ETHANOL REGULATION OF NMDA R2B GENE TRANSCRIPTION

NMDA R2B 基因转录的乙醇调控

• 批准号: 6509032
• 负责人: MAHARAJ K TICKU
• 金额: $ 28.9万
• 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 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6509032
• 关键词: DNA footprinting; DNA methylation; NMDA receptors; age difference; cAMP response element binding protein; calcium; cerebral cortex; chromatin; deoxyribonuclease I; developmental genetics; developmental neurobiology; embryo /fetus cell /tissue; enzyme activity; ethanol; gel mobility shift assay; genetic promoter element; genetic regulatory element; genetic transcription; laboratory mouse; nucleic acid structure; plasmids; receptor expression; transcription factor; transfection

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.

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，是乙醇的重要作用位点。 体内和体外长期乙醇处理上调 NMDA 受体数量和功能，同时体外 R1 和 R2B 多肽水平增加。体内长期乙醇治疗后R2B多肽水平上调是由于大脑皮层中的R2B mRNA水平增加了约40%，海马中的R2B mRNA水平增加了约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。 长期乙醇治疗导致 R2B mRNA 水平增加的分子机制是 NMDA R2B 基因转录率增加。 NMDA R2B 受体亚基在酒精介导的大脑变化中的重要性在于，乙醇对 R2B 亚基的影响在成人和胎儿组织中均发生，并且酒精影响的强度在这两种情况下是相同的。 该项目的长期计划是（i）确定是否在成人和胎儿组织中使用替代启动子； (ii) 检查甲基化在 R2B 基因表达中的作用； (iii) 通过脱氧核糖核酸酶 I 超敏分析鉴定 R2B 基因启动子区域中的乙醇反应性顺式控制调节元件； (iv)通过缺失转染分析鉴定足以显示对乙醇作用的反应的“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R2B基因表达并精确绘制最小顺式作用DNA序列内有多少核苷酸足以结合上文鉴定的核因子的核蛋白因子，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 反应元件结合蛋白磷酸化的特定信号通路，而环 AMP 反应元件结合蛋白反过来又与 5' 侧翼中的环 AMP 反应元件结合R2B 基因区域导致 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 亚基。 更深入地了解乙醇改变 NMDA R2B 基因转录速率的相关分子机制可能有助于设计针对酒精相关疾病的新治疗方法。