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Ethanol-induced NMDA R1 mRNA Stabilization

乙醇诱导 NMDA R1 mRNA 稳定

基本信息

批准号：
7867211
负责人：
MEENA KUMARI
金额：
$ 1.2万
依托单位：
KANSAS STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-20 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7867211
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

项目摘要

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. -

NR1亚基是单个基因的产物，是功能性NMDA受体所没有的关键亚基