Transient Receptor Potential Channels and Retinal Ganglion Cell Death in Glaucoma

描述(申请人提供)：我们的长期目标是了解青光眼中导致视网膜神经节细胞(RGC)及其轴突死亡的早期分子事件。青光眼的主要特征是对眼压(IOP)敏感，高眼压是该病的重要危险因素。从药物上降低眼压是减缓疾病的标准治疗方法，但由于RGC变性与压力之间的神经生物学机制仍未解决，因此没有治愈方法。青光眼中视网膜节细胞的死亡显示了其他退行性疾病中神经元死亡的关键方面，最突出的是通过细胞凋亡和轴突变性造成的躯体(细胞体)损失。在其他疾病中，躯体和轴突变性通常与细胞内钙离子升高有关，钙依赖级联反应也可能导致青光眼的RGC变性。我们的研究表明，对于在培养中暴露于高压下的视网膜节细胞，迅速增加的细胞内钙离子预示着躯体和轴突的丢失。这些观察提出了这样的问题：压力诱导的RGC死亡是否依赖于细胞内钙离子的增加，如果是，这种依赖的机制是什么？我们假设，压力诱导的RGC变性涉及机械敏感通道的激活，该通道直接门控细胞内钙离子的增加。为了支持这一假说，我们最近在视网膜节细胞中发现了辣椒素敏感的香草素-1瞬时受体电位(TRPV1)通道。TRPV1的特点是具有强大的钙电导，这有助于在其他系统中引起压力敏感性和钙依赖的细胞死亡。在这里，我们将使用优化用于研究躯体变性的纯化RGC的体外制备和优化用于体外研究轴突变性的视网膜外植体制备，来探讨压力诱导的细胞内钙变化、RGC死亡和TRPV1激活之间的关系。通过将药理学工具应用于这些系统，我们将(1)测试压力诱导的RGC变性的钙依赖性以及TRPV1在压力诱导的RGC细胞内钙升高中的作用，以及(2)确定压力诱导的RGC变性对TRPV1激活的依赖性。最后，通过应用我们实验室开发的基因抑制和过度表达的遗传工具和一只TRPV1基因敲除小鼠，我们将(3)测试TRPV1的表达与RGC对压力诱导的变性的易感性之间的关系。公共卫生相关性：。随着人口老龄化，到2020年，青光眼将困扰全球近8000万人，使其成为导致不可逆转失明的首要原因。青光眼仍然无法治愈，这在很大程度上是因为我们对压力敏感性如何转化为RGC变性的理解还不完整。这项工作将探索RGC对压力相关损伤易感性的可行分子机制，并测试其作为新的治疗靶点的相关性。

DESCRIPTION (provided by applicant): Our long-term objective is to understand the early molecular events leading to the death of retinal ganglion cells (RGCs) and their axons in glaucoma. The defining feature of glaucoma is sensitivity to intraocular pressure (IOP), and elevated IOP represents a significant risk factor for the disease. Lowering IOP pharmacologically is the standard treatment to slow the disease, but there is no cure because the neurobiological mechanisms linking RGC degeneration to pressure remain unresolved. The death of RGCs in glaucoma demonstrates key aspects of neuronal death in other degenerative diseases, most prominently somatic (cell body) loss via apoptosis and axonal degeneration. In other diseases, somatic and axonal degenerative are often linked to elevated intracellular Ca2+, and Ca2+-dependent cascades are also likely to contribute to RGC degeneration in glaucoma. Our studies demonstrate that for RGCs exposed to elevated pressure in culture, rapidly increased intracellular Ca2+ predicates both somatic and axonal loss. These observations raise the questions of whether pressure-induced RGC death is dependent on increased intracellular Ca2+ and, if so, what is the mechanism of this dependence. We have hypothesized that pressure-induced RGC degeneration involves the activation of a mechanosensitive channel that directly gates an increase in intracellular Ca2+. In support of this hypothesis, we recently identified in RGCs the capsaicin-sensitive, vanilloid-1 transient receptor potential (TRPV1) channel. TRPV1 is characterized by a robust Ca2+ conductance that contributes to pressure sensitivity and Ca2+-dependent cell death in other systems. Here we will probe the relationships between pressure-induced changes in intracellular Ca2+, RGC death and TRPV1 activation using an in vitro preparation of purified RGCs optimized for studying somatic degeneration and a retinal explant preparation optimized for studying axonal degeneration ex vivo. By applying pharmacological tools to these systems we will (1) test the Ca2+-dependence of pressure-induced RGC degeneration and the contribution of TRPV1 to pressure-induced increases in RGC intracellular Ca2+ and (2) determine the dependence of pressure-induced RGC degeneration on TRPV1 activation. Finally, by applying genetic tools for gene inhibition and over-expression developed in our laboratory and a TRPV1 knock-out mouse we will (3) test the relationship between TRPV1 expression and RGC susceptibility to pressure-induced degeneration. PUBLIC HEALTH RELEVANCE:. With the aging of the population, glaucoma will afflict nearly 80 million people worldwide by 2020, making the disease the leading cause of irreversible blindness. Glaucoma remains incurable, largely because our understanding of how pressure sensitivity translates to RGC degeneration is incomplete. The work proposed here will explore a viable molecular mechanism for contributing to RGC susceptibility to pressure-related injury and test its relevance as a novel therapeutic target.