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

 描述（由申请人提供）：视力丧失是美国十大残疾之一，给社会带来沉重的经济负担。为了解决这一重大问题，国家眼科研究所最近宣布了大胆的目标，即“再生眼睛和视觉系统中的神经元和神经连接”（：//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剂，我们正在研究斑马鱼，视网膜损伤刺激Müller神经胶质细胞增殖并产生神经元祖细胞，再生缺失的斑马鱼神经元。虽然人类视网膜也有缪勒神经胶质，但它们无法再生视网膜神经元。识别诱导斑马鱼Müller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视网膜再生反应的方法。我们最近确定肿瘤坏死因子α（TNF α）和Notch信号传导分别为Müller胶质细胞增殖的正性和负性调节因子。然而，TNFa和Notch的信号传导通路不太清楚。阐明这些途径可以通过产生一种在患有各种形式失明的个体中再生视网膜神经元的策略来显着推进NEI的大胆目标。 我们的长期目标是识别和 表征再生受损的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（TNF α）是在垂死的斑马鱼光感受器中产生的，并且是Müller胶质细胞增殖所必需和充分的。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TNF α，TNF α结合Müller胶质细胞上的受体并激活Stat 3。Stat 3然后调节Ascl 1a的表达以诱导Müller胶质细胞增殖。此外，视网膜损伤抑制Notch信号传导，以增加Ascl 1a和Müller胶质细胞增殖的表达，这可能是通过降低他/她基因的表达。 目的1将探索启动Müller胶质细胞增殖的TNF α信号通路的组成部分及其作用于哪些细胞，包括TNF α诱导Stat 3和Ascl 1a表达的能力，Stat 3在诱导Ascl 1a表达中的潜在作用，如果Stat 3必须在Müller胶质细胞或其他视网膜细胞类型中被激活以用于Müller胶质细胞增殖，以及TNF α是否直接或通过中间体如NF-κB（NF-κ B）、JNK或p38激活Stat 3。目的2将研究Notch信号通路在未受损视网膜中维持Müller胶质细胞处于静止（非增殖）状态中的作用。我们将确定Notch活性是否必须在Müller胶质细胞中才能维持静止，并确定Notch受体和配体的身份，这些受体和配体是防止Müller胶质细胞重新进入未受损视网膜细胞周期所必需的。 因此，该项目的预期结果将揭示TNF α和Notch作为Müller胶质细胞增殖的正调控因子和负调控因子的关系，以及Stat 3和Ascl 1a在受损的斑马鱼视网膜中的调节方式。我们预计，这项工作的影响将导致更好地了解是什么调节米勒胶质细胞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，这些方法使用内源性Müller神经胶质细胞来再生视力丧失个体中丢失的视网膜神经元。

 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.