Glutamate receptors, specifically of the N-methyl D-aspartate (NMDA) subtype, play crucial roles in development, learning, and cell survival. In terms of addictive behaviors, a neuronal circuit involving NMDA receptors of the prefrontal cortex could act to modify activation of neurochemical reward pathways occurring through the mesolimbic dopamine system, extending from the ventral tegmental area (VTA) in midbrain to the nucleus accumbens, prefrontal cortex, and dorsal striatium. In addition, recent studies have implicated glutamatergic interactions with stress-induced changes in the corticotropin-releasing hormone (CRH) system, in control of long-term alcohol responses. Specifically, we hypothesize that glutamate receptors activate plastic mechanisms, such as neurotrophin release and synthesis, to strengthen conditioned responses by neurons. Because neurotrophin synthesis appears to be regulated by interactions with glutamate receptors using signal transduction pathways that are activated either during learning processes or during remodeling (such as would occur following stress or injury), studies of neuronal plasticity and conditioned learning acting through glutamate and neurotrophin receptors are particularly justified. In addition, it is plausible that allelic variation (single nucleotide polymorphisms or SNPs) in glutamate receptors (and associated signal transduction pathways) may play a role in early responses (sensitization) or conditioned responses (addiction) and may be responsible for adaptive changes to stress influenced by alcohol or other drugs. As a first approach to understanding the molecular mechanisms underlying plastic responses in neurons, we determined the role of the transcription factor NF-kappaB in NMDA receptor-mediated induction of the neuronal protein, brain derived neurotrophic factor (BDNF). BDNF is known to be important in neuronal development, plasticity, and survival. As a model of BDNF-mediated function, we used primary cerebellar and hippocampal neurons from rats to determine the role of NF-kappaB in neuronal survival. We showed that NMDA exerts survival activity by activating neurotrophin signaling and inducing BDNF synthesis. NMDA also induced steady state BDNF mRNA levels. Of the four known promoters that control BDNF mRNA synthesis, those located upstream of exons 1 and 3 are responsible for BDNF synthesis in brain. We discovered an NF-kappaB candidate sequence within the 5-prime flanking region of exon 3 and investigated its role in NMDA-mediated neuronal survival. We first determined the kinetics of NMDA-mediated induction of NF-kappaB using an NF-kappaB double-stranded DNA target based on a candidate site we observed within the 5-prime flanking region of exon 3 from the rat BDNF gene. Increased DNA-protein binding activity was observed within 40 min and the DNA binding constituents contained both p65 and p50 subunits. Furthermore, the NMDA-mediated DNA-binding activity detected with a rat BDNF NF-kappaB target sequence was abolished in the presence of excess unlabeled target DNA, indicating the specificity of the DNA-protein interaction. It should be noted that the NF-kappaB target DNA sequence did not overlap a previously known cyclic AMP response element (CRE), ruling out the possibility that another transcription factor, CREB, was directly binding to the NF-kappaB site. Because NMDA receptors are only expressed by neurons in this culture system, the activation of NF-kappaB is strictly neuronal. Thus, the contribution of NF-kappaB activation in astrocytes, which represent less than 5% of the total cells present in the neuronal culture, but produce neurotrophic factors that could potentially protect neurons from excitotoxic concentrations of glutamate, does not play a role in our study. A characteristic biochemical step in the functional activation of NF-kappaB is the receptor-mediated activation of IkappaB kinases that catalyze the phosphorylation of IkappaB proteins. To determine if NMDA receptors mediated phosphorylation of IkappaB-alpha, we performed immunoblots using an antibody specific for IkappaB-alpha phosphorylated at Serine 32 versus cytoplasmic protein preparations from neurons incubated with different concentrations of NMDA. As expected, 100 microM NMDA greatly increased the level of immunoreactive phosphorylated IkappaB-alpha compared to untreated neurons. Thus, NMDA receptors activate a signaling pathway(s) leading to the phosphorylation of IkappaB-alpha. The relationship between NMDA receptor-mediated neuronal survival and NF-kappaB activation was shown by pre-treating rat cerebellar granule cell neurons with a double stranded target DNA containing an NF-kappaB binding site derived from the rat BDNF gene. Once the target DNA is internalized, it binds activated NF-kappaB located in the cytoplasm, preventing it from entering the nucleus and binding to NF-kappaB binding motifs. Our results show that NMDA receptor-mediated survival was completely abolished by pre-treating the cells with the NF-kappaB target DNA. Cell survival observed with the NF-kappaB target DNA was significantly lower compared to the levels of surviving neurons treated with NMDA only or for neurons treated with the scramble DNA alone, suggesting that NF-kappaB was necessary and sufficient for this effect. In addition, the NF-kappaB target DNA inhibited NMDA-mediated NF-kappaB DNA binding activity and abolished BDNF exon 3-specific mRNA induction. Taken together, these data support the idea that NF-kappaB is required for NMDA-mediated neuronal survival. Because the target DNA was prepared from an NF-kappaB candidate sequence in the rat BDNF gene, our results provide strong support to the idea that activated NF-kappaB translocates to the nucleus, binds to the NF-kappaB DNA site in the 5-flanking flanking region of exon 3 of the BDNF gene, to activate transcription. To better understand NMDA receptor and BDNF gene function, we used denaturing high performance liquid chromatography (dHPLC) to identify candidate SNPs in PCR amplified products obtained from a panel of genomic DNAs from 500 unrelated individuals of diverse clinical and ethnic background. DNA sequence variants were characterized by DNA sequencing. Using this approach, we identified seven gene variants in NR1 and NR2B, one of which resulted in an amino acid change. We also discovered nucleotide variants in the promoter region of the NR2B and BDNF genes. All of the sequence variants were previously unidentified. We are currently using these sequence variants (which have frequencies of 5-10%) in genetic association studies with alcohol susceptibility or other possible maladaptive behavioral responses. In parallel experiments, we are screening the new gene variants for functional changes using high throughput quantitative reporter gene and quantitative gene-specific mRNA assays.
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