Hippo and Wnt signaling in Müller Glia-mediated Retinal Regeneration

1项目摘要（限制：30行文本） 2随着年龄的增长，青光眼、糖尿病视网膜病变和年龄相关性黄斑变性（AMD）等疾病 3导致视网膜神经元的不可逆损失，从而导致视力下降，这导致生活质量下降， 4、临床负担重。虽然正在追求几种治疗方法，但基因治疗的最新进展 5疗法显示出治疗导致视网膜变性的遗传性疾病的前景。也是 如果类似的方法可以用于疾病的视网膜再生策略， 7 AMD，没有明显的遗传原因。视网膜再生的一个潜在来源可能是在 8例哺乳动物Müller胶质细胞（MG）。与哺乳动物不同，斑马鱼MG具有以下能力： 9产生多能细胞，可以分化成新的视网膜神经元，能够恢复视力。我们实验室正在 10目前正在研究可能阻断哺乳动物MG细胞的细胞和分子机制。 11介导的视网膜再生，我们已经发现了Hippo信号通路的这种作用。因此，我们认为， 12我的建议旨在使用腺相关病毒（AAV）介导的基因传递来操纵河马。 13通路，并评估其再生潜力。 14 Hippo信号通路已被证明抑制心肌细胞增殖和破坏心肌细胞。 15 Hippo信号显著促进哺乳动物心脏的再生。在我的第一个共同第一作者 16出版物，通过使用转录辅因子雅普的组成型活性形式的转基因表达 17（YAP 5SA），我能够绕过Hippo信号传导的负调控。这些河马无反应的MG 18自发进入细胞周期，并重新编程为一个身份，更让人想起多能视网膜 19个祖细胞（RPC）。最近，研究表明Wnt通路效应子β-连环蛋白的过度表达， 20还将MG重新编程为RPC样状态，然后可以重新分化为功能性杆， 21恢复视力。在其他系统中，已经显示雅普和β-连环蛋白在功能上协同作用， 22转录因子复合体。综上所述，这些数据使我假设雅普和β-连环蛋白 23协同驱动MG重编程为多能状态，能够分化为视网膜神经元。 本项目的主要目标是确定雅普和β-连环蛋白是否类似地重编程 25毫克，导致相同的神经原性电位。首先，我将确定雅普之间的物理相互作用 26和MG中的β-连环蛋白。然后，使用AAV介导的基因递送到β-连环蛋白突变体MG，我将确定 27 YAP 5SA介导的MG重编程是否需要活性β-连环蛋白。最后，我将决定是否 28 YAP 5SA重编程MG确实是多潜能的，因此能够分化成几种 29个视网膜神经元的身份。该提案的成功完成将可能导致AAV的未来发展 30种方法来操纵Hippo和Wnt通路以实现真正的视网膜再生。

1 Project Summary (limit: 30 lines of text) 2 As we age, diseases such as glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD) 3 result in irreversible loss of retinal neurons and thus sight, which leads to a reduced quality of life and a 4 significant clinical burden. While several therapeutic approaches are being pursued, recent advances in gene 5 therapy show promise for treating genetic disorders leading to retinal degeneration. It would also be 6 advantageous if a similar approach could be employed in retinal regenerative strategies for diseases such as 7 AMD, which has no obvious genetic cause. One potential source of retinal regeneration may be found within 8 mammalian Müller glial cells (MGs). Unlike mammalian counterparts, zebrafish MGs have the ability to 9 generate multipotent cells that can differentiate into new retinal neurons capable of restoring vision. Our lab is 10 currently investigating the cellular and molecular mechanisms that potentially block mammalian MG-cell 11 mediated retinal regeneration and we have uncovered such a role for the Hippo signaling pathway. Therefore, 12 my proposal aims to use Adeno-associated virus (AAV)-mediated gene delivery to manipulate the Hippo 13 pathway within mouse MGs and assess their regenerative potential. 14 The Hippo signaling pathway has been shown to restrain cardiomyocyte proliferation and disruption of 15 Hippo signaling significantly promotes regeneration of the mammalian heart. In my first co-first author 16 publication, by using transgenic expression of a constitutively active form of the transcription cofactor YAP 17 (YAP5SA), I was able to bypass the negative regulation of Hippo signaling. These Hippo non-responsive MGs 18 spontaneously entered the cell cycle and reprogrammed to an identity more reminiscent of multipotent retinal 19 progenitor cells (RPCs). Recently, it was shown that overexpression of the Wnt pathway effector ß-CATENIN 20 also reprogrammed MGs into a RPC-like state, which could then be re-differentiated into functional rods to 21 restore vision. In other systems, YAP and ß-CATENIN have been shown to functionally cooperate in a 22 transcription factor complex. Taken together, these data have led me to hypothesize that YAP and ß-CATENIN 23 cooperatively drive MG reprogramming to a multipotent state, capable of differentiating into retinal neurons. 24 The main objective of this project is to determine whether YAP and ß-CATENIN similarly reprogram 25 MGs, resulting in the same neurogenic potential. First, I will determine the physical interaction between YAP 26 and ß-CATENIN in MGs. Then, using AAV-mediated gene delivery to ß-Catenin mutant MGs, I will determine 27 whether YAP5SA-mediated MG reprogramming requires active ß-CATENIN. Finally, I will determine whether 28 YAP5SA-reprogrammed MGs are indeed multipotential and thereby capable of differentiation into several 29 retinal neuron identities. Successful completion of this proposal will likely lead to future development of AAV 30 approaches to manipulate both the Hippo and Wnt pathways for bona fide retinal regeneration.