Ethanol-induced NMDA R1 mRNA Stabilization

乙醇诱导 NMDA R1 mRNA 稳定

• 批准号: 7535013
• 负责人: MEENA KUMARI
• 金额: $ 25.52万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7535013
• 关键词: Acids; Alcohol dependence; Alcohols; Antibodies; Biological Assay; Brain; Cell Culture Techniques; Cell Nucleus; Cells; Chronic; Code; Complementary DNA; Computer software; Cytosol; Dactinomycin; Data; Development; Disease; Ethanol; Exons; Genes; Genetic Transcription; Goals; Grant; Half-Life; In Situ; In Situ Hybridization; In Vitro; Incubated; Lead; Length; Mediating; Memory; Messenger RNA; Methionine; Molecular; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NR1 gene; Neurons; Oryctolagus cuniculus; Pharmacology; Physiology; Plasmids; Play; Process; Protein Biosynthesis; Proteins; RNA; RNA Interference; RNA Probes; RNA Splicing; RNA-Protein Interaction; Regulation; Reporter Genes; Research Personnel; Reticulocytes; Ribonucleases; Role; Scintillation Counting; Site; System; Testing; Transfection; Translations; Untranslated Regions; Variant; Western Blotting; alcohol effect; alcohol exposure; alcoholism therapy; chronic alcohol ingestion; design; fetal; immunocytochemistry; in vivo; internal control; knock-down; mRNA Stability; neurotransmission; novel therapeutic intervention; polypeptide; programs; receptor; time interval; translation assay

The NR1 subunit, a product of a single gene, is the key subunit as functional NMDA receptors are not formed without it. NMDA receptors are an important site of action of ethanol. Chronic ethanol exposure up regulates NMDA receptor number/function in vivo and in vitro with a concomitant increase in NR1 and NR2B protein levels. Out of four NR1 splice variants, chronic ethanol favors the expression of only one NR1splice variant (NR1-4a) at the mRNA & polypeptide levels in cultured fetal cortical neurons (FCN) (Kumari, 2001) uggesting an altered physiology and pharmacology of NMDA receptors in ethanol treated neurons. Furthermore, chronic ethanol-mediated increase in NR1 mRNA half-life in vitro requires de novo protein synthesis. A closer molecular examination identified two cis-acting regions in the NR1 3' UTR. One cis- acting region interacts with three trans-acting proteins and chronic ethanol up regulates one trans-acting protein in FCN (Anji et al., 2004). Long term plan of this project is to elucidate molecular mechanism(s) involved in ethanol-mediated regulation of NR1 subunit in FCN. In this grant, the following are proposed: (1) delineate mechanism(s) including splicing in the nucleus that alter mRNA levels of NR1 splice variants in ethanol treated FCN; determine the importance of RNA-protein interactions on NR1 mRNA (2) stability; (3) transport and localization; and (4) translation. These goals will be achieved by (a) determining half-lives of NR1 splice variants and splicing of exon 5 in FCN; (b) generating & testing chimeric plasmids, by RNAi, transient transfection or stable transfection to determine role of cis and trans factors on NR1 mRNA stability, NR1 mRNA transport (half-life study using actinomycin-D); transport and localization (by in situ hybridiztion & immunocytochemistry); and translation (using an in vitro translation system). A more thorough understanding of the pertinent molecular mechanisms through which ethanol modulates R1 mRNA stability/translation may permit the design of novel therapeutic approaches to alcohol-related diseases. NMDA receptors mediate neurotransmission and play important roles in memory formation. These receptors are also effected by a chronic alcohol consumption and play a role in development alcohol dependence. A better understanding of the effects of alcohol on NMDA receptors regulation in the brain will lead to better treatment for alcoholism. -

NR 1亚基是单个基因的产物，是关键亚基，而功能性NMDA受体不是 NMDA受体是乙醇作用的重要部位。慢性乙醇暴露 调节体内和体外NMDA受体数量/功能，同时增加NR 1和NR 2B 蛋白质水平在四种NR 1剪接变异体中，慢性乙醇仅有利于一种NR 1剪接的表达 培养的胎儿皮质神经元（FCN）中mRNA和多肽水平的变体（NR 1 -4a）（Kumari，2001） 提示乙醇处理的神经元中NMDA受体的生理学和药理学改变。 此外，慢性乙醇介导的体外NR 1 mRNA半衰期增加需要从头蛋白质 合成.进一步的分子检测在NR 1 3' UTR中鉴定出两个顺式作用区域。一个顺- 作用区与三个反式作用蛋白相互作用，慢性乙醇上调一个反式作用蛋白， FCN中的蛋白质（Anji等人，2004年）。本计画的长期计画是阐明分子机制 参与乙醇介导的FCN中NR 1亚基的调节。在这项补助金中，提出了以下建议：（1） 描述机制，包括在细胞核中剪接，改变NR 1剪接变体的mRNA水平， 乙醇处理的FCN;确定RNA-蛋白质相互作用对NR 1 mRNA的重要性（2）稳定性;（3） 运输和本地化;（4）翻译。这些目标将通过以下方式实现：（a）确定 （B）通过RNAi产生和测试嵌合质粒， 瞬时转染或稳定转染以确定顺式和反式因子对NR 1 mRNA稳定性的作用， NR 1 mRNA转运（使用放线菌素D的半衰期研究）;转运和定位（通过原位杂交和 免疫细胞化学）;和翻译（使用体外翻译系统）。更彻底的 了解乙醇调节R1 mRNA的相关分子机制 稳定性/翻译可能允许设计酒精相关疾病的新治疗方法。 NMDA受体介导神经传递，在记忆形成中起重要作用。 这些受体也受到慢性酒精消耗的影响，并在酒精的发展中发挥作用。 依赖更好地了解酒精对大脑中NMDA受体调节的影响将有助于我们更好地了解酒精对大脑中NMDA受体调节的影响。 从而更好地治疗酒精中毒。-