microRNA-204 and microRNA-211 regulation of RPE phagocytosis

microRNA-204 和 microRNA-211 对 RPE 吞噬作用的调节

• 批准号: 10388983
• 负责人: Samuel Wang Du
• 金额: $ 4.22万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-07 至 2027-01-06
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388983
• 关键词: 11 cis Retinal; Age related macular degeneration; Binding; Biological Assay; Biology; Blindness; Cell physiology; Cells; Cessation of life; Complement; Complex; Cytomegalovirus; Cytoskeleton; Development; Diagnostic; Disease; Disease model; Down-Regulation; Electrophysiology (science); Endosomes; Epithelial; Exhibits; Exposure to; Extracellular Matrix; Eye; Functional disorder; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Recombination; Genetic Transcription; Health; Histologic; Homeostasis; Hour; Human; Human body; Image; Impairment; In Vitro; Intraperitoneal Injections; Joints; Knock-in; Knock-in Mouse; Knockout Mice; Knowledge; Lead; Leber' s amaurosis; Light; Link; Luciferases; Lysosomes; Maintenance; Mediating; Medical; Messenger RNA; MicroRNAs; Mitotic; Mus; Nucleotides; Nutrient; Ocular Physiology; Organelles; Pathogenesis; Pathway interactions; Pattern; Periodicity; Phagocytes; Phagocytosis; Phenotype; Photoreceptors; Phototransduction; Physiology; Play; Process; Property; Proteins; Quantitative Reverse Transcriptase PCR; RNA Polymerase II; RNA-Induced Silencing Complex; RPE65 protein; Regulation; Research; Retina; Retinal Degeneration; Retinal Photoreceptors; Retinal Pigments; Rodent Diseases; Role; Seeds; Signal Transduction; Structure of retinal pigment epithelium; System; Tamoxifen; Testing; Therapeutic; Tissue-Specific Gene Expression; Tissues; Untranslated RNA; Untranslated Regions; Up-Regulation; Vertebrate Photoreceptors; Vision; Visual; Visual Acuity; Work; age related; aged; base; cell type; chromophore; circadian; circadian pacemaker; circadian regulation; combat; differential expression; disability; ezrin; human disease; induced pluripotent stem cell; intraperitoneal; knock-down; lipid metabolism; melanoma; monolayer; overexpression; photoreceptor degeneration; pleiotropism; single-cell RNA sequencing; social; therapeutic development; trafficking

PROJECT SUMMARY/ABSTRACT Age-related macular degeneration (AMD) is a disease that leads to the loss of visual acuity, resulting in a substantial medical and social burden. Many unique pathways lead to the pathogenesis of AMD. The retinal pigment epithelium (RPE) is a post-mitotic epithelial monolayer which sits directly adjacent to the photoreceptor outer segments (POS) and is responsible for the maintenance of photoreceptor health. Among its functions include the generation of 11-cis-retinal chromophore for phototransduction, nutrient delivery, and diurnal phagocytosis of spent POS. Degeneration and dysfunction in the RPE is linked to the subsequent decline in photoreceptors seen in diseases such as AMD and Leber’s congenital amaurosis. Therefore, understanding RPE dysfunction is critical to understanding overall retinal health, but regulation of these key RPE roles is still incompletely understood. Recent research has pointed to the important role of microRNA (miR) regulation of gene expression, and miRs are critical for ophthalmic development and homeostasis. Of these miRs, miR-204 and miR-211 are among the most highly expressed miRs in the RPE and have been shown to both maintain its epithelial properties and modulate endosomal/lysosomal processing. These two miRs share the same seed sequence and are hypothesized to regulate many of the same genes, and are themselves regulated by light and the circadian clock. Previous studies have implicated the expression of these miRs in the control of RPE phagocytosis, but this has not been fully tested in a RPE specific manner. Thus, this proposal will examine the role(s) of miR-204 and miR-211 in an RPE specific manner and test the hypothesis that these two miRs regulate RPE phagocytosis. To pursue these objectives, we have generated miR-204fl/fl and miR-211fl/fl double knock-in mice. Induction of recombination in double knock-in mice will be achieved by (1) crossing them with RPE65-ERT2-cre mice and inducing recombination in the resultant triple knock-in mice by intraperitoneal injection of tamoxifen; and (2) subretinal delivery of AAV1-CMV-cre-GFP. The miR double knockout mice will be aged and assessed for phenotypic, electrophysiological, and histological changes. RPE and retina from these mice will also be collected and analyzed through bulk and single cell RNA sequencing to examine changes in gene expression resulting from miR-204/miR-211 deletion. The direct target genes of these two miRs will be assessed through the application of Halo-enhanced Ago2 pulldown (HEAP). We will also perform the in vitro culture of double knock-in mouse RPE and challenge them with POS to assess their phagocytic capacity. Lastly, we will correlate the results for mouse RPE to analogous results for human RPE through the use of RPE cultures derived from induced pluripotent stem cells. Overall, these studies will seek to define the role(s) of miR-204 and miR-211 in the RPE, identify their regulated genes, and suggest further genes and pathways to target in the development of AMD therapeutics and diagnostics.

项目总结/摘要 视网膜相关性黄斑变性（AMD）是一种导致视力丧失的疾病， 沉重医疗和社会负担。许多独特的途径导致AMD的发病机制。 视网膜色素上皮（RPE）是有丝分裂后的上皮单层，其直接邻近视网膜色素上皮（RPE）。 光感受器外节（POS），负责维持光感受器的健康。之间 其功能包括产生用于光传导的11-顺式-视网膜发色团、营养递送， 消耗POS的昼夜吞噬作用。视网膜色素上皮的变性和功能障碍与随后的 在AMD和利伯氏先天性黑蒙等疾病中可见的光感受器下降。 因此，了解RPE功能障碍对于了解整体视网膜健康至关重要，但RPE功能障碍的调节 这些关键的RPE角色仍然不完全清楚。最近的研究表明， microRNA（miR）调节基因表达，miR对眼发育和眼内分泌至关重要。 体内平衡。在这些miR中，miR-204和miR-211是RPE中最高表达的miR 并且已经显示既保持其上皮性质又调节内体/溶酶体加工。 这两个miR共享相同的种子序列，并且被假设调节许多相同的基因， 它们自身也受光和生物钟的调节。先前的研究表明， 这些miR在RPE吞噬作用的控制中起重要作用，但这还没有以RPE特异性方式进行充分测试。 因此，本提案将以RPE特异性方式检查miR-204和miR-211的作用，并测试miR-204和miR-211在RPE特异性方式中的作用。 假设这两种miR调节RPE吞噬作用。为了实现这些目标，我们 miR-204 fl/fl和miR-211 fl/fl双敲入小鼠。将在双敲入小鼠中诱导重组。 通过（1）将它们与RPE 65-ERT 2-cre小鼠杂交并在所得三联体中诱导重组来实现 通过腹膜内注射他莫昔芬敲入小鼠;和（2）视网膜下递送AAV 1-CMV-cre-GFP。的 将对miR双敲除小鼠进行老化并评估表型、电生理学和组织学 变化还将收集这些小鼠的RPE和视网膜，并通过大量和单细胞RNA进行分析 测序以检查由miR-204/miR-211缺失引起的基因表达的变化。直接 这两种miR的靶基因将通过应用Halo增强的Ago 2 pulldown来评估 （堆）。我们还将进行双基因敲入小鼠RPE的体外培养，并用POS进行激发 来评估它们的吞噬能力最后，我们将把小鼠RPE的结果与 通过使用衍生自诱导多能干细胞的RPE培养物来培养人RPE。总的来说，这些 研究将试图确定miR-204和miR-211在RPE中的作用，鉴定其调控基因， 提出了在AMD治疗和诊断的开发中靶向的其他基因和途径。