ETHANOL REGULATION OF NMDA R2B GENE TRANSCRIPTION

N-甲基-D-天冬氨酸（NMDA）受体是脑内的一种兴奋性神经递质， 乙醇的重要作用部位。体内和体外慢性乙醇处理上调 NMDA受体数量和功能，伴随R1和R2 B多肽水平的增加 体外体内慢性乙醇处理后R2 B多肽水平的上调是由于 R2 B mRNA水平在大脑皮层中增加约40%，在海马中增加约30%。 在体外培养的胎儿皮层神经元中，R2 B mRNA水平也出现类似的增加。的 慢性乙醇引起R2 B mRNA水平升高的分子机制 治疗的结果是NMDA R2 B基因转录速率增加（Kumari和Ticku，1998）。的 NMDA R2 B受体亚单位在酒精介导的大脑变化中的重要性在于， 乙醇对R2 B亚基的影响在成人和胎儿组织中都有发生， 酒精在这两种情况下的效果是一样的。本项目的长期计划是（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 R2 B基因表达;（iii）鉴定R2 B基因表达中的乙醇响应性顺式调节元件。 通过脱氧核糖核酸酶I超敏分析R2 B基因的启动子区;（iv）鉴定 “最小顺式作用DNA序列”足以显示对乙醇作用的响应， 缺失转染分析;（v）鉴定可能与最小c/s相互作用的核蛋白因子。 作用于DNA序列以改变R2 B基因表达，并精确地绘制R2 B基因中 在最小的顺式/反式作用的DNA序列足以结合核因子鉴定 最后（vi）确定乙醇介导的细胞内钙增加是否激活 导致环AMP反应元件结合磷酸化的特异性信号通路 一种蛋白质，该蛋白质又与R2 B基因5 ′侧翼区的环AMP反应元件结合 导致R2 B基因转录速率增加。我们建议利用小鼠胎儿皮质 在培养的前7天，胎儿皮层神经元表达 主要是R2 B亚基。通过对相关分子机制的了解， 乙醇改变NMDA R2 B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。

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.