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: 10000925
• 负责人: Minghao Jin
• 金额: $ 36.75万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000925
• 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; 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; Subfamily lentivirinae; 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/摘要 视紫红质和视锥色素的11-顺式-视网膜（11 cRAL）发色团的连续再生是 对维持光敏感性和光感受器的存活至关重要。RPE 65是一种关键的维甲酸异构酶， 负责再生11 cRAL的视觉周期。RPE 65功能在视力和 视网膜健康反映在RPE 65基因中超过100种不同的突变与以下事实有关： Leber先天性黑蒙（LCA）和视网膜色素变性（RP）。拟议的长期目标 研究是1）破译调节正常和非正常表达的表达，稳定性和活性的机制， 致病突变型RPE 65 s，2）鉴定导致视网膜光感受器死亡的分子途径， 患有RPE 65突变的患者和3）开发新的治疗干预以预防或延迟视力丧失 在病人身上。在最近的和初步的研究中，我们发现脂肪酸转运蛋白4（FATP 4）， 极长链脂肪酸1（VL 1）和26 S蛋白酶体非ATP酶调节亚基13的延伸 （PSMD 13）是RPE 65的负调节因子。FATP 4和FATPVL 1抑制11-顺式-视黄醇的合成 PSMD 13通过泛素化促进RPE 65的降解，而PSMD 13通过泛素化促进RPE 65的降解。 视网膜色素上皮（RPE）中的依赖性蛋白酶体途径。我们注意到，大多数 致病性错义突变定位在RPE 65的非活性位点上，并且这些突变体中的许多 由于错误折叠，经历了PSMD 13介导的蛋白酶体降解。使用基于活细胞的测定， 我们发现化学伴侣如4-苯基丁酸酯（PBA）和甘油可以挽救稳定性， 膜结合和异构酶功能的许多致病性RPE 65与非活性位点突变。 重要的是，PBA（FDA批准的药物）改善了LCA小鼠模型中的视锥细胞存活和功能 R91 W RPE 65是最常见的LCA相关RPE 65突变体。此外，我们的初步 研究表明，在R91 W基因敲入（KI）小鼠中缺失FATP 4显著提高了视锥细胞存活率， 和功能为了充分利用这些研究结果，我们建议在拟议的 项目具体目标1是确定FATP 4缺乏如何改善锥状细胞的分子机制。 在LCA的KI小鼠模型中的存活和视力。为此，我们将分析视觉周期和 导致KI小鼠视锥细胞死亡的致病途径。我们还将测试FATP 4抑制剂和PBA是否 可对KI小鼠的锥视长期保存发挥协同作用。具体目标2是测试 假设RPE特异性敲除ESTVL 1增加了视网膜中可见发色团的合成， 小鼠与野生型或突变RPE 65，从而改善KI小鼠的视锥细胞存活和功能。我们 还将分析BMPVL 1抑制11-顺式-视黄醇合成的分子机制， RPE 65。这个创新项目的结果将确定FATP 4和FATPVL 1作为新的治疗靶点， 为将来开发针对RPE 65突变患者的新疗法提供知识基础。