Cholesterol homeostasis in the vertebrate retina

脊椎动物视网膜中的胆固醇稳态

基本信息

批准号：
10580969
负责人：
Steven J. Fliesler
金额：
$ 39万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2028-02-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10580969
关键词：
7-dehydrocholesterol 7-dehydrocholesterol reductase AY9944 Ablation Affect Age Alleles Antioxidants Astrocytes Autophagocytosis Biological Assay Biological Models Biology Blood Cell Culture Techniques Cell Death Cell Line Cells Cholesterol Cholesterol Homeostasis Cholesterol Synthesis Inhibition Defect Dehydrocholesterols Development Diet Disease Enterobacteria phage P1 Cre recombinase Equilibrium Eukaryotic Cell Exhibits Exons Family Future Gender Genes Genetic Glial Fibrillary Acidic Protein Goals Hepatocyte Histology Homeostasis Human In Vitro Inherited Knock-out Knockout Mice Lipoproteins Liver LoxP-flanked allele Measures Metabolic Diseases Methods Modeling Molecular Monitor Morphology Muller&apos s cell Mus Mutation Neural Retina Neuroglia Niacinamide Online Mendelian Inheritance In Man Optic Nerve Oxidative Stress Oxidoreductase Pathology Patients Phagocytes Phagosomes Photoreceptors Physiological Process Rare Diseases Rattus Reporter Resources Retina Retinal Degeneration Retinal Diseases Rod Outer Segments Role Serum Sirolimus Smith-Lemli-Opitz Syndrome Source Sterols Structure Testing Therapeutic Intervention Time Tissues Wild Type Mouse cell type cholesterol biosynthesis disease-in-a-dish drug candidate improved in vivo induced pluripotent stem cell inhibitor mouse model myelination novel outcome prediction retinal neuron retinal rods rho stable isotope sterol homeostasis therapeutically effective uptake

项目摘要

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.

抽象的：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。新合成、含胆脂蛋白的摄取和胆汁流出（即“胆汁稳态”）。对视网膜胆碱稳态的了解仍处于初级阶段。胆碱合成的遗传性缺陷。包括一系列严重的、通常致命的代谢性疾病，例如史密斯-莱姆利-奥皮茨综合征 (SLOS)。 SLOS 涉及 7-脱氢胆固醇 (7DHC) 向 Chol 的缺陷转化，由 DHCR7 (7- 用 DHCR7 抑制剂（例如 AY9944）治疗大鼠会导致进行性、不可逆的视网膜变性：感光细胞 (PR) 优先退化并死亡，RPE 细胞也表现出自噬/异噬缺陷，但是 DHCR7 抑制剂的脱靶效应不能被抑制。避免了，并且没有可行的 SLOS 基因小鼠模型可用，我们已经产生了两种新颖的、可行的、 SLOS 的条件等位基因模型，允许定向消融 Dhcr7 外显子 8 或单独的外显子 9，以我们在选择性细胞类型和组织中部分或完全阻断 DHCR7，从头解决了这个问题。仅靠视网膜神经元的合成不足以维持其活力和功能；相反，它们依赖于视网膜神经元的合成。从血源性（肝源性）脂蛋白、Müller 神经胶质细胞和/或 RPE 摄取胆碱以满足其甾醇的要求此外，正常胆固醇稳态的破坏会引起吞噬溶酶体生物学的缺陷。 RPE 和视网膜。我们将按如下方式检验我们的假设：在目标 1 中，我们将选择性地敲除视杆细胞中的 Dhcr7。 PR 和，分别在 Müller 胶质细胞中（或串联），然后评估对外部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，我们将生成全视网膜敲除 (KO) Dhcr7 或单独（并串联）在肝脏（肝细胞）中，然后评估对甾醇的影响我们将使用这些资源和稳定同位素方法来估计视网膜、肝脏和血液的组成。视网膜甾醇合成、摄取和周转率；在目标 3 中，我们将使用 RPE 和肝脏特异性 Dhcr7 KO 我们将用小鼠评估从头合成 7DHC 及其摄取对 RPE 吞噬功能的影响。使用患者 iPSC 衍生的 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 和吞噬细胞缺陷。我们将使用新型体内测定法，使用串联标记的自噬报告小鼠模型来系统地阐明Chol合成在视网膜吞噬溶酶体生物学中的作用将获得显着的结果。两者都增进了我们对脊椎动物视网膜中胆固醇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学，并提供新的易于处理的模型系统以便未来测试针对 SLOS 和相关孤儿疾病的更有效的治疗干预措施。