Stimulation of α7 nicotinic acetylcholine receptor regulates glutamate transporter GLAST via basic fibroblast growth factor production in cultured cortical microglia.

The α7 nicotinic acetylcholine (nACh) receptor expressed in microglia has a crucial role in neuroprotection. Simulation of α7 nACh receptor leads to increased expression of glutamate/aspartate transporter (GLAST), which in turn decreases synaptic glutamate levels. However, the upregulation of GLAST in cultured rat cortical microglia appears long after (over 18 h) stimulation of the α7 nACh receptor with nicotine. Thus, the current study elucidated the pathway responsible for the induction of GLAST expression in cultured cortical microglia. Nicotine-induced GLAST mRNA expression was significantly inhibited by cycloheximide pretreatment, indicating that a protein intermediary, such as a growth factor, is required for GLAST expression. The expression of fibroblast growth factor-2 (FGF-2) mRNA in cortical microglia was significantly increased 6 and 12h after treatment with nicotine, and this increase was potently inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The treatment with nicotine also significantly increased FGF-2 protein expression. Furthermore, treatment with recombinant FGF-2 increased GLAST mRNA, protein expression and (14)C-glutamate uptake, a functional measurement of GLAST activity. Conversely, pretreatment with PD173074, an inhibitor of FGF receptor (FGFR) tyrosine kinase, significantly prevented the nicotine-induced expression of GLAST mRNA, its protein and (14)C-glutamate uptake. Reverse transcription polymerase chain reaction confirmed FGFR1 mRNA expression was confined to cultured cortical microglia. Together, the current findings demonstrate that the neuroprotective effect of activation of microglial α7 nACh receptors could be due to the expression of FGF-2, which in turn increases GLAST expression, thereby clearing glutamate from synapse and decreasing glutamate neurotransmission.

The activation of P2Y6 receptor in cultured spinal microglia induces the production of CCL2 through the MAP kinases-NF-κB pathway.

Rat primary cultures of spinal microglia were stimulated by UTP, a known P2Y2/4 receptor agonist, which resulted in the production and release of the C-C chemokine CCL2 (monocyte chemoattractant protein-1; MCP-1) measured by real-time PCR and ELISA, respectively. In an in vitro preparation of rat spinal microglia, with regard to the P2Y subtypes, the expression of P2Y1, 2, 6, 12, 13 and P2Y14, but not P2Y4, were detected by RT-PCR. The subtype of microglial P2Y receptor which could be involved in the production of CCL2 was also determined. The UTP-induced production of CCL2 was significantly blocked by pretreatment with reactive blue 2 and suramin, nonselective P2Y receptor antagonists, and MRS2578, a selective P2Y6 receptor antagonist. By contrast, knockdown of the P2Y2 receptor by RNA interference had no effect. The stimulatory effect of UTP was inhibited by phospholipase C (PLC) inhibitor U73122 and Src tyrosine kinase inhibitor PP2. A potential role of mitogen activated protein kinases was suggested since UTP-induced CCL2 production was significantly blocked by both U0126 and SB 202190, which are potent inhibitors of extracellular signal-regulated kinase (ERK) and p38, respectively. Moreover, UTP-stimulated phosphorylation of these kinases involved the activation of the P2Y6 receptor. Lastly, activation of nuclear factor-κB (NF-κB) by UTP is likely to be essential in the expression of CCL2. Together, these findings suggest that stimulation of spinal microglia P2Y6 receptors induce the production of CCL2 through either PLC-mediated ERK or p38 phosphorylation and the subsequent activation of NF-κB.

Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice.

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.

Noradrenaline induces clock gene Per1 mRNA expression in C6 glioma cells through beta(2)-adrenergic receptor coupled with protein kinase A - cAMP response element binding protein (PKA-CREB) and Src-tyrosine kinase - glycogen synthase kinase-3beta (Src-GSK-3beta).

Astrocytes in the hypothalamic suprachiasmatic nucleus, site of the master circadian pacemaker, play an essential role in the regulation of systemic circadian rhythms. To evaluate involvement of noradrenergic systems in regulation of circadian variation of clock-genes in astrocytes, we investigated effects of noradrenaline (NA) on expression of several clock genes in C6 glioma cells by using real-time PCR analysis. Treatment with NA (10 microM) induced transient expression of Per1 mRNA, but not of Per2, Bmal1, Clock, Cry1, or Cry2 mRNA, through activation of beta(2) adrenoceptors. Action of NA was partially blocked by H-89 [protein kinase A (PKA) inhibitor] or KG-501 [inhibitor of cAMP response element binding protein (CREB)]. We found that pretreatment with genistein or PP2 (general or Src tyrosine kinase inhibitors, respectively) or LiCl [inhibitor of glycogen synthase kinase-3beta (GSK-3beta)] significantly inhibited NA-induced Per1 mRNA expression. In addition, treatment with H-89 and either genistein or LiCl completely blocked NA stimulatory effects. NA markedly induced tyrosine phosphorylation of Src and GSK-3beta via activation of beta(2) adrenoceptors. Phosphorylation of GSK-3beta by NA was completely eliminated by genistein or PP2. These results primarily suggest that two distinct NA-mediating pathways, PKA-CREB and Src-GSK-3beta, play crucial roles in regulation of Per1 expression in astroglial cells.