Morioka Norimitsu, Harano Sakura, Tokuhara Masato, Idenoshita Yuko, Zhang Fang Fang, Hisaoka-Nakashima Kazue, Nakata Yoshihiro
Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
Department of Pharmacology, Hiroshima University Graduate School of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
Brain Res. 2015 Nov 2;1625:111-20. doi: 10.1016/j.brainres.2015.08.029. Epub 2015 Aug 29.
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.
小胶质细胞中表达的α7烟碱型乙酰胆碱（nACh）受体在神经保护中起关键作用。模拟α7 nACh受体会导致谷氨酸/天冬氨酸转运体（GLAST）表达增加，进而降低突触谷氨酸水平。然而，在用尼古丁刺激α7 nACh受体后很长时间（超过18小时），培养的大鼠皮质小胶质细胞中GLAST才出现上调。因此，本研究阐明了培养的皮质小胶质细胞中诱导GLAST表达的途径。环己酰亚胺预处理可显著抑制尼古丁诱导的GLAST mRNA表达，表明GLAST表达需要一种蛋白质中间体，如生长因子。用尼古丁处理后6小时和12小时，皮质小胶质细胞中成纤维细胞生长因子-2（FGF-2）mRNA的表达显著增加，而这种增加被选择性α7 nACh受体拮抗剂甲基lycaconitine预处理有效抑制。用尼古丁处理也显著增加了FGF-2蛋白的表达。此外，用重组FGF-2处理可增加GLAST mRNA、蛋白表达以及（14）C-谷氨酸摄取，这是GLAST活性的一种功能测定。相反，FGF受体（FGFR）酪氨酸激酶抑制剂PD173074预处理可显著阻止尼古丁诱导的GLAST mRNA、其蛋白表达以及（14）C-谷氨酸摄取。逆转录聚合酶链反应证实FGFR1 mRNA表达局限于培养的皮质小胶质细胞。总之，目前的研究结果表明，小胶质细胞α7 nACh受体激活的神经保护作用可能归因于FGF-2的表达，FGF-2进而增加GLAST表达，从而从突触中清除谷氨酸并减少谷氨酸神经传递。
