Cholesterol homeostasis in the vertebrate retina

摘要： 所有真核细胞都需要胆固醇(Chol)才能生存，并通过平衡胆固醇来调节其稳态水平。 新的合成、含胆固醇的脂蛋白的摄取和胆固醇的外流(即“胆固醇稳态”)。我们目前的情况 对视网膜内胆碱稳态的了解仍处于初级阶段。胆固醇合成中的遗传性缺陷 包括一系列严重的、往往是致命的代谢紊乱，例如Smith-Lemli-Opitz综合征(SLOS)。 SLOS涉及7-脱氢胆固醇(7DHC)到胆固醇(Chol)的缺陷转化，这是由DHCR7(7-DHC)催化的。 脱氢胆固醇还原酶)。用DHCR7抑制剂(例如，AY9944)治疗大鼠会导致进行性， 不可逆性视网膜变性：光感受器(PR)优先变性死亡，RPE细胞 也表现出自噬/异噬缺陷。然而，DHCR7抑制剂的非靶点效应不能 目前尚无SLOS的遗传性小鼠模型可用。我们产生了两个新颖的，可行的， SLOS的条件等位基因模型，允许靶向消融Dhcr7外显子8或单独的外显子9，以 部分或完全阻断选择性细胞类型和组织中的DHCR7。我们假设从头开始 仅靠视网膜神经元的合成不足以维持其活性和功能；相反，它们依赖于 从血液(肝源性)脂蛋白、Müller胶质细胞和/或RPE摄取Chol以满足其甾醇 要求。此外，正常胆固醇稳态的破坏会导致吞噬小体生物学缺陷。 RPE和视网膜。我们将检验我们的假设如下：在目标1中，我们将选择性地敲除ROD中的Dhcr7 分别在Müller胶质细胞中(或串联)，然后评估对外周血甾醇组成的影响 节段和视神经，以及视网膜结构/功能；在目标2中，我们将产生全视网膜敲除 (Ko)Dhcr7或，单独(和串联)，在肝脏(肝细胞)中，然后评估对固醇的影响 视网膜、肝脏和血液的成分。利用这些资源和稳定的同位素方法，我们将估计 视网膜固醇合成、摄取和周转率；在目标3中，我们将使用RPE和肝脏特异的Dhcr7 KO 小鼠评价从头合成7DHC及其摄取对RPE吞噬功能的影响。我们会 使用患者IPSC来源的RPE细胞(多个克隆，具有 适当的对照)，并筛选候选药物以改善观察到的EMT和吞噬缺陷。 我们将使用新的体内测试方法，使用串联标记的自噬报告鼠模型来系统地 阐明Chol合成在视网膜吞噬小体生物学中的作用。所获得的结果将显著地 促进我们对脊椎动物视网膜胆碱稳态及其机制的基本理解 与Chol合成缺陷相关的潜在视网膜病理，并提供新的易处理的模型系统 用于未来对SLOS和相关孤儿疾病进行更有效的治疗干预的测试。

ABSTRACT: All eukaryotic cells require cholesterol (Chol) for survival, and regulate their steady-state levels by balancing de novo synthesis, uptake of Chol-containing lipoproteins, and Chol efflux (i.e., “Chol homeostasis”). Our current understanding of Chol homeostasis in the retina remains rudimentary. Hereditary defects in Chol synthesis comprise a family of severe, often lethal, metabolic disorders, e.g., Smith-Lemli-Opitz Syndrome (SLOS). SLOS involves defective conversion of 7-dehydrocholesterol (7DHC) to Chol, which is catalyzed by DHCR7 (7- dehydrocholesterol reductase). Treating rats with a DHCR7 inhibitor (e.g., AY9944) causes progressive, irreversible retinal degeneration: the photoreceptors (PRs) preferentially degenerate and die, and RPE cells also exhibit autophagy/heterophagy defects. However, off-target effects of DHCR7 inhibitors cannot be obviated, and no viable genetic mouse models of SLOS are available. We have generated two novel, viable, conditional allele models of SLOS, allowing targeted ablation of either Dhcr7 exon 8, or separately exon 9, to partially or completely block DHCR7 in selective cell types and tissues. We hypothesize that de novo Chol synthesis by retinal neurons alone is insufficient to maintain their viability and functionality; rather, they rely upon Chol uptake from blood-borne (liver-derived) lipoproteins, Müller glia, and/or the RPE to meet their sterol demands. Also, that disruption of normal Chol homeostasis provokes defective phagolysosomal biology in the RPE and retina. We will test our hypothesis as follows: In Aim 1, we will selectively knock out Dhcr7 in rod PRs and, separately, in Müller glia (or in tandem) and then assess the impact on sterol composition of outer segments and optic nerve, as well as retinal structure/function; in Aim 2, we will generate panretinal knock-out (KO) of Dhcr7 or, separately (and in tandem), in liver (hepatocytes) and then assess the impact on sterol composition retina, liver, and blood. Using these resources, and a stable isotope approach, we will estimate retinal sterol synthesis, uptake and turnover rates; and in Aim 3, we will use RPE and liver-specific Dhcr7 KO mice to assess the effect of de novo 7DHC synthesis and its uptake on RPE phagocytic function. We will model the observed SLOS RPE pathology using patient iPSC-derived RPE cells (multiple clones, with appropriate controls), and screen for candidate drugs to improve the observed EMT and phagocytic defects. We will use novel in vivo assays using a tandem-tagged autophagy reporter mouse model to systematically elucidate the role of Chol synthesis in retinal phagolysosomal biology. The results obtained will significantly advance both our fundamental understanding of Chol homeostasis in the vertebrate retina and mechanisms underlying retinal pathology associated with Chol synthesis defects, and provide new tractable model systems for future testing of more effective therapeutic interventions for SLOS and related orphan diseases.