ANTISENSE INHIBITION OF RETINAL GLUTAMATE METABOLISM

这个K08项目的目的是测试目前关于谷氨酸在青光眼视网膜神经节细胞(RGC)死亡中的作用的两个主要假说之一。根据这一假说，眼压升高的一个结果是视网膜节细胞谷氨酸代谢周期紊乱。这种紊乱导致谷氨酸兴奋毒性，在青光眼的发病机制中起主要作用。本项目的目标1和目标2检验这一假设的第二个组成部分；目标3检验第一个组成部分。目的1是研究视网膜谷氨酸代谢紊乱对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视网膜神经节细胞的谷氨酸代谢和摄取。它们的效果将在组织培养中确定，然后在玻璃体内给药大鼠以评估RGC的存活、GDH的活性和数量。GS和GLAST，玻璃体内谷氨酸浓度，以及视网膜电描记术的视觉功能。目的2检测谷氨酸拮抗剂和抗细胞凋亡药物能否逆转反义寡核苷酸引起的视网膜谷氨酸循环紊乱。目的3确定在慢性高眼压青光眼大鼠模型中是否存在类似于使用GDH、GS和GLAST反义寡核苷酸诱导的谷氨酸代谢周期的变化。这位K08候选人最近完成了青光眼领域的临床研究，并开始了在Mt.西奈医学院。虽然他确实有一些先前的基础研究经验，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，以及将用于测试动物和评估结果的一些具体方法。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.