ABSTRACT: Nuclear factor-kappa B (NF-?B) is a ubiquitous transcription factor that regulates immune and cell-survival signaling pathways. NF-?B has been reported to be present in neurons wherein it reportedly responds to immune and toxic stimuli, glutamate, and synaptic activity. However, because the brain contains many cell types, assays specifically measuring neuronal NF-?B activity are difficult to perform and interpret. To address this, we compared NF-?B activity in cultures of primary neocortical neurons, mixed brain cells, and liver cells, employing Western blot of NF-?B subunits, electrophoretic mobility shift assay (EMSA) of nuclear ?B DNA binding, reporter assay of ?B DNA binding, immunofluorescence of the NF-?B subunit protein p65, quantitative real-time polymerase chain reaction (PCR) of NF-?B-regulated gene expression, and enzyme-linked immunosorbent assay (ELISA) of produced proteins. Assay of p65 showed its constitutive presence in cytoplasm and nucleus of neurons at levels significantly lower than in mixed brain or liver cells. EMSA and reporter assays showed that constitutive NF-?B activity was nearly absent in neurons. Induced activity was minimal--many fold lower than in other cell types, as measured by phosphorylation and degradation of the inhibitor I?B?, nuclear accumulation of p65, binding to ?B DNA consensus sites, NF-?B reporting, or induction of NF-?B-responsive genes. The most efficacious activating stimuli for neurons were the pro-inflammatory cytokines tumor necrosis factor ? (TNF?) and interleukin-beta (IL-?). Neuronal NF-?B was not responsive to glutamate in most assays, and it was also unresponsive to hydrogen peroxide, lipopolysaccharide, norepinephrine, ATP, phorbol ester, and nerve growth factor. The chemokine gene transcripts CCL2, CXCL1, and CXCL10 were strongly induced via NF-?B activation by TNF? in neurons, but many candidate responsive genes were not, including the neuroprotective genes SOD2 and Bcl-xL. Importantly, the level of induced neuronal NF-?B activity in response to TNF? or any other stimulus was lower than the level of constitutive activity in non-neuronal cells, calling into question the functional significance of neuronal NF-?B activity.
