Department of Geriatrics, University of Arkansas, Little Rock, AR, USA.
Neurotox Res. 2013 Apr;23(3):201-13. doi: 10.1007/s12640-012-9327-6. Epub 2012 May 8.
Neuronal excitation is mediated by the activation of NMDA receptor and associated with the formation of reactive oxygen species due to the activation of NADPH oxidase complex proteins. The activation of Gs protein coupled receptors (GPCRs) induces neuronal activation in the cAMP-dependent protein kinase A (PKA)-mediated signal cascade and regulates NADPH oxidase activity. However, it is unknown whether PKA regulates NADPH oxidase gene expression in neurons and microglia. In the present research, the NADPH oxidase gene expression was studied in rat cortical neurons and microglia in vitro. Purified microglial cells were identified with OX-42 antibody and they also expressed apolipoprotein E (ApoE). The time-dependent effect of cytokine interleukin-4 (IL-4) (20 ng/ml) in NADPH oxidase gene expression was studied in microglial cells. The levels of mRNA were determined by quantitative RT-PCR. The expression of NOX1, NOX2, and NCF2 was upregulated after IL-4 treatment for 4 h, but it was downregulated after 8-24 h. The expression of NCF1 was suppressed during any time of cytokine effect. IL-4 upregulated arginase1 (Arg1) and serine racemase1 (SRR1) gene expressions in microglia. Amyloid beta (Ab) suppressed NOX2, NCF1, and NCF2 gene expressions and upregulated glutamate cystine transporter (xCT), although IL-4 attenuated the effect of Ab (500 μM) in the upregulation of xCT gene expression. The activation of PKA with agonist dibutyryl cAMP (dbcAMP) (100 μM) induced the upregulation of Arg1 gene expression in microglia involving in the process of microglial activation. The transcription of NOX1, NOX2, and NCF1 was suppressed in microglial cells after dbcAMP treatment within 24 h. Neurons were identified with the microtubule-associated protein tau. The uniform distribution of tau along axons was established in normal neurons. Tau protein was redistributed after PKA agonist dbcAMP treatment for 24 h. L-glutamate (50 μM) caused the apoptotic processes and the accumulation of tau in the soma of neurons and along axons. The activation of PKA for 24 h induced the transcriptional upregulation of NOX1 and NCF1 in cortical neurons. However, L-glutamate suppressed NOX1 gene expression in neurons. These data demonstrate that the effects of IL-4 and dbcAMP are similar in the regulation of SRR1, Arg1, and NADPH oxidase complex gene expressions in neurons and microglia. IL-4 prevents glutamate release from microglia suppressing xCT expression induced by Ab. These findings suggest that the activation of GPCR in PKA-mediated pathway leads to transcriptional regulation of NADPH oxidase complex. The modulation of GPCR activation may inhibit the oxidative stress in neurons.
神经元的兴奋是通过 NMDA 受体的激活介导的，由于 NADPH 氧化酶复合物蛋白的激活，导致活性氧物质的形成。Gs 蛋白偶联受体 (GPCR) 的激活诱导 cAMP 依赖性蛋白激酶 A (PKA) 介导的信号级联中的神经元激活，并调节 NADPH 氧化酶活性。然而，尚不清楚 PKA 是否调节神经元和小胶质细胞中的 NADPH 氧化酶基因表达。在本研究中，研究了体外培养的大鼠皮质神经元和小胶质细胞中的 NADPH 氧化酶基因表达。用 OX-42 抗体鉴定纯化的小胶质细胞，它们还表达载脂蛋白 E (ApoE)。研究了细胞因子白细胞介素-4 (IL-4) (20ng/ml) 在小胶质细胞中对 NADPH 氧化酶基因表达的时间依赖性影响。通过定量 RT-PCR 测定 mRNA 水平。IL-4 处理 4 小时后，NOX1、NOX2 和 NCF2 的表达上调，但处理 8-24 小时后下调。在细胞因子作用的任何时间，NCF1 的表达均受到抑制。IL-4 上调小胶质细胞中精氨酸酶 1 (Arg1) 和丝氨酸消旋酶 1 (SRR1) 的基因表达。淀粉样蛋白-β (Ab) 抑制 NOX2、NCF1 和 NCF2 基因表达，并上调谷氨酸胱氨酸转运蛋白 (xCT)，尽管 PKA 激动剂二丁酰环磷酸腺苷 (dbcAMP) (100μM) 减弱了 Ab(500μM) 对 xCT 基因表达的上调作用。PKA 的激活用激动剂二丁酰环磷酸腺苷 (dbcAMP) (100μM) 诱导小胶质细胞中 Arg1 基因表达上调，这涉及小胶质细胞激活过程。dbcAMP 处理后 24 小时内，NOX1、NOX2 和 NCF1 的转录在小胶质细胞中受到抑制。神经元用微管相关蛋白 tau 鉴定。在正常神经元中建立了 tau 沿着轴突均匀分布的模式。PKA 激动剂 dbcAMP 处理 24 小时后，tau 蛋白重新分布。L-谷氨酸 (50μM) 导致神经元体和沿轴突的 tau 聚集的凋亡过程。PKA 激活 24 小时诱导皮质神经元中 NOX1 和 NCF1 的转录上调。然而，L-谷氨酸抑制神经元中 NOX1 基因的表达。这些数据表明，IL-4 和 dbcAMP 在调节神经元和小胶质细胞中 SRR1、Arg1 和 NADPH 氧化酶复合物基因表达方面的作用相似。IL-4 通过抑制 Ab 诱导的 xCT 表达来阻止小胶质细胞中谷氨酸的释放。这些发现表明，GPCR 在 PKA 介导的途径中的激活导致 NADPH 氧化酶复合物的转录调节。GPCR 激活的调节可能抑制神经元中的氧化应激。
