核因子-κB(NF-κB)是一种普遍存在的转录因子,可调节免疫和细胞存活信号通路。据报道,NF-κB存在于神经元中,据报道,它对免疫和毒性刺激有反应。谷氨酸,和突触活动。然而,因为大脑包含许多细胞类型,特异性测量神经元NF-κB活性的测定难以执行和解释。为了解决这个问题,我们比较了原代新皮层神经元培养物中的NF-κB活性,混合脑细胞,和肝细胞,采用NF-κB亚基的蛋白质印迹,核κBDNA结合的电泳迁移率变化测定(EMSA),κBDNA结合的报告基因测定,NF-κB亚基蛋白p65的免疫荧光,NF-κB调节基因表达的定量实时聚合酶链反应(PCR),和产生的蛋白质的酶联免疫吸附测定(ELISA)。p65的测定显示其在神经元的细胞质和细胞核中的组成性存在,其水平显着低于混合的脑或肝细胞。EMSA和报告基因分析显示,神经元中几乎没有组成型NF-κB活性。诱导活性是最低的-比其他细胞类型低很多倍,通过抑制剂IκBα的磷酸化和降解来测量,p65的核积累,与κBDNA共有位点结合,NF-κB报告,或诱导NF-κB反应基因。对神经元最有效的激活刺激是促炎细胞因子肿瘤坏死因子α(TNFα)和白介素-β(IL-β)。在大多数试验中,神经元NF-κB对谷氨酸无反应,它对过氧化氢也没有反应,脂多糖,去甲肾上腺素,ATP,佛波酯,和神经生长因子.趋化因子基因转录物CCL2,CXCL1和CXCL10通过TNFα激活NF-κB而强烈诱导神经元,但是许多候选反应基因不是,包括神经保护基因SOD2和Bcl-xL。重要的是,响应TNFα或任何其他刺激的诱导神经元NF-κB活性水平低于非神经元细胞的组成活性水平,质疑神经元NF-κB活性的功能意义。
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.