BESTROPHIN AND RETINAL DISEASE

黄斑素和视网膜疾病

• 批准号: 6384911
• 负责人: ALAN D MARMORSTEIN
• 金额: $ 35.8万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384911
• 关键词: autosomal dominant trait; chimeric proteins; electron microscopy; electrophysiology; electroretinography; fluorescence microscopy; gene expression; green fluorescent proteins; histopathology; human tissue; immunoaffinity chromatography; immunoelectron microscopy; in situ hybridization; intracellular transport; iron metabolism; laboratory mouse; laboratory rat; light microscopy; macular degeneration; membrane proteins; northern blottings; organ culture; protein binding; protein structure function; retinal pigment epithelium; visual photosensitivity

Best's macular dystrophy (BMD), a disease with many similarities to age-related macular degeneration (AMD). AMD is the leading cause of blindness in the western world and is currently untreatable. By understanding how mutations in bestrophin cause BMD we will gain insights into the causes of AMD with the expectation that the knowledge gained will lead to effective therapies for this disease. A distinguishing symptom of BMD is a depressed light response in the electro-oculogram without alterations in the electro-retinogram, a condition mimicked by the toxic responses induced by ocular iron overload, and drugs that affect iron metabolism. Thus the studies outlined in this proposal are designed to determine a function for bestrophin guided by the hypothesis that bestrophin is involved in the regulation of iron homeostasis or transcytotic transport by the retinal pigment epithelium (RPE). This hypothesis is reinforced by the presence of several potential iron binding motifs in the amino acid sequence of bestrophin. Specific aims focus on: The subcellular localization and developmental expression of bestrophin; determination of bestrophin function in vitro; determination of bestrophin function in vivo; and biochemical characterization of bestrophin. The localization of bestrophin will be determined by immunofluorescence microscopy using antibodies raised against bestrophin, or bestrophin fused with green fluorescent protein. The function of bestrophin will be tested in assays of iron binding, ferroxidase/ferrireductase activity, and transport and uptake of iron by the RPE. The effects of mutant bestrophin on the phagocytic pathway of RPE cells, such as delayed kinetics of outer segment degradation and altered pH of endosomal/phagosomal compartments will be tested. BMD associated mutants of bestrophin will be expressed in the RPE of mice and rats using adenovirus mediated gene transfer, followed by histologic, electrophysiologic, and biochemical analysis of the effects on lipofuscin accumulation and retinoid processing.

贝斯特黄斑营养不良（BMD），一种与年龄相关性黄斑变性（AMD）有许多相似之处的疾病。 AMD是西方世界失明的主要原因，目前无法治愈。 通过了解bestrophin突变如何导致BMD，我们将深入了解AMD的原因，并期望获得的知识将导致这种疾病的有效治疗。 BMD的一个显著症状是眼电图中的光反应降低，而视网膜电图没有改变，这是一种由眼部铁过载和影响铁代谢的药物诱导的毒性反应所模拟的病症。 因此，本提案中概述的研究旨在确定由以下假设指导的bestrophin的功能：bestrophin参与调节视网膜色素上皮细胞（RPE）的铁稳态或胞吞转运。 这一假设得到加强的存在下，几个潜在的铁结合基序的氨基酸序列的雌激素。 具体目标侧重于：雌激素的亚细胞定位和发育表达;雌激素的体外功能测定;雌激素的体内功能测定;雌激素的生化特性。 使用针对雌激素或与绿色荧光蛋白融合的雌激素产生的抗体，通过免疫荧光显微镜测定雌激素的定位。 将在铁结合、铁氧化酶/铁还原酶活性以及RPE对铁的转运和摄取的测定中检测雌激素样肽的功能。 将测试突变体雌激素对RPE细胞的吞噬途径的影响，例如外节降解的延迟动力学和内体/吞噬体隔室的改变的pH。使用腺病毒介导的基因转移，在小鼠和大鼠的RPE中表达雌激素样蛋白的BMD相关突变体，然后进行对脂褐质积累和类维生素A加工的影响的组织学、电生理学和生物化学分析。