些数据导致了一个总体假设 角膜内皮积极代谢谷氨酰胺，SLC 4A 11的缺失改变了谷氨酰胺 代谢，导致线粒体功能障碍，ROS和最终的凋亡。初步数据表明 SLC 4A 11既是一种质膜蛋白，又是一种线粒体膜蛋白，这导致了新的假说 SLC 4A 11是线粒体解偶联剂使用多种体外和体内互补方法， 假设将在三个目标进行测试。目的1将确定谷氨酰胺代谢是如何促进角膜 内皮线粒体。假设Slc 4a 11是一种NH3敏感的线粒体解偶联剂， 与解偶联蛋白-2和潜在的线粒体缓冲剂牛磺酸一起促进谷氨酰胺 猫目的2将检查ROS的来源和ROS作为Slc 4a 11 KO中细胞凋亡的刺激物。我们 一种假设是，细胞凋亡是由ROS加速的，ROS是由NH3与一种 激发电子传递链，并通过SLC 4A 11解偶联还原。目的3将确定角膜炎的原因 Slc 4a 11 KO小鼠的水肿。我们将检验Slc 4a 11的缺失继发性诱导下调的假设 促进乳酸盐运输的关键蛋白质。这项研究的完成将确定SLC 4A 11的作用， 谷氨酰胺在内皮细胞代谢中作用，为谷氨酰胺代谢机制提供新的认识 还减轻NH3诱导的ROS产生，其将被转移到广泛的谷氨酰胺代谢中， 组织;并为开发内皮营养不良的疗法提供见解。