Mechanisms that regulate RPE65 isomerase in normal and blindness-associated RPE

正常和失明相关 RPE 中 RPE65 异构酶的调节机制

• 批准号: 10249271
• 负责人: Minghao Jin
• 金额: $ 35.65万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249271
• 关键词: 11 cis Retinal; 11-cis-Retinol; 26S proteasome; 9-cis-retinal; Acceleration; Age related macular degeneration; Biological Assay; Blindness; Canis familiaris; Cause of Death; Cell Death; Cells; Cessation of life; Chemicals; Coenzyme A; Cone; Data; Defect; Development; Disease; Esters; FDA approved; Fatty Acids; Future; Genes; Glycerol; Goals; Health; Heart; Human; Isomerase; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Leber' s amaurosis; Lentivirus; Light; Link; Maps; Measures; Mediating; Membrane; Missense Mutation; Molecular; Molecular Chaperones; Mus; Mutation; Natural regeneration; Opsin; Pathogenicity; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Phenylbutyrates; Photophobia; Photoreceptors; Population; Predisposition; RPE65 protein; Recombinants; Recovery; Reproducibility; Research; Research Project Grants; Retina; Retinal Degeneration; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Role; Skin; Stargardt' s disease; Structure of retinal pigment epithelium; Testing; Therapeutic Intervention; Ubiquitin; Ubiquitination; Very Long Chain Fatty Acid; Virulence Factors; Vision; Visual; base; chromophore; cold temperature; cytotoxic; endoplasmic reticulum stress; fatty acid-transport protein; improved; innovation; knowledge base; mouse model; multicatalytic endopeptidase complex; mutant; new therapeutic target; novel therapeutic intervention; novel therapeutics; preservation; prevent; response; retinol isomerase; small hairpin RNA; stem; trafficking; visual cycle

Project Summary/Abstract Continuous regeneration of the 11-cis-retinal (11cRAL) chromophore of rhodopsin and cone visual pigments is essential for sustaining light-sensitivity and survival of photoreceptors. RPE65 is a key retinoid isomerase in the visual cycle responsible for regenerating 11cRAL. The importance of the RPE65 function in vision and retinal health is reflected by the facts that over 100 different mutations in the RPE65 gene are associated with Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). The long-term goals of the proposed research are 1) to decipher the mechanisms that regulate the expression, stability and activity of normal and disease-causing mutant RPE65s, 2) to identify the molecular pathway leading to photoreceptor death in patients with RPE65 mutations and 3) to develop a new therapeutic intervention to prevent or delay vision loss in the patients. In the recent and preliminary studies, we found that fatty acid transport protein 4 (FATP4), elongation of very long chain fatty acids 1 (ELOVL1) and 26S proteasome non-ATPase regulatory subunit 13 (PSMD13) are negative regulators of RPE65. FATP4 and ELOVL1 inhibited synthesis of 11-cis-retinol catalyzed by RPE65 while PSMD13 promoted degradation of misfolded RPE65 via the ubiquitination- dependent proteasomal pathway in the retinal pigment epithelium (RPE). We observed that the majority of pathogenic missense mutations are mapped on the non-active sites of RPE65 and many of these mutants underwent the PSMD13-mediated proteasomal degradation due to misfolding. Using a living cell-based assay, we discovered that chemical chaperones such as 4-phenylbutyrate (PBA) and glycerol can rescue the stability, membrane-association and the isomerase function of many pathogenic RPE65s with non-active site mutations. Importantly, PBA (a FDA-approved medication) improved cone survival and function in a mouse model of LCA caused by R91W RPE65, the most frequent LCA-associated RPE65 mutant. In addition, our preliminary studies showed that deletion of FATP4 in the R91W knock-in (KI) mouse dramatically improved cone survival and function. To capitalize on these findings, we propose to accomplish two specific aims in the proposed project. Specific Aim 1 is to identify the molecular mechanisms of how FATP4-deficiency improves cone survival and vision in the KI mouse model of LCA. To this end, we will analyze the visual cycle and the pathogenic pathways leading to cone death in the KI mouse. We will also test if FATP4 suppressor and PBA can exert synergistic effects on long-term preservation of cone vision in the KI mouse. Specific Aim 2 is to test the hypothesis that RPE-specific knockout of ELOVL1 increases synthesis of the visual chromophores in mouse with wild-type or the mutant RPE65, thereby improving cone survival and function in the KI mouse. We will also analyze the molecular mechanisms by which ELOVL1 inhibits synthesis of 11-cis-retinol catalyzed by RPE65. The results of this innovative project will identify FATP4 and ELOVL1 as new therapeutic targets, providing a knowledge-base for future development of new therapies for patients with RPE65 mutations.

项目总结/文摘