Significance of CYP46A1 and other P450s in retinal function

说明(申请人提供)：胆固醇是哺乳动物生命所必需的。然而，长期过量的胆固醇是心血管疾病和阿尔茨海默氏症以及可能的老年性黄斑变性(AMD)的危险因素。随着年龄的增长，胆固醇和其他类脂在视网膜色素上皮和Bruchs膜之间积累，但在AMD患者中，这种积累更加突出，表明胆固醇的清除存在缺陷。胆固醇的去除通常涉及两种机制，脂蛋白介导的转运和细胞色素P450酶(CYP)包括细胞色素P450酶(CYP)27A1和46A1的分解代谢。虽然最近的遗传学研究揭示了AMD与影响高密度脂蛋白形成的基因之间的关联，但酶促视网膜胆固醇去除的意义仍不清楚。我们只知道在人类中，CYP27A1缺乏症的眼部表现包括视网膜过早衰老和胆固醇沉积，而伏立康唑抑制CYP46A1会导致视力障碍。为了开始了解视网膜中的胆固醇分解代谢，我们分析了身体的眼睛，发现视网膜胆固醇主要由细胞色素P27A1代谢，而另一种P450亚型，细胞色素P46A1，起次要作用。我们还发现，视网膜中CYP27A1蛋白的水平与其代谢物的水平没有相关性 5-胆汁酸，并确定了这一差异的基础。我们证明了由花生四烯酸产生的异uglandins对CYP27A1进行了有害的翻译后修饰。我们还发现，缺乏一个(CYP27A1)或两个(CYP27A1和46A1)胆固醇分解酶的小鼠会出现结构性视网膜病变、血流改变和病理性视网膜血管形成。这些发现证实了酶促胆固醇清除是视网膜正常功能的重要因素。在这场相互竞争的更新中，我们建议进一步探索我们的发现。这项应用的具体目的是：1)描述胆固醇分解代谢受损小鼠的眼部表型；2)确定调节视网膜CHO水平的主要机制；3)检测视网膜中的等LG-内收。将使用先进的诊断仪器、最先进的质谱学方法、人类和小鼠标本的生化和分子生物学分析，以及小鼠的饮食和药物干预措施来实现该项目的目标。所获得的数据将极大地促进我们对胆固醇在视觉中的作用以及视网膜病理性血管形成机制的理解。这些研究可能有助于确定预防和治疗新生血管性AMD的治疗靶点。

DESCRIPTION (provided by applicant): Cholesterol is essential for life in mammals. Yet, chronic excess of cholesterol is a risk factor for cardiovascular and Alzheimer's diseases and likely age-related macular degeneration (AMD). Cholesterol and other lipids accumulate with age between the retinal pigment epithelium and Bruch's membrane, but in people with AMD this accumulation is more prominent suggesting defects in cholesterol removal. Cholesterol removal always involves two mechanisms, lipoprotein-mediated transport and catabolism to oxysterols by cytochrome P450 enzymes (CYP) including CYPs 27A1 and 46A1. While recent genetic studies revealed an association between AMD and genes affecting the formation of high density lipoproteins, the significance of the enzymatic cholesterol removal from the retina is still unknown. We only know that in humans, ocular manifestations of CYP27A1 deficiency include premature retinal senescence and cholesterol deposition, and that inhibition of CYP46A1 with voriconazole causes visual disturbances. To begin to gain insight into cholesterol catabolism in the retina, we analyzed cadaveric human eyes and established that retinal cholesterol is mainly metabolized by CYP27A1 while the other P450 isoform, CYP46A1, plays a lesser role. We also found that retinal levels of the CYP27A1 protein do not correlate with the levels of its metabolite 5-cholestenoic acid and ascertained the basis for this discrepancy. We demonstrated that CYP27A1 is subjected to a deleterious post-translational modification by isolevuglandins generated from arachidonic acid. We also found that mice lacking one (CYP27A1) or two (CYPs 27A1 and 46A1) cholesterol-catabolizing enzymes have structural retinal pathologies, altered blood flow and pathologic retinal vascularization. These findings confirm that enzymatic cholesterol elimination is an important contributor to the normal function of the retina. In this competing renewal, we propose to further explore our findings. The Specific Aims of this application are: 1) to characterize the ocular phenotype of mice with impaired cholesterol catabolism; 2) to identify the major mechanisms regulating retinal CHO levels; and 3) to examine the isoLG-adduction in the retina. Advanced diagnostic instrumentation, state-of-the-art mass spectrometry methodologies, biochemical and molecular biology analyses of human and mouse specimens, as well as dietary and pharmacological interventions in mice will be used to address the goals of the project. The data obtained will significantly advance our understanding of the role of cholesterol in vision and the mechanisms for pathologic vascularization in the retina. These studies may lead to the identification of therapeutic targets for prevention and treatment of neovascular AMD.