ANTISENSE INHIBITION OF RETINAL GLUTAMATE METABOLISM

本K08项目的目的是验证目前关于谷氨酸在青光眼视网膜神经节细胞（RGC）死亡中的作用的两种主要假设之一。根据这一假设，IOP升高的一个结果是RGCs中谷氨酸代谢周期的紊乱。这种紊乱导致谷氨酸兴奋性毒性，在青光眼的发病机制中起主要作用。这个项目的目标1和目标2检验这个假设的第二个组成部分；目标3测试第一个组件。目的1是确定视网膜中紊乱的谷氨酸代谢周期对RGC功能和存活的影响。谷氨酸代谢酶，谷氨酸脱氢酶（GDH）和谷氨酰胺合成酶（GS）以及谷氨酸转运体（GLAST）的反义寡核苷酸将被用来抑制rgc的谷氨酸代谢和摄取。它们的作用将在组织培养中确定，然后在玻璃体给药后评估RGC存活、活性和GDH的量。GS和GLAST，玻璃体内谷氨酸浓度，视网膜电图视觉功能。目的2测试谷氨酸拮抗剂和抗凋亡药物是否可以逆转由反义寡核苷酸引起的视网膜谷氨酸周期紊乱的影响。目的3是确定在慢性IOP升高的青光眼大鼠模型中，谷氨酸代谢周期的变化是否类似于使用GDH、GS和GLAST的反义寡核苷酸所诱导的变化。K08候选人最近完成了青光眼领域的临床研究，并开始了西奈山医学院的终身教职生涯。虽然他之前确实有一些基础研究经验，但为了开始拟议的研究，他必须首先加强他在反义设计和药代动力学领域的技能，以及一些将用于动物测试和评估结果的特定方法。这些领域的资深科学家和专家将提供指导。

The objective of this K08 project is to test one of the two major current hypotheses about the role of glutamate in glaucomatous retinal ganglion cell (RGC) death. According to this hypothesis one result of elevated IOP is a derangement of the glutamate metabolic cycle in RGCs. This derangement results in glutamate excitotoxicity that has a primary role in the pathogenesis of glaucoma. Aim 1 and 2 of this project test the second component of this hypothesis; aim 3 tests the first component. Aim 1 is to determine the effects of a deranged glutamate metabolic cycle in the retina on the RGC function and survival. Antisense oligonucleotides to the glutamate metabolizing enzymes, glutamate dehydrogenase (GDH) and glutamine synthetase (GS) and to glutamate transporters (GLAST) will be used to inhibit glutamate metabolism and uptake in RGCs. Their effects will be determined in tissue culture and then following intra-vitreal administration rats to assess RGC survival, activities and amounts of GDH. GS and GLAST, intra-vitreal glutamate concentrations, and visual function by electroretinography. Aim 2 tests whether glutamate antagonists and anti-apoptosis agents can reverse the effects of a deranged glutamate cycle in the retina caused by the antisense oligonucleotides. Aim 3 is to determine whether changes in the glutamate metabolic cycle similar to those induced by the use of antisense oligonucleotides to GDH, GS and GLAST are present in a chronically elevated IOP model of glaucoma in rats. The K08 candidate has recently completed a clinical fellowship in the field of glaucoma and has begun a tenure track academic career in Mt. Sinai School of Medicine. While he does have some prior fundamental research experience, in order to embark upon the proposed studies he must first strengthen his skills in the areas of antisense design and pharmacokinetics and in some specific methods that will be used in testing the animals and assessing the outcomes. Mentoring will be provided by senior scientists, experts in these areas.