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，是乙醇的重要作用部位。 慢性乙醇治疗在体内和体外上调NMDA受体的数量和功能，伴随着增加R1和R2 B多肽水平在体外。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。 慢性乙醇处理引起R2 B mRNA水平增加的分子机制是NMDA R2 B基因转录速率增加。 NMDA R2 B受体亚单位在酒精介导的脑变化中的重要性在于，在成人和胎儿组织中均观察到酒精对R2 B亚单位的作用，并且在两种情况下酒精作用的强度相同。 本项目的长期计划是（i）鉴定成人和胎儿组织中是否使用了替代启动子;（ii）检测甲基化在R2 B基因表达中的作用;（iii）通过脱氧核糖核酸酶I超敏分析鉴定R2 B基因启动子区域中乙醇响应性顺式调控元件;（iv）通过缺失转染分析鉴定足以显示对乙醇作用的响应的“最小顺式作用DNA序列”;（v）鉴定可与最小顺式作用DNA序列相互作用以改变R2 B基因表达的核蛋白因子，并精确定位最小顺式作用DNA序列内有多少核苷酸，作用DNA序列足以结合上述核因子，最后（vi）确定乙醇介导的细胞内钙的增加是否激活导致环AMP应答元件结合蛋白磷酸化的特异性信号通路，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层神经元来实现这些目标，因为在培养的前7天，胎儿皮层神经元主要表达R2 B亚基。 更深入地了解乙醇改变NMDA R2 B基因转录速率的相关分子机制，可能会设计出治疗酒精相关疾病的新方法。

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 approximately 40 percent in cerebral cortex and by approximately 30 percent 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. 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 cis-controlling regulatory elements in the promoter region of the R2B gene by deoxyribonuclease I hypersensitive analysis; (iv) identify "minimal cis-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 cis-acting DNA sequences to alter R2B gene expression and to precisely map how many nucleotides within minimal cis-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.