Regeneration of rod photoreceptors from Muller glial cells in adult mouse retina

 描述(申请人提供)：穆勒胶质细胞(MGs)是脊椎动物视网膜的主要支持细胞。在斑马鱼等冷血脊椎动物中，MGs是干细胞的来源，因为它们可以很容易地重新进入细胞周期，补充丢失的神经元，建立强大的自我修复机制。然而，在哺乳动物中，MG天生就是静止的，缺乏再生能力。光感受器是哺乳动物视网膜中最丰富的细胞，它们调节视觉的第一步。光感受器的死亡是导致包括老年性黄斑变性(AMD)和视网膜色素变性(RP)在内的主要视网膜退行性疾病视力障碍和失明的主要原因。旨在恢复MGs在哺乳动物中的再生能力的广泛研究努力收效甚微。目前MG来源的光感受器再生的策略依赖于视网膜损伤和各种因素对整个视网膜的治疗。视网膜损伤首先会杀死视网膜神经元。对整个视网膜的整体治疗可能会导致非靶向细胞的不良副作用。我们研究的长期目标是了解MG来源的光感受器再生的分子和细胞途径，并开发策略来激活哺乳动物MG的再生能力以进行视网膜自我修复。我们建议在体内重新编程成年小鼠MG，以在不损伤视网膜的情况下再生杆状感光细胞：目的1)研究Wnt信号是否为损伤诱导的激活MG增殖的信号通路。我们将研究神经毒性损伤是否激活Wnt信号，并进一步测试抑制Wnt信号是否抑制损伤诱导的MG增殖。为了针对MGs细胞类型特异性，我们将开发一种针对MGs细胞类型特异性的基因转移方法。目的2)在不引起视网膜损伤的情况下，通过激活Wnt信号，恢复MGs的视网膜祖细胞/干细胞状态。我们将研究β-catenin基因转移是否在不损伤视网膜的情况下激活Wnt信号和MG的增殖。Gsk3β通过β-连环蛋白的磷酸化来调节Wnt信号，导致其降解。我们将研究在没有视网膜损伤的情况下，GSK3β的缺失是否激活了Wnt信号和MG的增殖。目的3)引导MG来源的视网膜祖细胞/干细胞向视杆细胞分化 光感受器。我们将通过基因转移转录因子来指导MG来源的视网膜前体/干细胞的分化，这些转录因子对于视杆细胞在视网膜发育过程中的命运和分化至关重要，并测试MG来源的新视杆是否具有天然视杆的分子、结构和功能特性。我们的研究将极大地促进我们对成年哺乳动物视网膜中MG衍生杆感光细胞再生的基本机制和功能的理解，并将为以光感受器退化为典型特征的主要视网膜退行性疾病的视网膜自我修复奠定基础。

 DESCRIPTION (provided by applicant): Müller glial cells (MGs) are the primary support cells in the vertebrate retina. In cold-blooded vertebrates such as zebrafish, MGs are a source of stem cells for they can readily re-enter the cell cycle and replenish lost neurons, establishing a powerful self-repair mechanism. In mammals, however, MGs are naturally quiescent and lack regenerative capability. Photoreceptors are the most abundant cells in the mammalian retina and they mediate the first step in vision. The death of photoreceptors is a leading cause of vision impairment and blindness in major retinal degenerative diseases including age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Extensive research efforts aimed at restoring the regenerative capability of MGs in mammals have met with little success. Current strategies for MG-derived photoreceptor regeneration rely on retinal injury and treatment of the whole retina with various factors. Retinal injury kills retinal neurons in the first place. Global treatment of the entire retina may lead to undesirable side effects in untargeted cells. The long-term goal of our research is to understand the molecular and cellular pathways underlying MG-derived photoreceptor regeneration, and to develop strategies to activate the regenerative capability of mammalian MGs for retinal self-repair. We propose to reprogram adult mouse MGs, in vivo, for regeneration of rod photoreceptors without retinal injury, through the following Aims: Aim 1) Investigate whether Wnt signaling is an injury-induced signaling pathway to activate MG proliferation. We will examine whether neurotoxic injury activates Wnt signaling and further test whether inhibition of Wnt signaling suppressed injury-induced MG proliferation. To target MGs cell-type-specifically, we will develop a gene transfer method targeting MGs cell-type-specifically. Aim 2) Restore the retinal progenitor/stem cell status of MGs through activation of Wnt signaling, without introduction of retinal injury. We will investigate whether gene transfer of β-catenin activates Wnt signaling and MG proliferation without retinal injury. GSk3β regulates Wnt signaling by phosphorylation of β-catenin leading to its degradation. We will examine whether deletion of GSK3β activates Wnt signaling and MG proliferation without retinal injury. Aim 3) Guide the differentiation of MG-derived retinal progenitor/stem cells to rod photoreceptors. We will guide the differentiation of MG-derived retinal progenitor/stem cells by gene transfer of transcription factors that are essential for rod photoreceptor cell fate determination and differentiation during retinal development, and test whether MG-derived new rods develop molecular, structural, and functional properties of native rods. Our proposed research will significantly advance our understanding of the basic mechanisms and functional implications of MG-derived rod photoreceptor regeneration in adult mammalian retina, and will set the stage for retinal self-repair in a major group of retinal degenerative diseases typically characterized by photoreceptor degeneration.