Dysregulation of Glutamine Synthetase in Human Temporal Lobe Epilepsy

描述(由申请人提供)：人类颞叶癫痫谷氨酰胺合成酶的失调：这项K08提案的主要目的是促进申请人在基础科学/翻译研究中发展成为一名独立的医生-科学家研究员。这将通过一系列补充的教育和指导研究培训经验来实现，在这些培训中，申请者将学习分子生物学、蛋白质组学、细胞培养和流式细胞术的最新方法。这些方法解决的科学问题是：内侧颞叶癫痫患者海马区谷氨酰胺合成酶下调的机制是什么？这个问题很重要，因为实验上抑制动物海马区的谷氨酰胺合成酶会导致癫痫发作，然后一旦酶恢复，癫痫发作就会停止。这项研究的具体目的是：1)利用激光捕获显微切割、实时定量聚合酶链式反应和原位杂交技术分析手术切除的人类癫痫海马区谷氨酰胺合成酶的转录调控。2)用双向凝胶电泳法、免疫学技术和质谱法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体系，采用急性和慢性培养的人致痫海马区星形胶质细胞，结合培养细胞的流式细胞仪分析。外行人摘要：患有颞叶癫痫的人大脑谷氨酰胺合成酶水平较低，谷氨酰胺合成酶是一种将谷氨酸转化为无毒化学物质谷氨酰胺的酶。其目的是了解该酶下调的原因，以便开发针对谷氨酰胺合成酶的新的抗癫痫治疗方法。

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.