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的失调：本K 08提案的主要目的是促进申请人作为基础科学/转化研究中的医生-科学家研究者的独立发展。这将通过一系列补充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的最先进方法。这些方法所解决的科学问题是：内侧颞叶癫痫患者海马谷氨酰胺合成酶下调的机制是什么？这个问题很重要，因为实验性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。本研究的具体目的是：1）利用激光捕获显微切割、真实的定量PCR和原位杂交技术，对手术切除的人癫痫脑组织中谷氨酰胺合成酶的转录调控进行分析。2)通过2D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)应用急性和慢性培养的人癫痫脑星形胶质细胞，结合流式细胞术分析，建立了谷氨酰胺合成酶调节的体外实验研究体系。Layman的摘要：颞叶癫痫患者的大脑谷氨酰胺合成酶水平较低，这种酶将谷氨酸转化为无毒的化学谷氨酰胺。我们的目标是了解为什么这种酶被下调，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.