Lipid Trafficking and Metabolism in the Retinal Pigment Epithelium

摘要：老年性黄斑变性(AMD)以视网膜色素上皮(RPE)脂质代谢异常为特征。 动态平衡，包括病理性的细胞外脂肪堆积，线粒体脂质代谢受损，以及 增加脂质过氧化，这是脂质氧化损伤的一种有毒形式。RPE通常在以下方面发挥重要作用 视网膜脂肪平衡，消耗和分泌大量的脂肪。脂肪的消耗量在 每天大量脂质负荷的形式，包括从脉络膜摄取脂蛋白和吞噬富含脂质的细胞 感光细胞外节(OS)。其中一些脂质在线粒体中被氧化代谢，产生 能量支持光感受器的代谢物。脂肪氧化代谢也能最大限度地减少葡萄糖的使用 通过RPE，允许未使用的葡萄糖从脉络膜传递到高度糖酵解的光感受器。脂肪分泌 主要是通过脂蛋白，其中一些支持OS的再生，一些积累为 典型的AMD细胞外沉积。细胞面临大量脂类负荷的一个特征是形成脂类 液滴(LD)，细胞内脂类储存细胞器，动态调节脂类的运输、代谢和 脂质过氧化。特定的LD功能在不同的细胞类型之间差别很大。RPE LD已在 视觉周期生物学的背景，但它们在维持非视黄酸类脂平衡中的生理学和作用 RPE基本上是未被开发的。这项建议考察了LD的组成、生理和在人类中的作用 RPE脂质动态平衡。最重要的假设是双重的：a)LD可以暂时存储大范围的 来自RPE每日脂质负荷的脂质，防止其脂质过氧化；以及b)LD通过 特定的酶优先在线粒体中代谢，而不是分泌。通过促进脂质氧化 代谢超过脂质分泌，RPE LD可能增强对光感受器的代谢支持，降低 AMD中可见病理性细胞外脂积聚。为了验证这一假设，将给不同的脂肪负荷 原代和诱导多能干细胞(IPSC)来源的人RPE培养，并评估LD成分 通过质谱分析。从LD释放的脂质的命运将通过显微镜，生化方法， 和脂质组学。主要研究人员的长期目标是发展专业知识来定义细胞内RPE 促进RPE脂质动态平衡和降低病理性细胞外脂的脂质转运途径 沉积，为AMD开辟了新的治疗途径。为了实现这一目标，首席调查员的 职业发展计划(CDP)专注于RPE脂质生物学，建立IPSC-RPE培养方面的专业知识， 放射性标记脂类示踪、高分辨率显微镜、生物能量分析和脂类组学。CDP的 导师团队利用密歇根大学在脂类生物学和脂类组学方面的深厚专业知识 有成功指导K获奖者研究独立性的记录。这两种技术的结合 这项建议中的教学和实践经验确保了调查员将成为一名成功的AMD- 专注于独立基础科学项目的临床医生，利用RPE脂质生物学来了解AMD。

SUMMARY: Age-related macular degeneration (AMD) is marked by retinal pigment epithelial (RPE) lipid dys- homeostasis, including pathologic extracellular lipid accumulation, impaired mitochondrial lipid metabolism, and increased lipid peroxidation, a toxic form of lipid oxidative damage. The RPE normally plays an essential role in retinal lipid homeostasis, consuming and secreting prodigious amounts of lipid. Lipid consumption comes in the form of a large daily lipid load, including both lipoprotein uptake from the choroid and phagocytosis of lipid-rich photoreceptor outer segments (OS). Some of this lipid is oxidatively metabolized in the mitochondria, producing metabolites that energetically support photoreceptors. Lipid oxidative metabolism also minimizes glucose use by the RPE, allowing glucose to pass unused from the choroid to highly glycolytic photoreceptors. Lipid secretion is primarily through lipoprotein, some of which supports regeneration of OS and some of which accumulates as the extracellular deposits typical of AMD. A characteristic of cells facing large lipid loads is the formation of lipid droplets (LD), intracellular lipid storage organelles that dynamically regulate lipid trafficking, metabolism, and lipid peroxidation. Specific LD function varies widely between different cell types. RPE LD have been studied in the context of visual cycle biology, but their physiology and role in maintaining non-retinoid lipid homeostasis in the RPE is essentially unexplored. This proposal examines the composition, physiology, and role of LD in human RPE lipid homeostasis. The over-arching hypothesis is two-fold: a) LD can temporarily store a wide-range of lipids derived from the RPE’s daily lipid load, preventing their lipid peroxidation; and b) lipid released from LD by specific enzymes is preferentially metabolized in mitochondria rather than secreted. By facilitating lipid oxidative metabolism over lipid secretion, RPE LD may enhance metabolic support for photoreceptors and decrease the accumulation of pathologic extracellular lipid seen in AMD. To test the hypothesis, various lipid loads will be fed to primary- and induced pluripotent stem cell (iPSC)-derived human RPE cultures, with LD composition assessed by mass spectrometry. The fate of lipids released from LD will be tracked via microscopy, biochemical methods, and lipidomics. The principal investigator’s long-term goal is to develop the expertise to define intracellular RPE lipid trafficking pathways that enhance RPE lipid homeostasis and decrease pathologic extracellular lipid deposition, opening up new therapeutic avenues for AMD. To achieve this goal, the principal investigator’s career development plan (CDP) focuses on RPE lipid biology, establishing expertise in iPSC-RPE culture, radiolabeled lipid tracing, high-resolution microscopy, bio-energetic profiling, and lipidomics. The CDP’s mentorship team takes advantage of deep expertise in lipid biology and lipidomics at University of Michigan and has a track-record of successfully mentoring K awardees to research independence. The combination of didactics and hands-on experience in this proposal ensures the investigator will become a successful AMD- focused clinician with an independent basic science program harnessing RPE lipid biology to understand AMD.