Hippo Signaling in Regeneration of the Retinal Pigment Epithelium

项目总结 视网膜色素上皮(RPE)是位于视网膜和脉络膜之间的基本单层。 支持视网膜光感受器的功能和完整性。RPE在体内维持组织动态平衡 成体主要通过长期存活而不是细胞周转。中心性RPE变性和萎缩的原因 有害的慢性疾病老年性黄斑变性(AMD)，这是导致 老年人口中的失明。未来治疗干性AMD的可能性是通过刺激内源性 RPE再生，但对体内驱动RPE再生的机制知之甚少。一 候选途径是河马信号通路，因为它在调节细胞增殖和 在许多组织中再生，也是发展中的RPE细胞命运规范所必需的。在……里面 发育研究发现，神经纤维素2(NF2)对平衡RPE至关重要 生长和扩散。我的初步数据表明，河马信号通过NF2中断进行调制 在成人中刺激损伤后RPE的增殖和分化。这项提案旨在确定 河马信号的遗传操作如何促进体内的增殖和再生反应 哺乳动物的RPE。在目标1中，我将描述NF2在促进RPE损伤后再生中的作用。 成年人。我已经建立了一个损伤范例，在这个范例中，我对小鼠的RPE进行了化学损伤。再生将是 通过RPE和增殖标记物的表达、共定位和量化在细胞水平上进行评估， 并通过视网膜电描记术在功能水平上进行检测。在目标2中，我将确定YAP的监管机制 RPE中的RPE规范和维护。多项研究表明，RPE需要YAP 在胚胎和成体中的同一性；然而，其分子机制还不是很清楚。在这里，我会 探讨YAP和转录因子tead直接调控RPE-1表达的假说 用染色质免疫沉淀法和荧光素酶分析检测特异性基因。这些实验的结果 将揭示YAP如何调节RPE规范，以及河马信号通路如何被调制到 促进RPE再生。了解促进RPE的细胞和分子机制 再生将有助于确定和开发AMD的治疗策略。

PROJECT SUMMARY The retinal pigment epithelium (RPE) is an essential monolayer that lies between the retina and the choroid and supports the function and integrity of the retinal photoreceptors. The RPE maintains tissue homeostasis in the adult mainly via long-term survival instead of cell turnover. Degeneration and atrophy of the central RPE causes the detrimental chronic disease age-related macular degeneration (AMD), which is the leading cause of blindness in the elderly population. A possibility for treating dry AMD in the future is by stimulating endogenous RPE regeneration, but little is known about the mechanisms that can drive RPE regeneration in vivo. One candidate pathway is the Hippo signaling pathway, as it plays a role in regulating cell proliferation and regeneration in many tissues and is also required in the developing RPE for cell fate specification. In developmental studies, we and others have discovered that Neurofibromin 2 (NF2) is critical for balancing RPE growth and proliferation. My preliminary data suggests that modulation of Hippo signaling through Nf2 disruption in the adult stimulates proliferation and differentiation of the RPE following injury. This proposal aims to determine how genetic manipulation of Hippo signaling promotes an intrinsic proliferative and regenerative response in the mammalian RPE. In Aim 1, I will characterize the role of NF2 in promoting RPE regeneration after injury in the adult. I have established an injury paradigm, in which I chemically injure the RPE of mice. Regeneration will be assessed on a cellular level by expression, co-localization, and quantification of RPE and proliferative markers, and on a functional level by electroretinography. In Aim 2, I will determine the mechanism by which YAP regulates RPE specification and maintenance in the RPE. Several studies demonstrate that YAP is required for RPE identity in the embryo and adult; however, the molecular mechanism is not well understood. Here, I will investigate the hypothesis that YAP and the transcription factor TEAD directly regulate expression of RPE- specific genes by using chromatin immunoprecipitation and luciferase assays. The results of these experiments will reveal how YAP regulates RPE specification, and how the Hippo signaling pathway can be modulated to promote RPE regeneration. Understanding the cellular and molecular mechanisms that promote RPE regeneration will help identify and develop therapeutic strategies for AMD.