ETHANOL REGULATION OF NMDA R2B GENE TRANSCRIPTION

NMDA R2B 基因转录的乙醇调控

• 批准号: 7484040
• 负责人: MAHARAJ K TICKU
• 金额: $ 6.93万
• 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/7484040
• 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)受体是一种存在于脑内的兴奋性神经递质。 乙醇的重要作用部位。体内和体外慢性乙醇治疗上调血管内皮生长因子 NMDA受体的数量和功能，伴随着r1和r2b多肽水平的增加 在试管中。体内慢性乙醇处理后r2b多肽水平上调 使大脑皮层和海马区r2b基因表达水平分别增加约40%和30%。 在体外培养的胎儿皮质神经元中，r2b mRNA水平也出现了类似的增加。这个 慢性酒精刺激后r2b基因表达水平升高的分子机制 治疗方法是提高NMDAR2b基因的转录速率(Kumari和Ticku，1998)。这个 NMDAR2b受体亚单位在酒精引起的脑内改变中的重要性在于 酒精对r2b亚单位的影响在成人和胎儿组织中都有发生，而且酒精的强度 在这两种情况下，酒精的作用是相同的。该项目的长期计划是：(I)确定 在成人和胎儿组织中使用替代启动子；(Ii)检查甲基化在 R2b基因的表达；(Iii)确定乙醇反应的c/S调控元件 脱氧核糖核酸酶I超敏分析r2b基因启动子区域；(Iv)鉴定 “最小c/S作用dna序列”，足以显示对乙醇作用的反应。 缺失转染分析；(V)确定可能与最低c/S相互作用的核蛋白因子(S)- 作用于改变r2b基因表达的DNA序列和预先/纵向定位多少个核苷酸 在最小的c/S作用下，dna序列足以与已确定的核因子结合。 上面，最后(Vi)确定乙醇是否介导了细胞内钙的增加 导致环磷酸腺苷反应元件结合磷酸化的特定信号通路 进而与r2b基因5‘侧翼区的环状AMP反应元件结合的蛋白质 导致r2b基因转录速率增加。我们建议利用小鼠胚胎皮质 达到这些目标的神经元在培养的前7天，胎儿皮质神经元表达 主要是R2B亚基。通过对相关分子机制的更深入的了解 哪种乙醇改变NMDAR2b基因转录的速率可能允许设计新的 酒精相关疾病的治疗方法。