ETHANOL REGULATION OF NMDA R2B GENE TRANSCRIPTION

NMDA R2B 基因转录的乙醇调控

• 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
• 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' 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

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.

N-甲基-D-天冬氨酸（NMDA）受体是大脑中的一种兴奋性神经递质，是一种 乙醇的重要作用部位。体内和体外慢性乙醇治疗上调 NMDA 受体数量和功能，同时 R1 和 R2B 多肽水平增加 体外。体内长期乙醇处理后 R2B 多肽水平上调是由于 大脑皮层中的 R2B mRNA 水平增加约 40%，海马中的 R2B mRNA 水平增加约 30%。 在体外培养的胎儿皮质神经元中，R2B mRNA 水平也出现类似的增加。这 慢性乙醇导致 R2B mRNA 水平增加的分子机制 治疗方法是提高 NMDA R2B 基因转录率（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以下事实 乙醇对 R2B 亚基的影响同时发生在成人和胎儿组织中，并且其强度 酒精在这两种情况下的效果是相同的。该项目的长期计划是 (i) 确定是否 在成人和胎儿组织中使用替代启动子； (ii) 检查甲基化的作用 R2B基因表达； (iii) 确定乙醇反应性 c/s 控制调控元件 通过脱氧核糖核酸酶 I 超敏分析对 R2B 基因的启动子区域进行分析； (四) 确定 “最小顺式/顺式作用 DNA 序列”足以显示对乙醇作用的反应 缺失转染分析； (v) 识别可能与最小 c/s-相互作用的核蛋白因子 作用 DNA 序列来改变 R2B 基因表达并 prec/sely 绘制核苷酸数量图 最小的顺式/顺式作用 DNA 序列足以结合已识别的核因子 如上所述，最后 (vi) 确定乙醇介导的细胞内钙增加是否会激活 导致环 AMP 反应元件结合磷酸化的特定信号通路 反过来，该蛋白质又与 R2B 基因 5' 侧翼区域中的环 AMP 响应元件结合 导致R2B基因转录率增加。我们建议利用小鼠胎儿皮质 神经元实现这些目标，因为在培养的前 7 天内，胎儿皮质神经元表达 主要是R2B亚基。通过更多地了解相关的分子机制 乙醇改变 NMDA R2B 基因转录速率可能允许设计新的 酒精相关疾病的治疗方法。