Microsomal Transfer Protein Modulates Lipoprotein Metabolism and Retinal lipid Homeostasis

脂质处理是视网膜色素上皮（RPE）的最关键功能之一， 位于神经视网膜和布鲁赫膜-脉络膜之间的不活跃细胞层。视功能 依赖于RPE和神经视网膜之间密切的结构、功能和代谢相互作用。 在这个复合体中，脂质代谢受到严格的调节，其失调引发过量脂质的积累。 RPE和邻近的布鲁赫膜和脉络膜血管系统内的脂质。在人类视网膜中， 中性脂质沉积物的积累是衰老的特征，并且在疾病相关的损伤之前。一 这种中性脂质的很大一部分包封在含有载脂蛋白B100的脂蛋白（Blps）中 类似于心肌细胞相关的Blps。大量的脂质必须由 RPE通过富含脂质的外节的再循环和代谢以及循环脂蛋白的摄取， 预测为了防止脂质过载，RPE合成并分泌Blp。在这方面，RPE分享代谢 与心肌细胞相似;两者都利用脂肪酸作为能量来源，并且都分泌独特的富含EC的BLP。 虽然已经显示MTP介导的Blps分泌保护心肌细胞免于脂质积累， 关于RPE在体内组装/分泌Blp的研究很少。在这些研究中，我们将使用小鼠 模型和细胞培养来解释与Blp失调相关的病理后果 通过抑制或基因消除RPE中的Mttp基因来调节RPE的组装和分泌途径。微粒体转移 MTTP蛋白（MTP）是MTTP基因的产物，是内质网驻留的脂质转运蛋白 这是Blp组装和分泌所必需的。在第一个具体目标中，我们将测试MTP介导的 Blp的分泌是防止RPE脂质过载的机制。为了测试本地化的 通过RPE合成和分泌Blp对视网膜脂质稳态和细胞功能的影响， 系统中，我们产生了RPE特异性MTP缺陷（RPE特异性MTP缺陷）小鼠。在第二个具体目标中， 检验Blp组装受摄入OS脂质的每日负荷调节的假设。我们将使用RPE 从人类诱导多能干细胞（iPSC）分化，以研究脂质 通过外节吞噬作用吸收调节MTP活性和Blp产生。利用这些细胞，我们 将确定是否通过基因消融（iRPE中MTP的KD）或药物抑制， 导致RPE脂肪变性。总的来说，这些探索性研究将解决当地BLP的具体作用 组装在视网膜脂质稳态。Blp功能失调可引起非自主性变化， 对整个系统产生负面影响并导致视力丧失。这些研究可能是研究 在与年龄相关的变化，如炎症和变性的发展的后续步骤， 脂蛋白代谢失调

Lipid handling is one of the most critical functions of the retinal pigment epithelium (RPE) a single mitotically inactive cell layer that is situated between the neural retina and the Bruch's membrane-Choroid. Visual function depends on the intimate structural, functional and metabolic interactions between the RPE and the neural retina. Within this complex, lipid metabolism is tightly regulated, and its' dysregulation triggers accumulation of excess lipids within the RPE and the adjacent Bruch's membrane and choroidal vasculature. In the human retina the accumulation of neutral lipid deposits is a characteristic of aging and precedes disease associated-lesions. A significant proportion of this neutral lipid is enclosed within apolipoprotein B100 containing lipoproteins (Blps) that resemble cardiomyocyte associated Blps. The prodigious amount of lipid that must be processed by the RPE, through the recycling and metabolism of lipid rich outer segments and ingestion of circulating lipoproteins, predicts that to prevent lipid overload the RPE synthesize and secrete Blp. In this regard, RPE shares metabolic similarity with cardiomyocytes; both utilize fatty acids as an energy source and both secrete unique EC rich Blps. While it has been shown that MTP-mediated secretion of Blps protects cardiomyocytes from lipid accumulation, there is a paucity of studies regarding Blp assembly/secretion by RPE in vivo. In these studies we will use mouse models and cell culture to decipher the pathological consequences associated with dysregulation of Blp assembly and secretion pathway by inhibiting or genetic ablation of the Mttp gene in RPE. Microsomal transfer protein (MTP), the product of the MTTP gene, is an endoplasmic reticulum-resident lipid transfer protein necessary for Blp assembly and secretion. In the first specific aim we will test the hypothesis that MTP-mediated secretion of Blp is a mechanism for protecting against RPE lipid overload. To test the contribution of localized synthesis and secretion of Blp by the RPE to retinal lipid homeostasis and cell function in a metabolically intact system, we generated the RPE-specific MTP deficient (RPEMttp) mouse. In the second specific aim we will test the hypothesis that Blp assembly is modulated by the daily load of ingested OS lipids. We will use RPE differentiated from human induced pluripotent stem cells (iPSCs) to investigate the mechanism by which lipids taken up through outer segment phagocytosis regulate MTP activity and Blp production. Using these cells, we will determine if loss of MTP, either through gene ablation (KD of MTP in iRPE) or pharmacological inhibition, contributes to RPE steatosis. Collectively, these exploratory studies will address the specific role of local Blp assembly in retinal lipid homeostasis. Dysregulation of Blp function can cause non- autonomous changes that negatively affect the entire system and lead to vision loss. These studies maybe foundational to study subsequent steps in the development of age-related changes such as inflammation and degeneration associated with dysregulation of lipoprotein metabolism.