RPE65 IN RETINAL METABOLISM AND DEGENERATION

RPE65 在视网膜代谢和退化中的作用

• 批准号: 6795339
• 负责人: DEBRA A THOMPSON
• 金额: $ 37.42万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6795339
• 关键词: RNase protection assay; SDS polyacrylamide gel electrophoresis; aging; dogs; enzyme activity; gene expression; gene mutation; genetic translation; high performance liquid chromatography; human tissue; immunofluorescence technique; immunoprecipitation; isomerase; laboratory mouse; northern blottings; protein protein interaction; protein structure function; retina degeneration; retinal pigment epithelium; retinoids; site directed mutagenesis; tissue /cell culture; transcription factor; vitamin biosynthesis; vitamin metabolism; western blottings

DESCRIPTION (Adapted from the applicant's abstract): The retinal pigment epithelium (RPE) plays a critical role in the maintenance of normal photoreceptor functions and has been implicated in several visual disorders, including macular degenerations and dystrophies. The investigator has cloned and characterized the first known RPE-specific human gene, RPE65, and has shown that mutations in this gene are responsible for certain forms of autosomal recessive childhood-onset severe retinal dystrophy (arCSRD), a finding supported by reports of RPE65 defects in Leber's congenital amaurosis. A research program has been developed to study the function of RPE65 in the normal biology of the retina and in the disease state, based on the view that RPE65 is necessary for the isomerase activity involved in the conversion of vitamin A to 11-cis retinal. Four specific aims have been identified for the proposed funding period. (1) Recombinant protein-protein interactions and enzyme activity will be studied in cultured cells transfected with RPE65 expression constructs in order to distinguish between the two prevailing hypotheses about the specific role of RPE65 in RPE retinoid metabolism. (2) Site-directed mutagenesis will be used with assays of expression and protein function to elucidate the role of RPE65 mutations in the pathogenesis of arCSRD to test the hypothesis that disease-associated mutations in RPE65 result in functional null alleles that disrupt the 11-cis retinal biosynthetic pathway. This aim will include further characterization of mutations present in patient populations. (3) Because preliminary information indicates that RPE65 is down-regulated by a variety of factors that are known to be related to aging and disease processes, and because decreased levels of RPE65 are implicated in retinal degeneration, the mechanisms involved in this down-regulation will be investigated, as well as the effects of aging and other physiological conditions on RPE65 expression. (4) Effects of the RPE65 mutation in a large animal model of arCSRD will be characterized in assays of the biochemistry and enzymology of retinoid metabolism, to test the hypothesis that defects resulting from RPE65 mutations will be amenable to retinal replacement therapy. The long-term goals of this project are to elucidate the mechanisms by which RPE65 defects contribute to retinal degeneration, and to lay the groundwork for the development of therapeutic approaches to the disease.

描述（改编自申请人的摘要）：视网膜色素 视网膜上皮细胞（RPE）在维持正常的 光感受器功能并与几种视觉障碍有关， 包括黄斑变性和营养不良。调查员克隆了 并鉴定了第一个已知的RPE特异性人类基因RPE 65，并显示 该基因的突变导致某些形式的常染色体 隐性儿童期发作的严重视网膜营养不良（arCSRD），一项发现 Leber先天性黑蒙中RPE 65缺陷的报告支持了这一观点。一 已经开发了研究计划来研究RPE 65在 视网膜的正常生物学和疾病状态，基于这样的观点， RPE 65对于参与转化的异构酶活性是必需的。 维生素A到11-顺式视黄醇。已确定了四个具体目标， 拟议供资期。(1)重组蛋白-蛋白相互作用和 将在用RPE 65转染的培养细胞中研究酶活性 表达结构，以区分两种流行的 关于RPE 65在RPE类维生素A代谢中的特定作用的假说。（二） 定点诱变将与表达和蛋白质分析一起使用。 阐明RPE 65突变在arCSRD发病机制中的作用 为了检验RPE 65中疾病相关突变导致 破坏11-顺式视网膜生物合成途径的功能性无效等位基因。 这一目标将包括进一步表征患者中存在的突变。 人口。(3)因为初步信息表明RPE 65是 被已知与衰老相关的多种因素下调 和疾病过程，并且因为RPE 65水平的降低与 视网膜变性，参与这种下调的机制将是 研究，以及老化和其他生理的影响， RPE 65的表达。(4)RPE 65突变在大细胞中的作用 arCSRD的动物模型将在生物化学和 维生素A代谢的酶学，以测试缺陷的假设， 由RPE 65突变引起的视网膜病变将适用于视网膜替代疗法。 本项目的长期目标是阐明 RPE 65缺陷导致视网膜变性，并为视网膜病变奠定基础。 治疗方法的发展。