ETHANOL REGULATION OF NMDA R2B GENE TRANSCRIPTION

NMDA R2B 基因转录的乙醇调控

基本信息

批准号：
7193526
负责人：
MAHARAJ K TICKU
金额：
$ 31.72万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7193526
关键词：
5&apos Flanking Region Adult Alcohols Appearance Binding Biological Assay Brain Calcium Cerebral cortex Chromatin Structure Chronic CpG Islands Cyclic AMP Response Element Cyclic AMP-Responsive DNA-Binding Protein Cytosine DNA DNA Sequence Deoxyribonuclease I Disease Electrophoretic Mobility Shift Assay Elements Enhancers Ethanol Fetal Tissues Gene Expression Genes Genetic Transcription Goals Hippocampus (Brain)In Vitro Lead Maps Mediating Messenger RNA Methylation Molecular Mus N-Methyl-D-Aspartate Receptors N-Methylaspartate Neurons Neurotransmitters Nuclear Nuclear Protein Nuclear Proteins Nucleotides Numbers Pattern Phosphorylation Promoter Regions Rate Regulation Regulatory Element Role Scanning Signal Pathway Site Tissues Transfection Up-Regulation alcohol effect alcohol response day deletion analysis design fetal in vivo novel therapeutics polypeptide programs promoter receptor response

项目摘要

The N-methyl-D-aspartate (NMDA) receptor, an excitatory neurotransmitter in brain, is an important site of action of ethanol. Chronic ethanol treatment in vivo and in vitro upregulates the NMDA receptor number and function, with a concomitant increase in R1 and R2B polypeptide levels in vitro. An upregulation of R2B polypeptide levels following chronic ethanol treatment in vivo is due to an increase in R2B mRNA levels by ~ 40% in cerebral cortex and by ~ 30% in hippocampus. Similar increase in R2B mRNA levels is seen to occur in vitro in cultured fetal cortical neurons. The molecular mechanism underlying an increase in R2B mRNA levels in response to chronic ethanol treatment is an increase in NMDA R2B gene transcription rate (Kumari and Ticku, 1998). The importance of NMDA R2B receptor subunit in alcohol mediated changes in the brain lies in the fact that ethanol's effect on R2B subunit is seen to occur both in adult and fetal tissue and the intensity of alcohol's effect is same in both instances. Long term plans of this project are to (i) identify if alternative promoters are utilized in adult and fetal tissues; (ii) examine the role of methylation in R2B gene expression; (iii) identify ethanol-responsive c/s-controlling regulatory elements in the promoter region of the R2B gene by deoxyribonuclease I hypersensitive analysis; (iv) identify "minimal c/s-acting DNA sequences" that are sufficient to show response to ethanol's action by deletion transfection analysis; (v) identify nuclear protein factor(s) that may interact with minimal c/s- acting DNA sequences to alter R2B gene expression and to prec/sely map how many nucleotides within minimal c/s-acting DNA sequences are sufficient for the binding of nuclear factors identified above, and lastly (vi) determine if ethanol mediated increase in intracellular calcium activates specific signal pathways that lead to phosphorylation of cyclic AMP response element binding protein which in turn, binds to cyclic AMP response element in the 5' flanking region of the R2B gene resulting in an increase in R2B gene transcription rate. We propose to utilize mouse fetal cortical neurons to achieve these goals as during the first 7 days in culture, fetal cortical neurons express mainly R2B subunit. A more through understanding of the pertinent molecular mechanisms through which ethanol alters rate of NMDA R2B gene transcription may permit the design of novel therapeutic approaches to alcohol-related diseases.

The N-methyl-D-aspartate (NMDA) receptor, an excitatory neurotransmitter in brain, is an 乙醇的重要作用部位。慢性乙醇在体内和体外上调 NMDA受体的数量和功能，R1和R2B多肽水平随之增加体外。在体内慢性乙醇治疗后，R2B多肽水平的上调应归因于在脑皮质中，R2B mRNA水平的增加约40％，海马中的R2B mRNA水平增加了约30％。在培养的胎儿皮质神经元中，R2B mRNA水平的类似增加也会发生。这慢性乙醇的R2B mRNA水平增加的基本分子机制治疗是NMDA R2B基因转录率的增加（Kumari和Ticku，1998）。这 NMDA R2B受体亚基在酒精介导的大脑变化中的重要性在于事实乙醇对R2B亚基的影响既发生在成人和胎儿组织中，又发生在这两种情况下，酒精的效果都是相同的。该项目的长期计划是（i）确定是否替代启动子用于成人和胎儿组织；（ii）检查甲基化在 R2B gene expression; （iii）确定乙醇响应性C/S控制调节元素通过脱氧核糖核酸酶I超敏分析，R2B基因的启动子区域；（iv）识别 “最小C/S-S-Octing DNA序列”足以显示出对乙醇作用的反应缺失转染分析；（v）确定可能与最小C/S-相互作用的核蛋白质因子作用DNA序列改变R2B基因表达并绘制了多少核苷酸在最小的C/S作用DNA序列中足以结合所鉴定的核因子上面，最后（VI）确定乙醇介导的细胞内钙的增加是否增加导致环状AMP响应元件结合的磷酸化的特定信号途径蛋白质又与R2B基因5'侧翼区域中的环状AMP响应元件结合导致R2B基因转录率的增加。我们建议利用小鼠胎儿皮质神经元可以实现这些目标，就像在文化中的头7天一样，胎儿皮质神经元表达主要是R2B亚基。通过了解相关的分子机制通过哪种乙醇改变NMDA R2B基因转录的速率可能允许设计新颖酒精相关疾病的治疗方法。