ETHANOL REGULATION OF NMDA R2B GENE TRANSCRIPTION

N-甲基-D-天冬氨酸(NMDA)受体是乙醇的重要作用部位，是脑内的一种兴奋性神经递质。体内和体外慢性乙醇处理上调了NMDA受体的数量和功能，并伴随着体外r1和r2b多肽水平的增加。体内慢性乙醇处理后r2b多肽水平的上调是由于大脑皮层和海马区r2b mRNA水平分别增加了约40%和约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。慢性乙醇处理导致R2b基因转录水平升高的分子机制是NMDAR2b基因转录速率的提高。NMDAR2b受体亚基在酒精介导的大脑变化中的重要性在于，可以看到乙醇对R2b亚基的影响在成人和胎儿组织中都存在，而且酒精的影响强度在这两种情况下是相同的。该项目的长期计划是：(I)确定成人和胎儿组织中是否使用了替代启动子；(Ii)研究甲基化在r2b基因表达中的作用；(Iii)通过脱氧核糖核酸酶I超敏分析，确定r2b基因启动子区域的乙醇反应顺式调控元件；(Iv)通过缺失转染分析，确定足以显示对乙醇作用的“最小顺式作用DNA序列”；(V)确定可能与最小顺式作用DNA序列相互作用的核蛋白因子(S)，以改变r2b基因的表达，并精确定位最小顺式作用dna序列中有多少核苷酸足以与上述确定的核因子结合，最后(Vi)确定乙醇是否激活了特定的信号通路，从而导致cAMP反应元件结合蛋白的磷酸化，而cAMP反应元件结合蛋白又与r2b基因5‘侧翼区的cAMP反应元件结合，导致r2b基因转录速率增加。我们建议利用小鼠胚胎皮质神经元来实现这些目标，因为在培养的前7天，胎儿皮质神经元主要表达r2b亚单位。通过更深入地了解酒精改变NMDAR2b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.