Purines and the health of retinal ganglion cells

描述（由申请人提供）：青光眼是世界上致盲的主要原因之一，其特征是视网膜神经节细胞的丧失。该病常与眼压升高有关，但目前尚不清楚眼压升高是如何导致神经节细胞死亡的。目前的建议是基于一种新的假设，即眼压升高会触发ATP的释放。这种释放的ATP可以激活视网膜神经节细胞上的P2X7 ATP受体，导致NMDA受体的激活和兴奋性毒性细胞死亡。或者，释放的ATP可以通过外酶转化为腺苷，刺激腺苷的A3受体，保护神经节细胞。将使用三种方法来检验这一假设。首先，将对大鼠A3受体的神经保护能力进行药理学研究，因为这代表了最直接的治疗机会。受体的身份将通过激光捕获显微解剖分离的荧光标记视网膜神经节细胞扩增mRNA信息来确认。其次，通过检测NMDA拮抗剂预防P2X7刺激引起的细胞死亡的能力，探讨P2X7与NMDA受体通道之间的相互作用。这种相互作用的机制将通过测量P2X7刺激对谷氨酸外排的影响，以及确定P2X7通道本身对谷氨酸的通透性来研究。还将探索相互作用的间接途径，研究P2X7受体刺激是否足以使细胞去极化以打开NMDA通道或触发谷氨酸的囊泡释放。最后，增加压力对ATP释放的影响将通过体外和体内模型进行测试。升高的压力触发游离大鼠视网膜细胞释放ATP的能力将被证实，并将研究改变这种压力的大小和持续时间的影响。这一假设将在实验性青光眼大鼠模型中得到验证，通过将眼压升高的幅度和持续时间与ATP和调节酶的水平相关联。总之，这项研究将为青光眼神经节细胞死亡提供新的解释，并提出几种预防这种死亡的新方法。

DESCRIPTION (provided by applicant): Glaucoma is a major cause of blindness in the world and is characterized by a loss of retinal ganglion cells. The disease is frequently associated with elevated intraocular pressure, but it is unclear how this elevation leads to the death of ganglion cells. The current proposal is based upon the novel hypothesis that elevated intraocular pressure triggers the release of ATP. This released ATP can activate P2X7 ATP receptors on retinal ganglion cells, leading to activation of NMDA receptors and excitotoxic cell death. Alternatively, the released ATP can be converted into adenosine by ecto-enzymes and stimulate the A3 receptor for adenosine and protect ganglion cells. Three approaches will be used to test this hypothesis. First, the neuroprotective capacity of rat A3 receptors will be investigated pharmacologically, as this represents the most straightforward opportunity for treatment. Receptor identity will be confirmed by amplifying mRNA message from fluorescently-labeled retinal ganglion cells isolated using Laser-Capture Microdissection. Secondly, the interaction between P2X7 and NMDA receptor channels will be explored by examining the ability of NMDA antagonists to prevent the cell death caused by P2X7 stimulation. The mechanism underlying this interaction will be examined by measuring the effect of P2X7 stimulation on glutamate efflux, and determining the permeability of the P2X7 channel itself to glutamate. Indirect pathways for interaction will also be explored, investigating whether P2X7 receptor stimulation depolarizes cells sufficiently to open NMDA channels or trigger a vesicular release of glutamate. Finally, the effect of increased pressure on ATP release will be tested using in vitro and in vivo models. The ability of elevated pressure to trigger ATP release from dissociated rat retinal cells will be confirmed, and the effect of changing the magnitude and duration of this pressure will be investigated. The hypothesis will be tested with a rat model of experimental glaucoma by correlating the magnitude and duration of intraocular pressure elevation with levels of ATP and regulating enzymes. Together this research will provide a new explanation for ganglion cell death in glaucoma and suggests several new approaches to prevent this death.