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.

Best的黄斑营养不良（BMD），这种疾病与与年龄相关的黄斑变性（AMD）有许多相似之处。 AMD是西方世界失明的主要原因，目前无法治疗。 通过了解Bestrophin中的突变如何导致BMD，我们将深入了解AMD的原因，并期望获得的知识将导致对该疾病的有效疗法。 BMD的一个区别症状是电录制图中的光反应抑郁症，而没有改变电回图的改变，这是由眼铁超载引起的有毒反应模仿的疾病，以及影响铁代谢的药物。 因此，该提案中概述的研究旨在确定Bestrophin的功能，这是由Bestrophin参与视网膜色素上皮（RPE）的铁稳态或转胞膜转运的假设。 通过在Bestrophin的氨基酸序列中存在几种潜在的铁结合基序来加强该假设。 具体目的重点是：Bestrophin的亚细胞定位和发育表达；在体外确定BESTRophin功能；确定体内BESTROPHIN功能；和Bestrophin的生化特征。 Bestrophin的定位将通过使用针对Bestrophin的抗体或与绿色荧光蛋白融合的抗体来确定的免疫荧光显微镜。 将在铁结合，铁氧化酶/铁糖酶活性以及RPE对铁的转运和摄取中测定Bestrophin的功能。 突变蛋白对RPE细胞的吞噬途径的影响，例如外部段降解的延迟动力学和内体/吞噬体室的pH改变。 BMD相关的BESTROPHIN突变体将使用腺病毒介导的基因转移在小鼠和大鼠的RPE中表达，然后进行组织学，电生理学以及对脂肪蛋白蛋白积累和类类类类动物加工的影响的生化分析。