RPE65 IN RETINAL METABOLISM AND DEGENERATION

RPE65 在视网膜代谢和退化中的作用

• 批准号: 7124825
• 负责人: DEBRA A THOMPSON
• 金额: $ 31.3万
• 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/7124825
• 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(摘自申请者的摘要)：视网膜色素 上皮细胞(RPE)在维持正常状态中起着至关重要的作用 光感受器的功能，并与几种视觉障碍有关， 包括黄斑变性和营养不良。调查员已经克隆了 并鉴定了第一个已知的RPE特异性人类基因RPE65，并展示了 该基因的突变导致了某些形式的常染色体 儿童期隐性起病的严重视网膜营养不良(ArCSRD) 莱伯氏先天性黑色素RPE65缺陷的报道支持这一观点。一个 已经开发了研究程序来研究RPE65在 视网膜的正常生物学和疾病状态，基于这样的观点 RPE65是异构酶活性所必需的 维生素A到11顺式视网膜。已经确定了四个具体的目标 建议的资金期限。(1)重组蛋白质-蛋白质相互作用和 转RPE65的培养细胞中的酶活性将被研究 表达式构造，以便区分两种流行的 RPE65在RPE类维甲酸代谢中具体作用的假说。(2) 定点突变将与表达和蛋白质分析一起使用 阐明RPE65突变在ArCSRD发病机制中的作用 为了检验RPE65的疾病相关突变会导致 破坏11顺式视网膜生物合成途径的功能性零等位基因。 这一目标将包括对患者体内存在的突变进行进一步表征 人口。(3)因为初步信息表明RPE65是 受多种已知与衰老相关的因素的下调 和疾病过程，而且因为RPE65水平降低与 视网膜变性，参与这种下调的机制将是 调查，以及衰老和其他生理因素的影响 RPE65表达的条件。(4)RPE65突变对大鼠的影响 ArCSRD动物模型将在生化和生物化学检测方面进行表征 类视黄素代谢的酶学，以检验缺陷假说 由RPE65突变导致的视网膜替代治疗将是可行的。 这个项目的长期目标是阐明 RPE65缺陷导致视网膜退变，并为 这种疾病的治疗方法的发展。