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 NR1 splice variant (NR1-4a) at the mRNA & polypeptide levels in cultured fetal cortical neurons (FCN) suggesting 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. 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.
