Cholesterol homeostasis in the vertebrate retina

脊椎动物视网膜中的胆固醇稳态

• 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
• 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' 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

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.

摘要： 所有的真核细胞都需要胆固醇（Chol）来维持生存，并通过平衡胆固醇的浓度来调节其稳态水平。 新合成、含Chol脂蛋白的摄取和Chol流出（即，“Chol homeostasis”）。我们目前 对视网膜中Chol稳态的理解仍然是基本的。Chol合成的遗传缺陷 包括一系列严重的、通常致命的代谢紊乱，例如，Smith-Lemli-Opitz综合征（SLOS）。 SLOS涉及7-脱氢胆固醇（7 DHC）向Chol的有缺陷的转化，其由DHCR 7（7-DHC）催化。 脱氢胆固醇还原酶）。用DHCR 7抑制剂（例如，AY 9944）导致进行性， 不可逆的视网膜变性：光感受器（PR）优先退化和死亡，RPE细胞 也表现出自噬/异噬缺陷。然而，DHCR 7抑制剂的脱靶效应不能被抑制。 没有可行的SLOS遗传小鼠模型可用。我们创造了两个新颖可行的， SLOS的条件等位基因模型，允许靶向切除Dhcr 7外显子8或单独的外显子9， 部分或完全阻断选择性细胞类型和组织中的DHCR 7。我们假设， 单独由视网膜神经元合成不足以维持它们的活力和功能;相反，它们依赖于 从血液（肝源性）脂蛋白、Müller胶质细胞和/或RPE中摄取Chol后， 要求。此外，正常Chol体内平衡的破坏引起小鼠中有缺陷的吞噬溶酶体生物学， RPE和视网膜。我们将如下检验我们的假设：在目标1中，我们将选择性地敲除杆中的Dhcr 7 PR和，分别在Müller胶质细胞（或串联），然后评估对甾醇组成的影响， 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)或单独（和串联），在肝脏（肝细胞）中，然后评估对固醇的影响 组成视网膜，肝脏和血液。利用这些资源和稳定同位素方法，我们将估计 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Dhcr 7 KO 小鼠以评估从头7 DHC合成及其摄取对RPE吞噬功能的影响。我们将 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和吞噬缺陷。 我们将使用新的体内试验，使用串联标记的自噬报告小鼠模型，系统地 阐明Chol合成在视网膜吞噬溶酶体生物学中的作用。所取得的成果将显著 推进我们对脊椎动物视网膜中Chol稳态的基本理解和机制 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 用于未来测试SLOS和相关孤儿疾病的更有效的治疗干预。