Hippo Signaling in Regeneration of the Retinal Pigment Epithelium

项目摘要 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。视网膜色素上皮维持视网膜中的组织稳态。 成体主要通过长期存活而不是细胞更新。视网膜色素上皮中央变性和萎缩的原因 有害的慢性疾病年龄相关性黄斑变性（AMD），这是导致 老年人的失明。未来治疗干性AMD的可能性是通过刺激内源性 RPE再生，但很少有人知道的机制，可以驱动RPE再生在体内。一 候选途径是Hippo信号传导途径，因为它在调节细胞增殖中起作用， 它在许多组织中是再生的关键，并且在发育中的RPE中也是细胞命运特化所必需的。在 在发育研究中，我们和其他人发现神经纤维蛋白2（NF 2）对平衡RPE至关重要 生长和增殖。我的初步数据表明，通过破坏Nf 2来调节Hippo信号 在成人中刺激损伤后RPE的增殖和分化。该提案旨在确定 Hippo信号的遗传操纵如何促进了细胞内固有的增殖和再生反应， 哺乳动物RPE。在目标1中，我将描述NF 2在促进RPE损伤后再生中的作用。 成年人了我已经建立了一个损伤范例，在这个范例中，我用化学方法损伤了小鼠的RPE。再生将得以 通过RPE和增殖标记物的表达、共定位和定量在细胞水平上进行评估， 和视网膜电图的功能水平。在目标2中，我将确定雅普调节 RPE中的RPE规范和维护。几项研究表明，雅普是RPE所必需的 在胚胎和成人中的同一性;然而，分子机制还没有很好地理解。来，我来 研究雅普和转录因子TEAD直接调节RPE表达的假设。 通过染色质免疫沉淀和荧光素酶测定来检测特定基因。这些实验的结果 将揭示雅普如何调节RPE特化，以及Hippo信号通路如何被调节， 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.