Muller glial cell pathophysiology during glaucoma onset

项目摘要 本提案的目标是了解神经胶质细胞病理生理学如何促进疾病进展， 青光眼，一种以视网膜神经节细胞功能障碍和死亡为特征的神经退行性眼病 （RGC），视网膜的输出神经元。眼内压（IOP）升高是最常见的风险因素， 虽然高眼压和RGC丢失的机制仍然知之甚少。在这里，我们专注于 重要但以前未检查的疾病进展方面：Müller早期结构重塑 神经胶质细胞有助于RGCs的变性。在青光眼中，与其他视网膜疾病一样，Müller细胞经历了一个 各种分子和形态学变化称为反应性神经胶质增生。然而，人们对 Müller细胞如何在疾病早期对高眼压做出反应，或者神经胶质增生如何导致RGC变性。我们 将使用光学成像、转基因小鼠、病毒技术和膜片钳电生理学的组合 来填补我们知识上的空白。疾病期间异常的Ca 2+信号传导 发病可能是在Müller细胞神经胶质增生明显体征之前发生的重要上游事件。在目标1A中，我们 我将测试我们的初步发现所提出的假设，即压力升高会导致过量的Ca 2 + 视网膜的内丛状层（IPL）内的Müller细胞突触周围过程中的信号传导。我们将使用 双光子（2 P）显微镜，比较正常压力下眼睛与正常压力下眼睛Müller神经胶质中的Ca 2+信号。 其IOP在青光眼的微珠阻塞模型中长期升高。在目标1B中，我们将直接 测试Ca 2+信号传导的变化是否可以通过选择性地改变Müller细胞的形态学变化， 操纵Müller细胞内[Ca 2 +]。目标2A中的实验将使用微珠阻塞模型， 荧光标记的Müller胶质细胞和RGCs的2 P成像，以研究高眼压是否改变了视网膜神经节细胞的功能。 Müller胶质细胞细突起和RGC树突之间的物理关系。在我们的初步工作中， 慢性高眼压眼组织中Müller细胞的形态，我们发现Müller细胞 过程缩回到IPL的特定亚层内。我们将测试的假设，建议由这些初步的 结果，Müller细胞过程选择性地退出某些RGC亚型后不久，眼 高血压Müller细胞突触周围突起的一个重要功能是调节细胞外谷氨酸 靠近突触Müller胶质细胞的去除可以通过促进RGC而对疾病进展做出重要贡献 因兴奋性毒性损伤而死亡目标2B中的实验将通过使用双光子Ca 2+来检验这种可能性。 成像以检验IOP升高增加光诱发的N-甲基-D-天冬氨酸型谷氨酸的假设 受体活性的树突的特定RGC亚型。在这两个目标中，我们将特别侧重于早期阶段 通过在IOP升高后的前1-2周内进行实验来评估疾病。通过识别 Müller细胞的病理生理变化发生在疾病进展的早期，这项工作将有助于揭示 在RGC变性导致不可逆视力丧失之前诊断和治疗青光眼的新方法。

PROJECT SUMMARY The goal of this proposal is to understand how glial cell pathophysiology contributes to disease progression in glaucoma, a neurodegenerative eye disease characterized by dysfunction and death of retinal ganglion cells (RGCs), the output neurons of the retina. Elevated intraocular pressure (IOP) is the most common risk factor for glaucoma, yet the mechanisms linking high IOP and RGC loss remain poorly understood. Here, we focus on an important but previously unexamined aspect of disease progression: how early structural remodeling of Müller glial cells contributes to degeneration of RGCs. In glaucoma, as in other retinal diseases, Müller cells undergo a variety of molecular and morphological changes referred to as reactive gliosis. However, little is known regarding how Müller cells respond to ocular hypertension early in disease or how gliosis leads to RGC degeneration. We will use a combination of optical imaging, transgenic mice, viral techniques, and patch-clamp electrophysiology in an ex vivo eyecup preparation to fill these gaps in our knowledge. Aberrant Ca2+ signaling during disease onset may be an important upstream event that occurs prior to overt signs of Müller cell gliosis. In Aim 1A, we will test the hypothesis, suggested by our preliminary findings, that elevated pressure induces excessive Ca2+ signaling in Müller cell peri-synaptic processes within the inner plexiform layer (IPL) of the retina. We will use two-photon (2P) microscopy to compare Ca2+ signals in Müller glia in eyes with normal pressure vs. eyes in which IOP has been chronically elevated in a microbead occlusion model of glaucoma. In Aim 1B, we will directly test whether changes in Ca2+ signaling can drive morphological changes in Müller cells by selectively manipulating Müller cell intracellular [Ca2+]. Experiments in Aim 2A will use the microbead occlusion model and 2P imaging of fluorescently labeled Müller glia and RGCs to investigate whether ocular hypertension alters the physical relationship between Müller glial fine processes and RGC dendrites. In our preliminary work examining the morphology of Müller cells in tissue from eyes with chronically elevated IOP, we found that Müller cell processes retract within specific sublayers of the IPL. We will test the hypothesis, suggested by these preliminary findings, that Müller cell processes selectively withdraw from certain RGC subtypes soon after onset of ocular hypertension. An important function of Müller cell peri-synaptic processes is to regulate extracellular glutamate near synapses. Withdrawal of Müller glia could contribute importantly to disease progression by promoting RGC death via excitotoxic damage. Experiments in Aim 2B will examine this possibility by using two-photon Ca2+ imaging to test the hypothesis that elevated IOP increases light-evoked N-methyl-D-aspartate-type glutamate receptor activity in the dendrites of specific RGC subtypes. In both Aims, we will focus specifically on early-stage disease by conducting experiments within the first 1-2 weeks after IOP elevation. By identifying pathophysiological changes in Müller cells that occur early in disease progression, this work will help reveal novel ways to diagnose and treat glaucoma before RGC degeneration leads to irreversible vision loss.