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: 10549728
• 负责人: Samuel Wang Du
• 金额: $ 4.38万
• 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/10549728
• 关键词: 11 cis Retinal; Age related macular degeneration; Binding; Biological Assay; Biology; Blindness; Cell physiology; Cells; Cessation of life; Compensation; Complement; Complex; Cytomegalovirus; Cytoskeleton; Development; Diagnostic; Disease; Disease model; Down-Regulation; Electrophysiology (science); Endosomes; Epithelium; 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; 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; Rodent Diseases; Role; 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; postmitotic; posttranscriptional; 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/文摘