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中视网膜神经节细胞（RGC）及其轴突死亡的早期分子事件。青光眼的定义特征是对眼内压（IOP）的敏感性，并且IOP升高代表该疾病的显著危险因素。降低眼压是减缓疾病的标准治疗方法，但由于将RGC变性与压力联系起来的神经生物学机制尚未解决，因此无法治愈。青光眼中RGC的死亡证明了其他退行性疾病中神经元死亡的关键方面，最显著的是通过细胞凋亡和轴突变性的体细胞（细胞体）损失。在其他疾病中，体细胞和轴突变性通常与细胞内Ca 2+升高有关，Ca 2+依赖性级联也可能导致青光眼中的RGC变性。我们的研究表明，对于暴露于培养中的高压的RGCs，细胞内Ca 2+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一个问题，是否压力诱导的RGC死亡是依赖于细胞内Ca 2+的增加，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变性涉及直接门控细胞内Ca 2+增加的机械敏感性通道的激活。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（TRPV 1）通道。TRPV 1的特征在于强大的Ca 2+电导，这有助于其他系统中的压力敏感性和Ca 2+依赖性细胞死亡。在这里，我们将探讨压力诱导的细胞内Ca 2+，RGC死亡和TRPV 1激活的变化之间的关系，使用体外制备的纯化RGC优化研究体细胞变性和视网膜外植体制备优化研究轴突变性离体。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Ca 2+依赖性和TRPV 1对压力诱导的RGC细胞内Ca 2+增加的贡献，以及（2）确定压力诱导的RGC变性对TRPV 1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TRPV 1敲除小鼠，我们将（3）测试TRPV 1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，使该疾病成为不可逆失明的主要原因。青光眼仍然无法治愈，主要是因为我们对压力敏感性如何转化为RGC变性的理解不完整。本文提出的工作将探索一种可行的分子机制，有助于RGC对压力相关损伤的易感性，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.