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Regulation of NADPH Oxidase Gene Expression with PKA and Cytokine IL-4 in Neurons and Microglia

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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.

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Amyloid beta


Apolipoprotein E




Chitinase 3-like 1


Dibutyryl cAMP


Glutamate cystine transporter


G-protein-coupled receptor








Nicotine adenine dinucleotide phosphate


NOX1 activator 1


NOX organizer 1




Protein kinase A


Reactive oxygen species


Serine racemase1


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Thanks to Prof. Barger for supervising me in this research at the University of Arkansas, Little Rock, AR. This work was supported by NIH Grants to S.W.B.

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Correspondence to Valentina L. Savchenko.

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Savchenko, V.L. Regulation of NADPH Oxidase Gene Expression with PKA and Cytokine IL-4 in Neurons and Microglia. Neurotox Res 23, 201–213 (2013).

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