Glutamate is a widespread excitatory neurotransmitter in the central nervous system (CNS) that has been shown to be involved in several important physiological processes, such as neuronal growth and differentiation during development and modulation of synaptic responses as part of learning and memory. However, glutamate also plays an important role in the neuropathology of epilepsy, ischemia, and traumatic brain injury by causing neurodegeneration through a process called excitotoxicity. The N-methyl-D-aspartate (NMDA) glutamate receptor subtype plays a major role in this neuropathology. A better understanding of the mechanisms that act in the transition between trophic and toxic glutamatergic inotropic receptor activation is needed in order to develop effective therapeutic strategies. We discovered that one of the downstream targets of the trophic pathway is activated NF-kappaB, which promotes transcription of exon 4 of the BDNF gene. Enhanced release and synthesis of BDNF plays a major role in NMDA-induced neuronal survival. To determine that activated NF-kappaB plays an important role in the expression levels of exon 4-specific BDNF mRNA levels in vivo, we performed a chromatin immunoprecipitation (ChIP) experiment. Cross-linked chromatin from hippocampal neuron cultures was sheared by sonication and subsequently incubated with antibodies specific to the p65 and p50 subunits of NF-kappaB and cAMP response element binding protein (CREB), which was used as a positive control, to recover proteins bound to chromatin. The immunoprecipitated DNA had the cross-linked proteins removed, followed by semi-quantitative PCR using primers that amplify the region of the BDNF promoter 4 region containing the NF-kappaB and CREB binding sites. Chromatin recovered with anti-p65, anti-p50 or anti-CREB antibodies resulted in an amplification product of the predicted size. These results show that there is a basal level of NF-kappaB and CREB occupancy of promoter 4 of the BDNF gene. NF-kappaB and CREB appear to be appropriate targets that underlie neuronal survival (Marini et al., 2006, Amino Acids, in press). These same transcription factors may also be appropriate targets in regulating addiction pathways.
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