Dysregulation of Glutamine Synthetase in Human Temporal Lobe Epilepsy

描述（由申请人提供）：人类颞叶癫痫中谷氨酰胺合成酶失调：本K08提案的主要目标是促进申请人在基础科学/转化研究中作为一名独立的医师科学家研究者的发展。这将通过一系列互补的教育和指导研究培训经验来实现，申请人将学习分子生物学，蛋白质组学，细胞培养和流式细胞术方面的最新方法。这些方法解决的科学问题是：内侧颞叶癫痫患者海马中谷氨酰胺合成酶下调的机制是什么？这个问题很重要，因为实验抑制动物海马中的谷氨酰胺合成酶会导致癫痫发作，一旦酶恢复，癫痫发作就会停止。本课题的具体目的是：1)利用激光捕获显微解剖、实时定量PCR和原位杂交技术分析手术切除的人癫痫海马中谷氨酰胺合成酶的转录调控。2)利用二维凝胶电泳、免疫学技术和质谱分析谷氨酰胺合成酶的翻译后修饰。3)建立谷氨酰胺合成酶调控的体外实验研究体系，采用急性和慢性培养人致痫海马星形胶质细胞，并对培养细胞进行流式细胞分析。摘要：颞叶癫痫患者的大脑谷氨酰胺合成酶水平较低，这种酶可以将谷氨酸转化为无毒的化学物质谷氨酰胺。我们的目标是了解谷氨酰胺合成酶下调的原因，从而开发出针对谷氨酰胺合成酶的新型抗癫痫治疗方法。

DESCRIPTION (provided by applicant): Dysregulation of Glutamine Synthetase in Human Temporal Lobe Epilepsy: The main objective of this K08 proposal is to facilitate the applicant's development towards independence as a physician-scientist investigator in basic science/translational research. This will be achieved through a series of complementary educational and mentored research training experiences in which the applicant will learn state-of-the-art methodologies in molecular biology, proteomics, cell culture and flow cytometry. The scientific question addressed by these methodologies is: what is the mechanism underlying the down-regulation of glutamine synthetase in the hippocampus of patients with mesial temporal lobe epilepsy? This question is important because experimental inhibition of glutamine synthetase in the hippocampus of animals causes epileptic seizures followed by cessation of the seizures once the enzyme has been restored. The specific aims of this proposal are: 1) Analysis of transcriptional regulation of glutamine synthetase using laser capture microdissection, quantitative real time PCR, and in situ hybridization on surgically resected human epilepsy hippocampi. 2) Analysis of posttranslational modification of glutamine synthetase by 2D gel electrophoresis, immunological techniques, and mass spectrometry. 3) Establish an in vitro system for experimental studies of glutamine synthetase regulation, using acute and chronic cultures of astrocytes from human epileptogenic hippocampi combined with flow cytometric analysis of the cultured cells. Layman's Abstract: People with temporal lobe epilepsy have low levels of brain glutamine synthetase, an enzyme that transforms glutamate to the non toxic chemical glutamine. The goal is to understand why the enzyme is down-regulated, so that new antiepileptic treatments targeting glutamine synthetase can be developed.