Roles of TNFa and Notch to Initiate Retinal Regeneration from Muller glia

 描述(申请人提供)：视力障碍是美国十大残疾之一，给社会造成了沉重的经济负担。为了解决这一重大问题，美国国家眼科研究所最近宣布了一项大胆的目标，即“再生眼睛和视觉系统中的神经元和神经连接”(网址：www.nei.nih.gov/audacious/)。为了实现这一大胆的目标，有必要确定“激活潜在的内源性细胞以取代丢失的宿主神经元”所需的分子信号。为了确定这些潜在的调节因子，我们正在研究斑马鱼，在斑马鱼中，视网膜损伤刺激Müler神经胶质细胞增殖，并产生神经元前体细胞，再生丢失的斑马鱼神经元。虽然人的视网膜也有Müler胶质细胞，但它们不能再生视网膜神经元。识别诱导斑马鱼Müler神经胶质细胞启动再生反应的分子开关，可能揭示在人类中诱导类似的视网膜再生反应的方法。我们最近发现肿瘤坏死因子α(TNFa)和Notch信号分别是Müller胶质细胞增殖的正性和负性调节因子。然而，TNFa和Notch的信号通路还不是很清楚。阐明这些途径可以极大地推进NEI的大胆目标，因为它可以产生一种策略，在患有各种形式失明的人中再生视网膜神经元。我们的长期目标是识别和 描述再生受损斑马鱼视网膜所需的分子和细胞事件。我们最近发现，肿瘤坏死因子-α(TNFa)是在濒临死亡的斑马鱼光感受器中产生的，是Müler神经胶质细胞增殖的必要条件和充分条件。我们还观察到，抑制Notch信号足以诱导Müller胶质细胞重新进入细胞周期，这表明Notch是启动再生反应的负调控因子。我们的中心假设是死亡的光感受器产生TNFa，它结合Müler神经胶质细胞上的受体并激活STAT3。然后，STAT3调节Ascl1a的表达，从而诱导Müler胶质细胞的增殖。此外，视网膜损伤抑制了Notch信号，增加了Ascl1a的表达和Müller细胞的增殖，可能是通过减少他/她的基因表达而实现的。 目的1将探讨启动Müler神经胶质细胞增殖的TnFa信号通路的组成部分及其作用于哪些细胞，包括TnFa诱导STAT3和Ascl1a表达的能力，STAT3在诱导Ascl1a表达中的潜在作用，STAT3是否必须在Müler胶质细胞或其他类型的视网膜细胞中被激活才能促进Müller胶质细胞的增殖，以及TnFa是否直接或通过如核因子-κB(NF-kappaB)、JNK或p38等中介激活STAT3。目的2将研究Notch信号通路在未受损视网膜中维持Müler胶质细胞处于静止(非增殖)状态的作用。我们将确定Notch活性是否必须在Müller胶质细胞中才能保持静止，并确定Notch受体和配体的身份，这些受体和配体是在未受损的视网膜中阻止Müler神经胶质细胞重新进入细胞周期所必需的。因此，该项目的预期结果将揭示TNFa和Notch作为Müller胶质细胞增殖的正负调控因子的关系，以及STAT3和Ascl1a在受损的斑马鱼视网膜中是如何调控的。我们预计，这项工作的影响将导致更好地理解是什么调节了Müler glia重新进入受损视网膜的细胞周期。这项工作还将有助于开发潜在的治疗方法，使用内源性Müler胶质细胞来再生视力丧失患者的视网膜神经元。

 DESCRIPTION (provided by applicant): Vision loss is among the top ten disabilities in the United States, which results in a heavy financial burden on society. To remedy this significant problem, the National Eye Institute recently announced the Audacious Goal to "regenerate neurons and neural connections in the eye and visual system" (://www.nei.nih.gov/audacious/). To accomplish this Audacious Goal, it is necessary to identify the molecular signals needed to "activate latent endogenous cells to replace lost host neurons". To identify these potential regulators, we are studying zebrafish, where retinal damage stimulates Müller glia to proliferate and produce neuronal progenitors that regenerate the missing zebrafish neurons. While the human retina also possesses Müller glia, they are unable to regenerate retinal neurons. Identifying the molecular switches that induce the zebrafish Müller glia to initiate the regeneration response may reveal approaches to induce a similar retinal regeneration response in humans. We recently identified tumor necrosis factor alpha (TNFa) and Notch signaling as positive and negative regulators of Müller glia proliferation, respectively. However, the signalin pathways of TNFa and Notch are less clear. Elucidating these pathways could significantly advance the NEI's Audacious Goal by yielding a strategy to regenerate retinal neurons in individuals who suffer from a variety of forms of blindness. Our long-term goal is to identify and characterize the molecular and cellular events required to regenerate the damaged zebrafish retina. We recently found that tumor necrosis factor-alpha (TNFa) is produced in the dying zebrafish photoreceptors and is necessary and sufficient for Müller glia proliferation. We also observed that repressing Notch signaling is sufficient to induce Müller glia to reenter the cell cycle, suggesting that Notch is a negative regulator of initiating the regeneration response. Our central hypothesis is that dying photoreceptors produce TNFa, which binds receptors on the Müller glia and activates Stat3. Stat3 then regulates the expression of Ascl1a to induce Müller glia proliferation. Additionally, retinal damage represses Notch signaling to increase expression of Ascl1a and Müller glia proliferation, likely through the decreased expression of his/her genes. Aim 1 will explore the components of the TNFa signaling pathway that initiates Müller glia proliferation and in what cells they act, including the ability of TNFa to induce expression of Stat3 and Ascl1a, the potential role of Stat3 in inducing Ascl1a expression, if Stat3 must be activated in Müller glia or another retinal cell type for Müller glia proliferation, and if TNFa activates Stat3 directly or through an intermediate such as NF-κB (NF-kappaB), JNK, or p38. Aim 2 will examine the role of the Notch signaling pathway to maintain Müller glia in a quiescent (non-proliferating) state in undamaged retinas. We will determine if Notch activity must be in the Müller glia to maintain quiescence and determine the identity of the Notch receptor and ligand that are required to keep the Müller glia from reentering the cell cycle in undamaged retinas. Thus, the expected outcomes of this project will reveal the relationships of TNFa and Notch as positive and negative regulators of Müller glia proliferation and how Stat3 and Ascl1a are regulated in the damaged zebrafish retina. We anticipate that the impact of this work will lead to a better understanding of what regulates Müller glia reentry into the cell cycle in the damaged retina. This work will also assist in the development of potential therapeutic approaches that use endogenous Müller glia to regenerate lost retinal neurons in individuals suffering from vision loss.