Lipid Trafficking and Metabolism in the Retinal Pigment Epithelium

视网膜色素上皮细胞（RPE）脂质异常是黄斑变性（AMD）的标志。 稳态，包括病理性细胞外脂质积聚，线粒体脂质代谢受损，以及 脂质过氧化增加，这是一种毒性形式的脂质氧化损伤。RPE通常在以下方面发挥着重要作用 视网膜脂质稳态，消耗和分泌大量脂质。脂肪消耗来自 一种大的每日脂质负荷的形式，包括从脉络膜摄取脂蛋白和吞噬富含脂质的 光感受器外节（OS）。其中一些脂质在线粒体中氧化代谢， 这些代谢物有力地支持光感受器。脂质氧化代谢也最大限度地减少葡萄糖的使用 通过视网膜色素上皮，允许葡萄糖从脉络膜传递到高度糖酵解的光感受器。脂质分泌 主要是通过脂蛋白，其中一些支持OS的再生，其中一些积累为 AMD典型的细胞外沉积物。面对大量脂质负荷的细胞的一个特征是脂质的形成 液滴（LD），细胞内脂质储存细胞器，动态调节脂质运输，代谢， 脂质过氧化特定的LD功能在不同的细胞类型之间变化很大。RPE LD已在 视觉周期生物学的背景下，但他们的生理学和在维持非维甲酸脂质稳态的作用， 视网膜色素上皮基本上还未被探索本研究旨在探讨LD在人体内的组成、生理学和作用。 RPE脂质稳态。过度存储假设是双重的：a）LD可以暂时存储大范围的 来自RPE的每日脂质负荷的脂质，防止它们的脂质过氧化;和B）由LD释放的脂质 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 因此，RPE LD可以增强光感受器的代谢支持，并降低光感受器的代谢。 在AMD中观察到病理性细胞外脂质积累。为了检验这一假设，将饲喂不同的脂质负荷， 至原代和诱导多能干细胞（iPSC）衍生的人RPE培养物，评估LD组成 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。主要研究者的长期目标是发展专业知识来定义细胞内RPE 增强RPE脂质稳态和减少病理性细胞外脂质的脂质运输途径 沉积，为AMD开辟了新的治疗途径。为了实现这一目标，首席研究员 职业发展计划（CDP）专注于RPE脂质生物学，建立iPSC-RPE培养的专业知识， 放射性标记的脂质示踪、高分辨率显微镜、生物能量分析和脂质组学。CDP的 导师团队利用密歇根大学在脂质生物学和脂质组学方面的深厚专业知识， 拥有成功指导K获奖者研究独立性的记录。的组合 教学和实践经验，在这个建议，确保调查员将成为一个成功的AMD- 专注于临床医生，拥有独立的基础科学项目，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.