RPE65 IN RETINAL METABOLISM AND DEGENERATION

RPE65 在视网膜代谢和退化中的作用

• 批准号: 6524954
• 负责人: DEBRA A THOMPSON
• 金额: $ 35.27万
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524954
• 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 上皮（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，已与几种视觉疾病有关， 包括黄斑变性和营养不良。调查员克隆了 并表征了第一个已知的RPE特异性人基因RPE65，并显示了 该基因中的突变负责某些形式的常染色体 隐性儿童期严重的视网膜营养不良（ARCSRD），这一发现 由Leber先天性瘤中RPE65缺陷的报道支持。一个 已经开发了研究计划来研究RPE65在 基于视网膜和疾病状态处的正常生物学 RPE65对于参与转化的异构酶活性是必需的 维生素A至11-CIS视网膜。已经确定了四个特定目标 拟议的资金期。 （1）重组蛋白 - 蛋白质相互作用和 将在用RPE65转染的培养细胞中研究酶活性 表达构造是为了区分这两个盛会 关于RPE65在RPE视乙他代谢中的特定作用的假设。 （2） 定点诱变将用于表达和蛋白质的测定 阐明RPE65突变在ARCSRD发病机理中的作用的功能 为了检验以下假设：rpe65中与疾病相关的突变导致 破坏11-CIS视网膜生物合成途径的功能无效等位基因。 该目标将包括进一步表征患者中存在的突变 人群。 （3）因为初步信息表明RPE65是 被已知与衰老有关的多种因素下调 和疾病过程，并且因为RPE65的水平降低与 视网膜变性，该下调所涉及的机制将是 研究了，以及衰老和其他生理的影响 RPE65表达的条件。 （4）大型RPE65突变的效果 ARCSRD的动物模型将以生物化学的测定和 视网膜定代谢的酶学，以检验缺陷的假设 由RPE65突变引起的视网膜替代疗法可以调节。 该项目的长期目标是阐明 RPE65缺陷有助于视网膜变性，并为 疾病治疗方法的发展。