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.
Lipsky
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RH
Laboratory of Neurogenetics, NIAAA, NIH, Rockville, Maryland 20852, USA. rlipsky@mail.nih.gov
Xu
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Zhu
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Kelly
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Terhakopian
A
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Novelli
A
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Marini
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D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D002531:Cerebellum / Q000166:cytology / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016291:Dizocilpine Maleate / Q000494:pharmacology*; D005091:Exons; D020672:I-kappa B Proteins; D007700:Kinetics; D016202:N-Methylaspartate / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000502:physiology*
D020121:5' Untranslated Regions; D019208:Brain-Derived Neurotrophic Factor; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D009838:Oligodeoxyribonucleotides; D016194:Receptors, N-Methyl-D-Aspartate; D000072000:NF-KappaB Inhibitor alpha; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate