Nuclear factor-kappa B (NF-κB) is a ubiquitous transcription factor that regulates immune and cell-survival signaling pathways. NF-κB 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-κB activity are difficult to perform and interpret. To address this, we compared NF-κB activity in cultures of primary neocortical neurons, mixed brain cells, and liver cells, employing Western blot of NF-κB subunits, electrophoretic mobility shift assay (EMSA) of nuclear κB DNA binding, reporter assay of κB DNA binding, immunofluorescence of the NF-κB subunit protein p65, quantitative real-time polymerase chain reaction (PCR) of NF-κB-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-κB 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 IκBα, nuclear accumulation of p65, binding to κB DNA consensus sites, NF-κB reporting, or induction of NF-κB-responsive genes. The most efficacious activating stimuli for neurons were the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-beta (IL-β). Neuronal NF-κB 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-κB activation by TNFα in neurons, but many candidate responsive genes were not, including the neuroprotective genes SOD2 and Bcl-xL. Importantly, the level of induced neuronal NF-κB activity in response to TNFα 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-κB activity.
Keywords: 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide; 2-amino-5-phosphonopentanoate; 6-cyano-7-nitroquinoxaline-2,3-dione; AP5; BDNF; BRN; CNQX; CxN; DMEM; Dulbecco’s modified Eagle’s medium; EDTA; EGTA; ELISA; EMSA; ERK; FBS; GAPDH; HBSS; HRP; Hanks balanced salt solution; IKK; IL; ISHH; IgG; IκB kinase; LCN2; LPS; LVR; NF-κB; NGF; NGS; NLS; PBS; PDTC; PMA; SDS; TBP; TBST; TLR; TNF; TNFα; TPCA; Tata binding protein; Tris-Buffered Saline containing 0.05% Tween-20; ammonium pyrrolidinedithiocarbamate; brain-derived neurotrophic factor; cortical neurons; electrophoretic mobility shift assay; enzyme-linked immunosorbent assay; ethylene glycol tetraacetic acid; ethylenediaminetetraacetic acid; extracellular signal-regulated kinase; fetal bovine serum; glyceraldehyde 3-phosphate dehydrogenase; horseradish peroxidase; immunoglobulin G; in situ hybridization histochemistry; interleukin; lipocalin 2; lipopolysaccharide; liver cells; mixed brain cells; nerve growth factor; neuroprotection; normal goat serum; nuclear factor-kappa B; nuclear localization signal; phorbol 12-myristate 13-acetate; phosphate-buffered saline; plasticity; qPCR; quantitative real-time polymerase chain reaction; sodium dodecyl sulfate; toll-like receptor; transcription factor; tumor necrosis factor α.
Published by Elsevier Ltd.