Deciphering the role of lipid metabolism pathways in the RPE and their implications for AMD

项目摘要 视网膜相关性黄斑变性（AMD）是美国老年人失明的主要原因。 States.高密度脂蛋白（HDL）胆固醇途径基因变体之间的强关联 （肝脂酶[LIPC]和胆固醇酯转移蛋白[CETP]）和晚期AMD被发现， 最近的全基因组关联研究。目前的建议旨在了解该机制 由LIPC和CETP基因中的SNP引起的潜在AMD，历史上已知与 与脂质生物合成途径。该研究计划测试了LIPC中的SNP的总体假设， 和CETP通过调节眼脂蛋白途径发挥其作用，这对 维持视网膜胆固醇稳态，从而引起视网膜病变。在目标1中，我们建议（i） 表征完整的脂蛋白分泌谱，包括顶侧和基底侧的HL和CETP 来源于2D极化的原代人胎儿视网膜色素上皮（hfRPE）培养物的条件培养基 在正常和胆固醇负荷条件下，（ii）研究HL和CETP敲低的影响，或 分别在hfRPE培养物中使用CRISPR/Cas9基因编辑和病毒基因递送的过表达， 并了解它们在维持RPE中脂质稳态途径中的功能。我们将确定 HL和CETP在这些培养物中的功能，通过进行脂质流出和流入试验，脂质分泌 在其他分析中。在目标2中，我们将确定AMD相关SNP对LIPC的影响， 通过表征这些基因中eQTL的存在来分析CETP转录物表达。我们建议使用 患者来源的诱导多能干细胞（iPSC）培养物在这些细胞中含有AMD相关SNP， 基因沿着与同基因对照。iPSC将分化成充分表征的RPE培养物。 将分析这些iPSC-RPE培养物以建立和鉴定LIPC和CETP中的任何SNP， 调节它们的表达。我们的研究结果将为理解分子生物学提供新的途径。 AMD的病理生理学，并解决HDL胆固醇代谢途径的重要性， 保持视网膜健康。了解基因在这些通路中的功能不仅可以让我们 确定治疗AMD的潜在治疗靶点，但也使我们能够使用和/或警告使用 现有的治疗动脉粥样硬化的药物。

PROJECT SUMMARY Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the United States. Strong association between variants in the high-density lipoprotein (HDL) cholesterol pathway genes (hepatic lipase [LIPC] and cholesterol ester transfer protein [CETP]) and advanced AMD were found in recent genome-wide association studies. The current proposal aims to understand the mechanism underlying AMD caused by SNPs in LIPC and CETP genes, which are historically known to be associated with lipid biogenesis pathways. The research proposal tests the overall hypothesis that the SNPs in LIPC and CETP exert their effects through modulating ocular lipoprotein pathways that are critical for the maintenance of retinal cholesterol homeostasis, thus causing retinal pathology. In Aim 1, we propose to (i) characterize the complete lipoprotein secretion profile including HL and CETP in the apical and basal conditioned media derived from 2D polarized primary human fetal retinal pigment epithelial (hfRPE) cultures in normal and cholesterol loaded conditions, (ii) study the effect of HL and CETP knockdown or overexpression using CRISPR/Cas9 gene editing and viral gene delivery, respectively in hfRPE cultures, and understand their function in maintenance of lipid homeostatic pathways in the RPE. We will determine the function of HL and CETP in these cultures by performing lipid efflux and influx assays, lipid secretion profile among other assays. In Aim 2, we will establish the effect of AMD-associated SNPs on LIPC and CETP transcript expression by characterizing the presence of eQTLs in these genes. We propose to use patient derived induced pluripotent stem cells (iPSC) cultures harboring AMD-associated SNPs in these genes along with isogenic controls. The iPSCs will be differentiated into well-characterized RPE cultures. These iPSC-RPE cultures will be analyzed to establish and identify any SNPs in LIPC and CETP that can regulate their expression. Outcome of our studies will open new avenues to understand the molecular pathophysiology of AMD and address the importance of HDL cholesterol metabolism pathways in maintaining retinal health. Understanding the function of genes in these pathways will not only allow us to determine potential therapeutic targets to treat AMD but also enable us to use and/or warn against the use of already available drugs for treatment of Atherosclerosis.