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

项目摘要/摘要 视紫红质和视锥视色素的11顺式视网膜(11环)生色团的持续再生 对维持光敏性和感光器的生存至关重要。RPE65是一种关键的维甲酸异构酶 负责再生的视觉周期。RPE65功能在视觉和免疫方面的重要性 RPE65基因的100多种不同突变与视网膜健康有关的事实反映了这一事实 Leber先天性黑色素(LCA)和视网膜色素变性(RP)。建议的长期目标 研究内容包括：1)破译调节正常和正常细胞的表达、稳定性和活性的机制 致病突变体RPE65s，2)鉴定导致光感受器死亡的分子途径 RPE65突变患者和3)开发一种新的治疗干预措施来预防或延缓视力丧失 在病人身上。在最近和初步的研究中，我们发现脂肪酸运输蛋白4(FATP4)， 超长链脂肪酸1(ELOVL1)和26S蛋白酶体非ATPase调节亚基13的延长 (PSMD13)是RPE65的负调控因子。FATP4和ELOVL1抑制11-顺式视黄醇的合成 由RPE65催化，而PSMD13通过泛素化促进错误折叠的RPE65的降解- 视网膜色素上皮(RPE)中的依赖蛋白酶体途径。我们观察到，大多数人 致病错义突变被定位在RPE65和许多这些突变体的非活性位点上 由于错误折叠，经历了PSMD13介导的蛋白酶体降解。使用基于活细胞的分析， 我们发现，4-苯基丁酸酯(PBA)和甘油等化学伴侣可以挽救稳定性， 许多具有非活性位点突变的致病RPE65的膜结合和异构酶功能。 重要的是，PBA(一种FDA批准的药物)改善了LCA小鼠模型的锥体存活和功能 由R91W RPE65引起，R91W RPE65是最常见的LCA相关RPE65突变。另外，我们初步的 研究表明，R91W敲入(KI)小鼠中FATP4的缺失显著提高了锥体存活 和功能。为善用这些研究结果，我们建议在建议的 项目。具体目的1是确定FATP4缺乏如何改善锥体细胞的分子机制 LCA Ki小鼠模型的存活率和视力。为此，我们将分析视觉循环和 导致KI小鼠锥体死亡的致病途径。我们还将测试FATP4抑制因子和PBA 对KI小鼠视锥细胞视觉的长期保存有协同作用。具体目标2是测试 RPE特异性敲除ELOVL1基因可增加视神经生色团合成的假说 将野生型或突变型RPE65与KI小鼠进行比较，从而提高KI小鼠的锥体存活率和功能。我们 我还将分析ELOVL1抑制11-顺式视黄醇合成的分子机制 RPE65。这一创新项目的结果将确定FATP4和ELOVL1为新的治疗靶点， 为未来开发针对RPE65突变患者的新疗法提供知识库。

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.