microRNA-204 and microRNA-211 regulation of RPE phagocytosis

项目摘要/摘要 老年性黄斑变性(AMD)是一种导致视力丧失的疾病，导致 沉重的医疗和社会负担。许多独特的途径导致了AMD的发病。 视网膜色素上皮(RPE)是一种有丝分裂后的单层上皮层，它直接位于 光感受器外节(POS)，负责维持光感受器健康。其中 它的功能包括产生11顺式视网膜发色团，用于光传导、营养输送和 用过的POS的日吞噬功能。RPE中的变性和功能障碍与随后的 在AMD和Leber‘s先天性黑猩猩等疾病中所见的光感受器功能下降。 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 这些关键的RPE角色仍然没有完全被理解。最近的研究已经指出， MicroRNA(MiR)调节基因表达，miR对眼球发育和 动态平衡。在这些miRs中，miR-204和miR-211是RPE中表达最高的miRs 并已被证明既能维持其上皮性，又能调节内体/溶酶体的加工。 这两个MIR共享相同的种子序列，并被假设为调节许多相同的基因， 它们自身受到光和生物钟的调节。此前的研究表明，这一表达与 这些MIR在控制RPE吞噬作用中的作用，但这还没有在RPE特有的方式中得到充分的测试。 因此，本提案将以RPE特定的方式审查MIR-204和MIR-211的作用(S)，并测试 假设这两个MIR调节RPE吞噬作用。为了实现这些目标，我们产生了 MiR-204fl/fl和miR-211fl/fl双敲入小鼠。双敲入小鼠的重组诱导将是 通过(1)将它们与RPE65-ERT2-cre小鼠杂交并在所得到的三元组中诱导重组 (2)视网膜下注射AAV1-CMV-cre-GFP。这个 MIR双基因敲除小鼠将被老化，并对其表型、电生理和组织学进行评估 改变。这些小鼠的RPE和视网膜也将通过散装和单细胞RNA收集和分析 测序以检测miR-204/miR-211缺失导致的基因表达变化。直接式 这两个miR的靶基因将通过Halo增强的Ago2 Pull Down进行评估 (堆)。我们还将进行双敲入小鼠RPE的体外培养，并用POS挑战它们 以评估它们的吞噬能力。最后，我们将小鼠RPE的结果与类似的结果相关联 通过使用来自诱导多能干细胞的RPE培养来获得人类RPE。总的来说，这些 研究将寻求确定miR-204和miR-211在RPE中的作用(S)，确定它们的调控基因，以及 建议在AMD治疗和诊断的发展中有更多的基因和途径作为靶点。

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.