Nuclear factor kappaB is a critical determinant in N-methyl-D-aspartate receptor-mediated neuroprotection

The role of a nuclear factor kappaB (NF-kappaB) in NMDA receptor-mediated neuroprotection is not known. A candidate sequence from the 5' flanking region of exon 3 of the rat brain-derived neurotrophic factor (BDNF) gene was used to show that exposure of rat cerebellar granule cells to 100 microM NMDA activated a specific DNA binding activity that was blocked by the NMDA receptor antagonist MK-801. Anti-p65 antibody or anti-p50 antibody 'supershifted' the DNA binding activity, suggesting that the DNA-protein complex was composed of p65 and p50 subunits. NMDA receptor-mediated neuroprotection was blocked when cerebellar neurons were transfected with a double-stranded oligonucleotide containing the BDNF gene NF-kappaB sequence. Furthermore, nuclear extracts prepared from neurons treated with NMDA and the double-stranded NF-kappaB oligonucleotide showed reduced DNA binding activity to the target sequence, supporting the idea that NF-kappaB may be involved in the transcriptional activation of the BDNF gene. To address this issue, we quantified the level of exon 3-specific BDNF mRNA. Relative to GAPDH mRNA levels and compared with untreated neurons, NMDA increased exon 3-specific BDNF mRNA twofold. In contrast, pretreatment of neurons with the NF-kappaB target DNA abolished the increase in BDNF mRNA following addition of NMDA. We also determined that BDNF itself induced an NF-kappaB DNA binding activity. Taken together, these data support a mechanism where NF-kappaB plays a critical role in NMDA-mediated neuroprotection.