Hippo and Wnt signaling in Müller Glia-mediated Retinal Regeneration

1个项目摘要(限制：30行文本) 随着年龄的增长，青光眼、糖尿病视网膜病变和老年性黄斑变性(AMD)等疾病 3会导致视网膜神经元不可逆转的丧失，从而导致视力下降，从而导致生活质量下降和视力下降。 4临床负担大。在寻求几种治疗方法的同时，基因治疗的最新进展 5治疗方法在治疗导致视网膜退化的遗传性疾病方面表现出了希望。它也会是 如果类似的方法可以应用于视网膜再生治疗疾病，如 AMD 7例，无明显遗传原因。视网膜再生的一个潜在来源可能是在 8个哺乳动物Müler神经胶质细胞(MGs)。与哺乳动物不同，斑马鱼MGs有能力 9产生多能细胞，可以分化为新的视网膜神经元，能够恢复视力。我们的实验室是 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介导了视网膜再生，我们已经发现了河马信号通路的这种作用。因此， 12我的提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，并评估其再生潜能。 14河马信号通路已被证明可以抑制心肌细胞的增殖和破坏 河马信号显著促进哺乳动物心脏的再生。在我的第一个联合第一作者中 16发表，通过使用转录辅因子Yap的构成活性形式的转基因表达 17(YAP5SA)，我能够绕过河马信号的负调控。这些河马没有反应的MG 18自发地进入细胞周期，并重新编程为更像多能视网膜的身份 19个祖细胞(RPC)。最近的研究表明，Wnt通路效应器？连环蛋白的过度表达 20还将MG重新编程为类似RPC的状态，然后可以重新分化为功能棒以 21恢复视力。在其他系统中，YAP和ç-catenin已被证明在功能上合作 22转录因子复合体。综上所述，这些数据让我假设YAP和ç-catenin 23协同驱动MG重新编程为多能状态，能够分化为视网膜神经元。 24这个项目的主要目标是确定YAP和？连环蛋白是否类似地重新编程 25毫克，导致相同的神经生成潜力。首先，我将确定YAP之间的物理交互 26和β-连环蛋白。然后，使用AAV介导的基因传递到ç-catenin突变体MGs，我将确定 27 YAP5SA介导的MG重编程是否需要活性？连环蛋白。最后，我会决定是否 28个YAP5SA-重新编程的MG确实是多潜能的，因此能够分化为几个 29个视网膜神经元身份。这项提案的成功完成将可能导致AAV的未来发展 控制河马和Wnt通路的30种方法，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.