Transient Receptor Potential Channels and Retinal Ganglion Cell Death in Glaucoma

描述（由申请人提供）：我们的长期目标是了解导致青光眼视网膜神经节细胞（RGCs）及其轴突死亡的早期分子事件。青光眼的定义特征是对眼压（IOP）的敏感性，而眼压升高是该疾病的一个重要危险因素。药理学上降低IOP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尚未解决，因此无法治愈。青光眼中RGCs的死亡证明了其他退行性疾病中神经元死亡的关键方面，最突出的是体细胞（细胞体）凋亡和轴突变性。在其他疾病中，体细胞和轴突变性通常与细胞内Ca2+升高有关，Ca2+依赖性级联也可能导致青光眼的RGC变性。我们的研究表明，对于暴露于高压培养的RGCs，细胞内Ca2+的迅速增加预示着体细胞和轴突的损失。这些观察结果提出了压力诱导的RGC死亡是否依赖于细胞内Ca2+增加的问题，如果是这样，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，该通道直接导致细胞内Ca2+的增加。为了支持这一假设，我们最近在RGCs中发现了辣椒素敏感的香草素-1瞬时受体电位（TRPV1）通道。TRPV1的特点是强大的Ca2+电导，有助于其他系统中的压力敏感性和Ca2+依赖性细胞死亡。在这里，我们将探索压力诱导的细胞内Ca2+变化、RGC死亡和TRPV1激活之间的关系，使用体外制备纯化的RGC来研究体细胞变性和体外研究轴突变性优化的视网膜外植体制备。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变性对Ca2+的依赖性，以及TRPV1对压力诱导的RGC胞内Ca2+增加的贡献，(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表达的遗传工具和TRPV1敲除小鼠，我们将(3)测试TRPV1表达与RGC对压力诱导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年全球将有近8000万人罹患青光眼，成为导致不可逆失明的主要原因。青光眼仍然无法治愈，很大程度上是因为我们对压力敏感性如何转化为RGC变性的理解尚不完整。本文提出的工作将探索促进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.