Significance of CYP46A1 and other P450s in retinal function

描述（由申请人提供）：胆固醇是哺乳动物生命所必需的。然而，慢性胆固醇过量是心血管疾病和阿尔茨海默病以及可能的年龄相关性黄斑变性（AMD）的危险因素。随着年龄的增长，胆固醇和其他脂质在视网膜色素上皮和Bruch膜之间积累，但在AMD患者中，这种积累更为突出，表明胆固醇去除存在缺陷。胆固醇的去除涉及两种机制，脂蛋白介导的转运和细胞色素P450酶（CYP）包括CYP 27A1和46A1分解代谢为氧甾醇。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联系，但酶促胆固醇从视网膜中去除的意义仍然未知。我们只知道，在人类中，CYP27A1缺乏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会引起视力障碍。为了开始深入了解视网膜中的胆固醇分解代谢，我们分析了尸体人眼，并确定视网膜胆固醇主要由CYP27A1代谢，而另一种P450亚型CYP46A1起较小的作用。我们还发现视网膜中CYP27A1蛋白的水平与其代谢物的水平无关

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.