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井上 浩一イノウエ コウイチ

所属部署医学研究科統合解剖学分野
職名准教授
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Last Updated :2020/06/03

研究活動情報

研究分野

  • ライフサイエンス, 神経科学一般
  • ライフサイエンス, 神経科学一般
  • ライフサイエンス, 精神神経科学
  • ライフサイエンス, 消化器外科学
  • ライフサイエンス, 麻酔科学

研究キーワード

    発達, cell sorting, 脳, マイクロアレイ, 移動, 神経, GABA, 細胞移動, クロライドトランスポーター, カハールレチウス細胞, 国際情報交換, ドイツ, タウリン, 皮質板細胞, グリシン, てんかん, イメージング, 皮質形成異常, クロライドイオン, 大脳皮質, Cl^-ホメオスタシス, Chrolide, プリン受容体, 小脳, 肝細胞癌, 生物進化, 電気穿孔法, 言語, クロラドトランスポーター, 化学発癌

論文

  • TRPM7 channels play a role in high glucose–induced endoplasmic reticulum stress and neuronal cell apoptosis, Huang Y, Leng TD, Inoue K, Tao Y, Liu M, Horgen FD, Fleig A, Li J, Xiong ZG, J Biol Chem, 93, (37) 14393 - 406,   2018年09月, 査読有り
  • Potential implication of SGK1-dependent activity change in BV-2 microglial cells., Asai H, Inoue K, Sakuma E, Shinohara Y, Ueki T, International journal of physiology, pathophysiology and pharmacology, 10, (2) 115 - 123,   2018年, 査読有り
  • Effect of Redox-Modifying Agents on the Activity of Channelrhodopsin-2, Bao-Ming Wu, Tian-Dong Leng, Koichi Inoue, Jun Li, Zhi-Gang Xiong, CNS NEUROSCIENCE & THERAPEUTICS, 23, (3) 216 - 221,   2017年03月, 査読有り, BackgroundThe algal protein Channelrhodopsin-2 (ChR2) has been widely used in recent years in optogenetic technique to investigate the functions of complex neuronal networks through minimally invasive and temporally precise photostimulation of genetically defined neurons. However, as with any other new technique, current optogentic approaches have various limitations. In addition, how ChR2 may behave in response to complex biochemical changes associated with various physiological/pathological conditions is largely unknown.AimIn this study, we investigated whether a change in redox state of the cell affects the activity of ChR2 channels.MethodsWhole-cell patch-clamp recordings were used to examine the effect of reducing and oxidizing agents on ChR2 currents activated by blue light.ResultsWe show that the reducing agent dithiothreitol (DTT) dramatically potentiates the ChR2 currents in a reversible and concentration-dependent manner. Glutathione, an endogenous reducing agent, shows a similar effect on ChR2 currents. The oxidizing agent 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) has no effect on ChR2 currents by itself; however, it completely reverses the potentiating effect of DTT. DTT also causes a shift in the current-voltage relationship by 23 4.31 mV, suggesting a change in ion selectivity.ConclusionTaken together, these data suggest that redox modification of ChR2 plays an important role in its sensitivity to the light stimulation. Our findings not only help for a better understanding of how ChR2 may behave in physiological/pathological conditions where changes in redox state are common, but also provide a new direction for further optimization of this important opsin.
  • ASIC1 promotes differentiation of neuroblastoma by negatively regulating Notch signaling pathway, Mingli Liu, Koichi Inoue, Tiandong Leng, An Zhou, Shanchun Guo, Zhi-gang Xiong, ONCOTARGET, 8, (5) 8283 - 8293,   2017年01月, 査読有り, In neurons, up-regulation of Notch activity either inhibits neurite extension or causes retraction of neurites. Conversely, inhibition of Notch1 facilitates neurite extension. Acid-sensing ion channels (ASICs) are a family of proton-gated cation channels, which play critical roles in synaptic plasticity, learning and memory and spine morphogenesis. Our pilot proteomics data from ASIC1a knock out mice implicated that ASIC1a may play a role in regulating Notch signaling, therefore, we explored whether or not ASIC1a regulates neurite growth during neuronal development through Notch signaling. In this study, we determined the effects of ASIC1a on neurite growth in a mouse neuroblastoma cell line, NS20Y cells, by modulating ASIC1a expression. We also determined the relationship between ASIC1a and Notch signaling on neuronal differentiation. Our results showed that down-regulation of ASIC1a in NS20Y cells inhibits CPT-cAMP induced neurite growth, while over expression of ASIC1a promotes its growth. In addition, down-regulation of ASIC1a increased the expression of Notch1 and its target gene Survivin while inhibitor of Notch significantly prevented the neurite extension induced by ASIC1a in NS20Y cells. These data indicate that Notch1 signaling may be required for ASIC1a-mediated neurite growth and neuronal differentiation.
  • Possible divergence of serum- and glucocorticoid-inducible kinase function in ischemic brain injury, Koichi Inoue, NEURAL REGENERATION RESEARCH, 11, (9) 1396 - 1397,   2016年09月, 査読有り
  • Serum- and glucocorticoid-inducible kinases in microglia, Koichi Inoue, Eisuke Sakuma, Hiroyuki Morimoto, Hayato Asai, Yoshinori Koide, Tiandong Leng, Ikuo Wada, Zhi-Gang Xiong, Takatoshi Ueki, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 478, (1) 53 - 59,   2016年09月, 査読有り, Microglia are derived from myelogenous cells and contribute to immunological and inflammatory responses in central nervous system. They play important roles not only in infectious diseases and inflammation after stroke, but also in psychiatric diseases such as schizophrenia. While recent studies suggest the significances of serum- and glucocorticoid-inducible kinases (SGKs) in other immune cells such as macrophages, T cells and dendritic cells, their role in microglia remains unknown. Here we, for the first time, report that SGK1 and SGK3 are expressed in multiple microglial cell lines. An SGK inhibitor, gsk650394, inhibits cell viability. In addition, lipopolysaccharide-induced expression of inflammatory regulators iNOS and TNF alpha was enhanced by gsk650394. Furthermore, translocation of NF-kappa B was enhanced by gsk650394. Taken together, these findings suggest that SGKs may play an important role in regulating microglial viability and inflammatory responses. (C) 2016 Elsevier Inc. All rights reserved.
  • Role of serum- and glucocorticoid-inducible kinases in stroke, Koichi Inoue, Tiandong Leng, Tao Yang, Zhao Zeng, Takatoshi Ueki, Zhi-Gang Xiong, JOURNAL OF NEUROCHEMISTRY, 138, (2) 354 - 361,   2016年07月, 査読有り, Increased expression of serum- and glucocorticoid-inducible kinase 1 (SGK1) can be induced by stress and growth factors in mammals, and plays an important role in cancer, diabetes, and hypertension. A recent work suggested that SGK1 activity restores damage in a stroke model. To further investigate the role of SGKs in ischemic brain injury, we examined how SGK inhibitors influence stroke outcome invivo and neurotoxicity invitro. Infarct volumes were compared in adult mice with middle cerebral artery occlusion, followed by 24h reperfusion, in the absence or presence of SGK inhibitors. Neurotoxicity assay, electrophysiological recording, and fluorescence Ca2+ imaging were carried out using cultured cortical neurons to evaluate the underlying mechanisms. Contrary to our expectation, infarct volume by stroke decreased significantly when SGK inhibitor, gsk650394, or EMD638683, was administrated 30min before middle cerebral artery occlusion under normal and diabetic conditions. SGK inhibitors reduced neurotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors, a leading factor responsible for cell death in stroke. SGK inhibitors also ameliorated Ca2+ increase and peak amplitude of NMDA current in cultured neurons. In addition, SGK inhibitor gsk650394 decreased phosphorylation of Nedd4-2 and inhibited voltage-gated sodium currents. These observations suggest that SGK activity exacerbates stroke damage and that SGK inhibitors may be useful candidates for therapeutic intervention.
  • Acid Sensing Ion Channels (ASICs) in NS20Y cells - potential role in neuronal differentiation, Zaven O'Bryant, Tiandong Leng, Mingli Liu, Koichi Inoue, Kiara T. Vann, Zhi-gang Xiong, MOLECULAR BRAIN, 9, (1) ,   2016年06月, 査読有り, Cultured neuronal cell lines can express properties of mature neurons if properly differentiated. Although the precise mechanisms underlying neuronal differentiation are not fully understood, the expression and activation of ion channels, particularly those of Ca2+-permeable channels, have been suggested to play a role. In this study, we explored the presence and characterized the properties of acid-sensing ion channels (ASICs) in NS20Y cells, a neuronal cell line previously used for the study of neuronal differentiation. In addition, the potential role of ASICs in cell differentiation was explored. Reverse Transcription Polymerase Chain Reaction and Western blot revealed the presence of ASIC1 subunits in these cells. Fast drops of extracellular pH activated transient inward currents which were blocked, in a dose dependent manner, by amiloride, a non-selective ASIC blocker, and by Psalmotoxin-1 (PcTX1), a specific inhibitor for homomeric ASIC1a and heteromeric ASIC1a/2b channels. Incubation of cells with PcTX1 significantly reduced the differentiation of NS20Y cells induced by cpt-cAMP, as evidenced by decreased neurite length, dendritic complexity, decreased expression of functional voltage gated Na+ channels. Consistent with ASIC1a inhibition, ASIC1a knockdown with small interference RNA significantly attenuates cpt-cAMP-induced increase of neurite outgrowth. In summary, we described the presence of functional ASICs in NS20Y cells and demonstrate that ASIC1a plays a role in the differentiation of these cells.
  • TRPM7 regulates vascular endothelial cell adhesion and tube formation., Zeng Z, Inoue K, Sun H, Leng T, Feng X, Zhu L, Xiong ZG, American journal of physiology. Cell physiology, 308, (4) C308 - 18,   2015年02月, 査読有り
  • IL-10 modulates antigen presentation by dendritic cells through regulation of NLRP3 inflammasome assembly during Chlamydia infection, Omosun Y, McKeithen D, Ryans K, Kibakaya C, Blas-machado U, Li D, Singh R, Inoue K, Xiong Z-G, Eko F, Black C, Igietseme J, He Q, Infect. Immun., 83, 4662 - 4672,   2015年, 査読有り
  • Zinc-permeable ion channels: effects on intracellular zinc dynamics and potential physiological/pathophysiological significance., Inoue K, O'Bryant Z, Xiong ZG, Current medicinal chemistry, 22, (10) 1248 - 1257,   2015年, 査読有り
  • Local Anesthetic Lidocaine Inhibits TRPM7 Current and TRPM7-Mediated Zinc Toxicity, Tian-Dong Leng, Jun Lin, Hua-Wei Sun, Zhao Zeng, Zaven O'Bryant, Koichi Inoue, Zhi-Gang Xiong, CNS NEUROSCIENCE & THERAPEUTICS, 21, (1) 32 - 39,   2015年01月, 査読有り, BackgroundPrevious study demonstrated that overstimulation of TRPM7 substantially contributes to zinc-mediated neuronal toxicity. Inhibition of TRPM7 activity and TRPM7-mediated intracellular Zn2+ accumulation may represent a promising strategy in the treatment of stroke. AimsTo investigate whether local anesthetics lidocaine could inhibit TRPM7 channel and TRPM7-mediated zinc toxicity. MethodsWhole-cell patch-clamp technique was used to investigate the effect of local anesthetics on TRPM7 currents in cultured mouse cortical neurons and TRPM7-overexpressed HEK293 cells. Fluorescent Zn2+ imaging technique was used to study the effect of lidocaine on TRPM7-mediated intracellular Zn2+ accumulation. TRPM7-mediated zinc toxicity in neurons was used to evaluate the neuroprotective effect of lidocaine. Results(1) Lidocaine dose dependently inhibits TRPM7-like currents, with an IC50 of 11.55 and 11.06mM in cultured mouse cortical neurons and TRPM7-overexpressed HEK293 cells, respectively; (2) Lidocaine inhibits TRPM7 currents in a use/frequency-dependent manner; (3) Lidocaine inhibits TRPM7-mediated intracellular Zn2+ accumulation in both cortical neurons and TRPM7-overexpressed HEK293 cells; (4) TRPM7-mediated Zn2+ toxicity is ameliorated by lidocaine in cortical neurons; (5) QX-314 has a similar inhibitory effect as lidocaine on TRPM7 currents when applied extracellularly; (6) Procaine also shows potent inhibitory effect on the TRPM7 currents in cortical neurons. ConclusionOur data provide the first evidence that local anesthetic lidocaine inhibits TRPM7 channel and TRPM7-mediated zinc toxicity.
  • Silencing TRPM7 in mouse cortical astrocytes impairs cell proliferation and migration via ERK and JNK signaling pathways., Zeng Z, Leng T, Feng X, Sun H, Inoue K, Zhu L, Xiong ZG, PloS one, 10, (3) ,   2015年, 査読有り
  • TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways, Mingli Liu, Koichi Inoue, Tiandong Leng, Shanchun Guo, Zhi-gang Xiong, CELLULAR SIGNALLING, 26, (12) 2773 - 2781,   2014年12月, 査読有り, Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults with median survival time of 14.6 months. A small fraction of cancer stem cells (CSC) initiate and maintain tumors thus driving glioma tumorigenesis and being responsible for resistance to classical chemo- and radio-therapies. It is desirable to identify signaling pathways related to CSC to develop novel therapies to selectively target them. Transient receptor potential cation channel, subfamily M, member 7, also known as TRPM7 is a ubiquitous, Ca2+ and Mg2+ permeable ion channels that are special in being both an ion channel and a serine/threonine kinase. In studies of glioma cells silenced for TRPM7, we demonstrated that Notch (Notch1, JAG1, Hey2, and Survivin) and STAT3 pathways are down regulated in glioma cells grown in monolayer. Furthermore, phospho-STAT3, Notch target genes and CSC markers (ALDH1 and CD133) were significantly higher in spheroid glioma CSCs when compared with monolayer cultures. The results further show that tyrosine-phosphorylated STAT3 binds and activates the ALDH1 promoters in glioma cells. We found that TRMP7-induced upregulation of ALDH1 expression is associated with increases in ALDH1 activity and is detectable in stem-like cells when expanded as spheroid CSCs. Finally, TRPM7 promotes proliferation, migration and invasion of glioma cells. These demonstrate that TRPM7 activates JAK2/STAT3 and/or Notch signaling pathways and leads to increased cell proliferation and migration. These findings for the first time demonstrates that TRPM7 (1) activates a previously unrecognized STAT3 -> ALDH1 pathway, and (2) promotes the induction of ALDH1 activity in glioma cells. (C) 2014 Elsevier Inc. All rights reserved.
  • Acid-sensing ion channels in mouse olfactory bulb M/T neurons, Ming-Hua Li, Selina Qiuying Liu, Koichi Inoue, Jinquan Lan, Roger P. Simon, Zhi-Gang Xiong, JOURNAL OF GENERAL PHYSIOLOGY, 143, (6) 719 - 731,   2014年06月, 査読有り, The olfactory bulb contains the first synaptic relay in the olfactory pathway, the sensory system in which odorants are detected enabling these chemical stimuli to be transformed into electrical signals and, ultimately, the perception of odor. Acid-sensing ion channels (ASICs), a family of proton-gated cation channels, are widely expressed in neurons of the central nervous system. However, no direct electrophysiological and pharmacological characterizations of ASICs in olfactory bulb neurons have been described. Using a combination of whole-cell patch-clamp recordings and biochemical and molecular biological analyses, we demonstrated that functional ASICs exist in mouse olfactory bulb mitral/tufted (M/T) neurons and mainly consist of homomeric ASIC1a and heteromeric ASIC1a/2a channels. ASIC activation depolarized cultured M/T neurons and increased their intracellular calcium concentration. Thus, ASIC activation may play an important role in normal olfactory function.
  • Role of TRPM7 Channels in Hyperglycemia-Mediated Injury of Vascular Endothelial Cells, Huawei Sun, Tiandong Leng, Zhao Zeng, Xiuren Gao, Koichi Inoue, Zhi-Gang Xiong, PLOS ONE, 8, (11) ,   2013年11月, 査読有り, This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.
  • PRE- AND POST-SYNAPTIC SWITCHES OF GABA ACTIONS ASSOCIATED WITH CL- HOMEOSTATIC CHANGES ARE INDUCED IN THE SPINAL NUCLEUS OF THE TRIGEMINAL NERVE IN A RAT MODEL OF TRIGEMINAL NEUROPATHIC PAIN, B. Wei, T. Kumada, T. Furukawa, K. Inoue, M. Watanabe, K. Sato, A. Fukuda, NEUROSCIENCE, 228, 334 - 348,   2013年01月, 査読有り, Although trigeminal neuropathic pain is one of the most common chronic pain syndromes, the etiology is still unknown. Here, a rat model was generated using chronic constrictive injury (CCI) with ligation of the infraorbital nerve to test the hypothesis that collapse of chloride homeostasis in trigeminal neurons causes impairment of gamma-aminobutyric acid-ergic (GABAergic) inhibition and induces trigeminal allodynia. Rats showed a reduction and increase in pain threshold and pain response scores, respectively, to mechanical stimulation, 1 and 3 weeks after CCI. In situ hybridization and immunohistochemical analysis showed that inward-directed Na+, K+-2Cl(-) cotransporter (NKCC1) mRNA and protein were upregulated in the small-sized and large-sized primary neurons in the injured side of the trigeminal ganglion and in the peripherin-positive terminal, respectively, for the first 2 weeks, while outward-directed K+-Cl- cotransporter (KCC2) mRNA and protein were downregulated in secondary relay neurons on the injured side of the spinal trigeminal nucleus caudalis (Sp5C). Optical imaging of evoked synaptic responses using a voltage-sensitive dye revealed that pre- and post-synaptic GABA actions were disinhibited and excitatory in the injured side, respectively, but inhibited in the sham-operated side of the Sp5C. This downregulation of KCC2 in the Sp5C may result in an excitatory switch by impairing postsynaptic GABA inhibition. GABA-mediated presynaptic disinhibition was attenuated by bumetanide, suggesting that NKCC1 upregulation in primary neurons may facilitate pain transmission by presynaptic GABAergic depolarization. Such Cl- homeostatic disruption resulting in perturbation of the inhibitory system possibly increases pain transmission, which may underlie the pathophysiology of trigeminal neuropathic pain. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Decreased Tonic Inhibition in Cerebellar Granule Cells Causes Motor Dysfunction in a Mouse Model of Angelman Syndrome, Kiyoshi Egawa, Kyoko Kitagawa, Koichi Inoue, Masakazu Takayama, Chitoshi Takayama, Shinji Saitoh, Tatsuya Kishino, Masatoshi Kitagawa, Atsuo Fukuda, SCIENCE TRANSLATIONAL MEDICINE, 4, (163) ,   2012年12月, 査読有り, Angelman syndrome is a neurodevelopmental disorder caused by loss of function of the UBE3A gene encoding a ubiquitin E3 ligase. Motor dysfunction is a characteristic feature of Angelman syndrome, but neither the mechanisms of action nor effective therapeutic strategies have yet been elucidated. We report that tonic inhibition is specifically decreased in cerebellar granule cells of Ube3a-deficient mice, a model of Angelman syndrome. As a mechanism underlying this decrease in tonic inhibition, we show that Ube3a controls degradation of gamma-aminobutyric acid (GABA) transporter 1 (GAT1) and that deficiency of Ube3a induces a surplus of GAT1 that results in a decrease in GABA concentrations in the extrasynaptic space. Administering low doses of 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP), a selective extrasynaptic GABA(A) receptor agonist, improves the abnormal firing properties of a population of Purkinje cells in cerebellar brain slices and reduces cerebellar ataxia in Ube3a-deficient mice in vivo. These results suggest that pharmacologically increasing tonic inhibition may be a useful strategy for alleviating motor dysfunction in Angelman syndrome.
  • Taurine Inhibits K+-Cl- Cotransporter KCC2 to Regulate Embryonic Cl- Homeostasis via With-no-lysine (WNK) Protein Kinase Signaling Pathway, Koichi Inoue, Tomonori Furukawa, Tatsuro Kumada, Junko Yamada, Tianying Wang, Rieko Inoue, Atsuo Fukuda, JOURNAL OF BIOLOGICAL CHEMISTRY, 287, (25) 20839 - 20850,   2012年06月, 査読有り, GABA inhibits mature neurons and conversely excites immature neurons due to lower K+-Cl- cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl- homeostasis, which is required for normal brain development.
  • CYSTEINE 149 IN THE EXTRACELLULAR FINGER DOMAIN OF ACID-SENSING ION CHANNEL 1B SUBUNIT IS CRITICAL FOR ZINC-MEDIATED INHIBITION, Q. Jiang, K. Inoue, X. Wu, C. J. Papasian, J. Q. Wang, Z. G. Xiong, X. P. Chu, NEUROSCIENCE, 193, 89 - 99,   2011年10月, 査読有り, Acid-sensing ion channel 1b (ASIC1b) is a proton-gated Na(+) channel mostly expressed in peripheral sensory neurons. To date, the functional significance of ASIC1b in these cells is unclear due to the lack of a specific inhibitor/blocker. A better understanding of the regulation of ASIC1b may provide a clue for future investigation of its functional importance. One important regulator of acid-sensing ion channels (ASICs) is zinc. In this study, we examined the detailed zinc inhibition of ASIC1b currents and specific amino acid(s) involved in the inhibition. In Chinese hamster ovary (CHO) cells expressing rat ASIC1b subunit, pretreatment with zinc concentration-dependently inhibited the ASIC1b currents triggered by pH dropping from 7.4 to 6.0 with a half-maximum inhibitory concentration of 26 mu M. The inhibition of ASIC1b currents by pre-applied zinc was independent of pH, voltage, or extracellular Ca(2+). Further, we showed that the effect of zinc is dependent on the extracellular cysteine, but not histidine residue. Mutating cysteine 149, but not cysteine 58 or cysteine 162, located in the extracellular domain of the ASIC1b subunit abolished the zinc inhibition. These findings suggest that cysteine 149 in the extracellular finger domain of ASIC1b subunit is critical for zinc-mediated inhibition and provide the basis for future mechanistic studies addressing the functional significance of zinc inhibition of ASIC1b. Published by Elsevier Ltd on behalf of IBRO.
  • Calcium-permeable ion channels involved in glutamate receptor-independent ischemic brain injury, Ming-hua Li, Koichi Inoue, Hong-fang Si, Zhi-gang Xiong, ACTA PHARMACOLOGICA SINICA, 32, (6) 734 - 740,   2011年06月, 査読有り, Brain ischemia is a leading cause of death and long-term disabilities worldwide. Unfortunately, current treatment is limited to thrombolysis, which has limited success and a potential side effect of intracerebral hemorrhage. Searching for new cell injury mechanisms and therapeutic interventions has become a major challenge in the field. It has been recognized for many years that intracellular Ca2+ overload in neurons is essential for neuronal injury associated with brain ischemia. However, the exact pathway(s) underlying the toxic Ca2+ loading remained elusive. This review discusses the role of two Ca2+-permeable cation channels, TRPM7 and acid-sensing channels, in glutamate-independent Ca2+ toxicity associated with brain ischemia.
  • Pathophysiologically Relevant Levels of Hydrogen Peroxide Induce Glutamate-Independent Neurodegeneration That Involves Activation of Transient Receptor Potential Melastatin 7 Channels, Emily Coombes, Jie Jiang, Xiang-Ping Chu, Koichi Inoue, Joshua Seeds, Deborah Branigan, Roger P. Simon, Zhi-Gang Xiong, ANTIOXIDANTS & REDOX SIGNALING, 14, (10) 1815 - 1827,   2011年05月, 査読有り, Stroke/brain ischemia is a leading cause of death and long-term disabilities. Increased oxidative stress plays an important role in the pathology of brain ischemia. Hydrogen peroxide (H2O2) is a major oxidant known to cause neuronal injury; however, the detailed mechanism remains unclear. Previous studies have suggested that H2O2-induced injury is associated with increased intracellular Ca2+, mediated by glutamate receptors or voltage-gated Ca2+ channels. Here, we demonstrate that, at concentrations relevant to stroke, H2O2 induces a Ca2+-dependent injury of mouse cortical neurons in the absence of activation of these receptors/channels. With the culture medium containing blockers of glutamate receptors and voltage-gated Ca2+ channels, brief exposure of neurons to H2O2 induced a dose-dependent injury. Reducing [Ca2+](e) inhibited whereas increasing [Ca2+](e) potentiated the H2O2 injury. Fluorescent Ca2+ imaging confirmed the increase of [Ca2+](i) by H2O2 in the presence of the blockers of glutamate receptors and voltage-gated Ca2+ channels. Addition of 2-aminoethoxydiphenyl borate, an inhibitor of transient receptor potential melastatin 7 (TRPM7) channels, or the use of TRPM7-small interference RNA, protected the neurons from H2O2 injury. In contrast, overexpressing TRPM7 channels in human embryonic kidney 293 cells increased H2O2 injury. Our findings indicate that H2O2 can induce Ca2+ toxicity independent of glutamate receptors and voltage-gated Ca2+ channels. Activation of TRPM7 channels is involved in such toxicity. Antioxid. Redox Signal. 14, 1815-1827.
  • Developmental change in the electrophysiological and pharmacological properties of acid-sensing ion channels in CNS neurons, Minghua Li, Eric Kratzer, Koichi Inoue, Roger P. Simon, Zhi-Gang Xiong, JOURNAL OF PHYSIOLOGY-LONDON, 588, (20) 3883 - 3900,   2010年10月, 査読有り, Acid-sensing ion channels (ASICs) are proton-gated cation channels that play important roles in the CNS including synaptic plasticity and acidosis-mediated neuronal injury. ASIC1a and ASIC2a subunits are predominant in CNS neurons, where homomultimeric and heteromultimeric channel configurations co-exist. Since ASIC1a and ASIC2a have dramatic differences in pH sensitivity, Ca2+ permeability and channel kinetics, any change in the level of individual subunits may have significant effects on the properties and functions of ASICs. Using patch-clamp recording, fluorescent Ca2+ imaging and molecular biological techniques, we show dramatic developmental changes in the properties of ASICs in mouse cortical neurons. For example, the amplitude of ASIC currents increases whereas desensitization decreases with neuronal maturation. Decreased H+ affinity and acid-evoked [Ca2+](i) but increased Zn2+ potentiation were also recorded in mature neurons. RT-PCR revealed significant increases in the ratio of ASIC2/ASIC1 mRNA with neuronal maturation. Thus, contributions of ASIC1a and ASIC2a to overall ASIC-mediated responses undergo distinct developmental changes. These findings may help in understanding the precise role of ASICs in physiological and pathological conditions at different developmental stages.
  • Low-concentration tributyltin perturbs inhibitory synaptogenesis and induces neuronal death in immature but not mature neurons, Junko Yamada, Koichi Inoue, Tomonori Furukawa, Atsuo Fukuda, TOXICOLOGY LETTERS, 198, (2) 282 - 288,   2010年10月, 査読有り, Tributyltin (TBT) has harmful effects on invertebrates. Reports indicate that intoxication of humans with organotin compounds could be associated with neurological symptoms such as epilepsy and amnesia; however, the toxicity mechanisms in mammals are unknown. TBT acts as a Cl(-)/OH(-) antiporter, and likely affects the GABAergic system by disturbing Cl(-) homeostasis. This study aimed to elucidate neurotoxic actions of TBT on mouse neocortical neurons during development. From 4 days in vitro (4 DIV) or 14 DIV in culture, cortical neurons were exposed to TBT continuously for 3 days. TBT-induced neuronal death at 30 nM during DIV 4-6, and at 50 nM during DIV 14-16. To further characterize this age-dependent cytotoxicity, miniature postsynaptic currents (mPSCs) were analyzed by whole-cell patch-clamp. The frequency of mPSCs was significantly reduced by treatment with 30 nM TBT during DIV 4-6, but not DIV 14-16. After TBT treatment during DIV 4-6, GABA(A) receptor-mediated reversal potentials (E(GABA)) were significantly shifted negatively. The TBT-induced E(GABA) shift and neuronal death were reversed by increment of extracellular Cl- concentration, suggesting that disruption of Cl(-) homeostasis underlies the disturbance of neuronal ontogeny induced by TBT. These data indicate that the TBT may affect synaptogenesis and neuronal survival, particularly in early development. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
  • Acid-sensing ion channels in acidosis-induced injury of human brain neurons, Minghua Li, Koichi Inoue, Deborah Branigan, Eric Kratzer, Jillian C. Hansen, Jeff W. Chen, Roger P. Simon, Zhi-Gang Xiong, JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM, 30, (6) 1247 - 1260,   2010年06月, 査読有り, Acidosis is a common feature of the human brain during ischemic stroke and is known to cause neuronal injury. However, the mechanism underlying acidosis-mediated injury of the human brain remains elusive. We show that a decrease in the extracellular pH evoked inward currents characteristic of acid-sensing ion channels (ASICs) and increased intracellular Ca2+ in cultured human cortical neurons. Acid-sensing ion channels in human cortical neurons show electrophysiological and pharmacological properties distinct from those in neurons of the rodent brain. Reverse transcriptase-PCR and western blot detected a high level of the ASIC1a subunit with little or no expression of other ASIC subunits. Treatment of human cortical neurons with acidic solution induced substantial cell injury, which was attenuated by the ASIC1a blockade. Thus, functional homomeric ASIC1a channels are predominantly expressed in neurons from the human brain. Activation of these channels has an important role in acidosis-mediated injury of human brain neurons. Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1247-1260; doi: 10.1038/jcbfm.2010.30; published online 10 March 2010
  • Zinc-induced Neurotoxicity Mediated by Transient Receptor Potential Melastatin 7 Channels, Koichi Inoue, Deborah Branigan, Zhi-Gang Xiong, JOURNAL OF BIOLOGICAL CHEMISTRY, 285, (10) 7430 - 7439,   2010年03月, 査読有り, Transient receptor potential melastatin 7 (TRPM7) channels are novel Ca(2+)-permeable non-selective cation channels ubiquitously expressed. Activation of TRPM7 channels has been shown to be involved in cellular Mg(2+) homeostasis, diseases caused by abnormal magnesium absorption, and in Ca(2+)-mediated neuronal injury under ischemic conditions. Here we show strong evidence suggesting that TRPM7 channels also play an important role in cellular Zn(2+) homeostasis and in Zn(2+)-mediated neuronal injury. Using a combination of fluorescent Zn(2+) imaging, small interfering RNA, pharmacological analysis, and cell injury assays, we show that activation of TRPM7 channels augmented Zn(2+)-induced injury of cultured mouse cortical neurons. The Zn(2+)-mediated neurotoxicity was inhibited by nonspecific TRPM7 blockers Gd(3+) or 2-aminoethoxydiphenyl borate, and by knockdown of TRPM7 channels with small interfering RNA. In addition, Zn(2+)-mediated neuronal injury under oxygen-glucose deprivation conditions was also diminished by silencing TRPM7. Furthermore, we show that overexpression of TRPM7 channels in HEK293 cells increased intracellular Zn(2+) accumulation and Zn(2+)-induced cell injury, while silencing TRPM7 by small interfering RNA attenuated the Zn(2+)-mediated cell toxicity. Thus, TRPM7 channels may represent a novel target for neurological disorders where Zn(2+) toxicity plays an important role.
  • The physiological roles of vesicular GABA transporter during embryonic development: a study using knockout mice, Kenzi Saito, Toshikazu Kakizaki, Ryotaro Hayashi, Hiroshi Nishimaru, Tomonori Furukawa, Yoichi Nakazato, Shigeo Takamori, Satoe Ebihara, Masakazu Uematsu, Masayoshi Mishina, Jun-ichi Miyazaki, Minesuke Yokoyama, Shiro Konishi, Koichi Inoue, Atsuo Fukuda, Manabu Fukumoto, Kenji Nakamura, Kunihiko Obata, Yuchio Yanagawa, MOLECULAR BRAIN, 3,   2010年, 査読有り, Background: The vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them. Results: VGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected. Conclusion: VGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes.
  • Silencing TRPM7 promotes growth/proliferation and nitric oxide production of vascular endothelial cells via the ERK pathway, Koichi Inoue, Zhi-Gang Xiong, CARDIOVASCULAR RESEARCH, 83, (3) 547 - 557,   2009年08月, 査読有り, The presence and potential function of transient receptor potential melastatin 7 (TRPM7), a Ca(2+)-permeable non-selective cation channel of the TRP channel superfamily in human vascular endothelial cells, were examined. Whole-cell patch-clamp recordings showed outward-rectifying currents in human umbilical vein endothelial cells (HUVECs), which was potentiated by removing the extracellular Ca(2+) and Mg(2+), but inhibited by non-specific TRPM7 blocker Gd(3+) or 2-aminoethoxydiphenyl borate (2-APB). TRPM7 mRNA was detected in HUVECs by RT-PCR, but TRPM6, its closest homologue, was not. Silencing TRPM7 by small interfering RNA (siRNA) decreased the level of TRPM7 mRNA and the TRPM7-like current. Interestingly, knockdown of TRPM7 with siRNA or inhibition of TRPM7 function with 2-APB increased the phosphorylation of extracellular signal-regulated kinase (ERK) and enhanced growth/proliferation of HUVECs. This enhanced cell growth/proliferation was abolished by an inhibitor of the ERK signalling pathway. In addition to cell growth/proliferation, silencing TRPM7 also increased expression of nitric oxide synthase and nitric oxide production in an ERK pathway-dependent manner. These observations suggest that TRPM7 channels may play an important role in the function of vascular endothelial cells.
  • In vivo and in vitro characterization of a novel neuroprotective strategy for stroke: ischemic postconditioning., Pignataro G, Meller R, Inoue K, Ordonez AN, Ashley MD, Xiong Z, Gala R, Simon RP, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 28, (2) 232 - 241,   2008年02月, 査読有り
  • Transient receptor potential melastatin 7-like current in human head and neck carcinoma cells: Role in cell proliferation, Jie Jiang, Ming-Hua Li, Koichi Inoue, Xiang-Ping Chu, Joshua Seeds, Zhi-Gang Xiong, CANCER RESEARCH, 67, (22) 10929 - 10938,   2007年11月, 査読有り, Ion channels are involved in normal physiologic processes and in the pathology of various diseases. In this study, we investigated the presence and potential function of transient receptor potential melastatin 7 (TRPM7) channels in the growth and proliferation of FaDu and SCC25 cells, two common human head and neck squamous carcinoma cell lines, using a combination of patch-clamp recording, Western blotting, immunocytochemistry, small interfering RNA (siRNA), fluorescent Ca2+ imaging, and cell counting techniques. Although voltage-gated K+ currents were recorded in all cells, none of FaDu cells express voltage-gated Na+ or Ca2+ currents. Perfusion of cells with NMDA or acidic solution did not activate inward currents, indicating a lack of NMDA receptor and acid-sensing channels. Lowering extracellular Ca2+, however, induced a large nondesensitizing current reminiscent of Ca2+ -sensing cation current or TRPM7 current previously described in other cells. This Ca2+ -sensing current can be inhibited by Gd3+, 2-aminoethoxydiphenyl borate (2-APB), or intracellular Mg2+, consistent with the TRPM7 current being activated. Immunocytochemistry, Western blot, and reverse transcription-PCR detected the expression of TRPM7 protein and mRNA in these cells. Transfection of FaDu cells with TRPM7 siRNA significantly reduced the expression of TRPM7 mRNA and protein as well as the amplitude of the Ca2+ -sensing current. Furthermore, we found that Ca2+ is critical for the growth and proliferation of FaDu cells. Blockade of TRPM7 channels by Gd3+ and 2-APB or suppression of TRPM7 expression by siRNA inhibited the growth and proliferation of these cells. Similar to FaDu cells, SCC25 cells also express TRPM7-like channels. Suppressing the function of these channels inhibited the proliferation of SCC25 cells.
  • Brain-type creatine kinase activates neuron-specific K(+)-Cl(-) co-transporter KCC2, K Inoue, J Yamada, S Ueno, A Fukuda, JOURNAL OF NEUROCHEMISTRY, 96, (2) 598 - 608,   2006年01月, 査読有り, GABA, a major inhibitory neurotransmitter in the adult CNS, is excitatory at early developmental stages as a result of the elevated intracellular Cl(-) concentration ([Cl(-)](i)). This functional switch is primarily attributable to the K(+)-Cl(-) co-transporter KCC2, the expression of which is developmentally regulated in neurons. Previously, we reported that KCC2 interacts with brain-type creatine kinase (CKB). To elucidate the functional significance of this interaction, HEK293 cells were transfected with KCC2 and glycine receptor alpha 2 subunit, and gramicidin-perforated patch-clamp recordings were performed to measure the glycine reversal potential (E(gly)), giving an estimate of [Cl(-)](i). KCC2-expressing cells displayed the expected changes in E(gly) following alterations in the extracellular K(+) concentration ([K(+)](o)) or administration of an inhibitor of KCCs, suggesting that the KCC2 function was being properly assessed. When added into KCC2-expressing cells, dominant-negative CKB induced a depolarizing shift in E(gly) and reduced the hyperpolarizing shift in E(gly) seen in response to a lowering of [K(+)](o) compared with wild-type CKB. Moreover, 2,4-dinitrofluorobenzene (DNFB), an inhibitor of CKs, shifted E(gly) in the depolarizing direction. In primary cortical neurons expressing CKB, the GABA reversal potential was also shifted in the depolarizing direction by DNFB. Our findings suggest that, in the cellular micro-environment, CKB activates the KCC2 function.
  • Characterization of newly cloned variant of rat glycine receptor alpha 1 subunit, K Inoue, S Ueno, J Yamada, A Fukuda, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 327, (1) 300 - 305,   2005年02月, 査読有り, Responses to glycine, a major inhibitory neurotransmitter within the nervous system, are mediated by glycine receptors (GlyRs). Here, we report the cloning and analysis of a novel splicing variant of the GlyRalpha1 subunit. This variant, named GlyRalpha1(del), has a truncated cytoplasmic region between transmembrane domains (TM)3 and TM4, and compared to other variants, the truncation is contributed by a different acceptor site in exon 9. We transfected GlyRalpha1 or GlyRalpha1(del) into HEK293 cells, and then examined the glycine-activated currents using a whole-cell patch-clamp recording technique. Maximal currents and current-voltage relationships showed no clear difference between GlyRalpha1(del) and GlyRalpha1. Moreover, dose-response curves indicated that the EC50 values for glycine differed significantly between the two GlyRalpha1 derivatives, although their Hill coefficients were similar. When present with other isoforms, GlyRalpha1(del) might alter the response to glycine or to other agonists, as this variant expands the potential heterogeneity among glycine receptors. (C) 2004 Elsevier Inc. All rights reserved.
  • Malfunction of respiratory-related neuronal activity in Na(+), K(+)-ATPase alpha 2 subunit-deficient mice is attributable to abnormal Cl(-) homeostasis in brainstem neurons, K Ikeda, H Onimaru, J Yamada, K Inoue, S Ueno, T Onaka, H Toyoda, A Arata, T Ishikawa, MM Taketo, A Fukuda, K Kawakami, JOURNAL OF NEUROSCIENCE, 24, (47) 10693 - 10701,   2004年11月, 査読有り, Na(+), K(+)-ATPase alpha2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2(-/-)) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity in Atp1a2(-/-) was investigated using a brainstem-spinal cord en bloc preparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective in Atp1a2(-/-) fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly in Atp1a2(-/-) than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain of Atp1a2(-/-). Lack of inhibitory neural activities in VLM of Atp1a2(-/-) was observed by optical recordings. High intracellular Cl(-) concentrations in neurons of theVLMof Atp1a2(-/-) were detected in gramicidin-perforated patch-clamp recordings. The alpha2 subunit and a neuron-specific K-Cl cotransporter KCC2 were coimmunoprecipitated in a purified synaptic membrane fraction of wild-type fetuses. Based on these results, we propose a model for functional coupling between the Na(+), K(+)-ATPase alpha2 subunit and KCC2, which excludes Cl(-) from the cytosol in respiratory center neurons.
  • Interaction of neuron-specific K(+)-Cl(-) cotransporter, KCC2, with brain-type creatine kinase, K Inoue, S Ueno, A Fukuda, FEBS LETTERS, 564, (1-2) 131 - 135,   2004年04月, 査読有り, gamma-Aminobutyric acid, a major inhibitory neurotransmitter within the adult central nervous system, is also known to be excitatory at early developmental stages due to the elevated intracellular Cl(-) concentration. This functional change is primarily attributable to a K(+)-Cl(-) cotransporter, KCC2, the expression of which is developmentally regulated in neurons. However, little detail information is available concerning the intracellular regulation of KCC2 function. Here, we identify an interaction between KCC2 and brain-type creatine kinase by means of yeast two-hybrid screening. This interaction, which was also detected in cultured cells and brain extracts, might contribute to KCC2-mediated modulation of Cl(-) homeostasis. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • TNF alpha-induced ATF3 expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells, K Inoue, T Zama, T Kamimoto, R Aoki, Y Ikeda, H Kimura, M Hagiwara, GENES TO CELLS, 9, (1) 59 - 70,   2004年01月, 査読有り, ATF3 (Activating transcription factor 3), a member of the CREB/ATF family, can be induced by stress and growth factors in mammalian cells, and is thought to play an important role in the cardiovascular system. However, little is currently known about how the induction of ATF3 is regulated, except that the JNK pathway is involved. Here, we investigated the differential roles of the MAPK pathways involved in TNFalpha (tumour necrosis factor alpha)-induced ATF3 expression in vascular endothelial cells. In human umbilical vein endothelial cells, the expression of constitutively active MKK7 (MAPK kinase 7) increased the number of ATF3-positive cells, and dominant negative MKK7 suppressed the TNFalpha-induced expression of ATF3, indicating a requirement for the JNK pathway. In contrast, the expression of constitutively active or dominant negative MEK1/2 (MAPK/ERK kinase 1/2) suppressed or enhanced TNFalpha-mediated induction of ATF3, respectively. In support of this, the MEK1/2 specific inhibitor U0126 enhanced the expression of ATF3 induced by TNFalpha. Furthermore, the ERK pathway inhibits the TNFalpha-mediated induction of ATF3 mRNA, but not its stability, suggesting the involvement of ERK activity in the transcriptional regulation of the ATF3 gene. Our results suggest that TNFalpha-induced ATF3 gene expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.
  • Identification of methylated proteins by protein arginine N-methyltransferase 1, PRMT1, with a new expression cloning strategy., Wada K, Inoue K, Hagiwara M, Biochimica et biophysica acta, 1591, (1-3) 1 - 10,   2002年08月, 査読有り
  • A novel dual specificity phosphatase SKRP1 interacts with the MAPK kinase MKK7 and inactivates the JNK MAPK pathway. Implication for the precise regulation of the particular MAPK pathway., Zama T, Aoki R, Kamimoto T, Inoue K, Ikeda Y, Hagiwara M, The Journal of biological chemistry, 277, (26) 23909 - 23918,   2002年06月, 査読有り
  • Scaffold role of a mitogen-activated protein kinase phosphatase, SKRP1, for the JNK signaling pathway, T Zama, R Aoki, T Kamimoto, K Inoue, Y Ikeda, M Hagiwara, JOURNAL OF BIOLOGICAL CHEMISTRY, 277, (26) 23919 - 23926,   2002年06月, 査読有り, Stress-activated protein kinase (SAPK) pathway-regulating phosphatase 1 (SKRP1) has been identified as a member of the mitogen-activated protein kinase (MAPK) phosphatase (MKP) family that interacts physically with the MAPK kinase (MAPKK) MKK7, a c-Jun N-terminal kinase (JNH) activator, and inactivates the MAPK JNK pathway. Although these findings indicated that SKRP1 contributes to the precise regulation of JNK signaling, it remains to be elucidated how SKRP1 is integrated into this pathway. We report that SKRP1 also plays a scaffold role for the JNK signaling, judged by the following observations. SKRP1 selectively formed the stable complexes with MKK7 but not with MKK4 and biphasically regulated the MKK7 activity and MKK7-induced gene transcription in vivo. Co-precipitation analysis between SKRP1 and MKK7-activating MAPKK kinases (MAPKKKs) revealed that SKRP1 also interacted with the MAPKKK, apoptosis signal-regulating kinase 1 (ASK1), but not with MAP kinase kinase kinase 1 (MEKK1). Consistent with these findings, SKRP1 expression increased the ASK1-MKK7 complexes in a dose-dependent manner and specifically enhanced the activation of MKK7 by ASK1. Thus, our findings are, to our knowledge, the first evidence to show that an MKP also functions as a scaffold protein for the particular MAPK signaling.

MISC

  • Novel RING finger proteins, Air1p and Air2p, interact with Hmt1p and inhibit the arginine methylation of Npl3p, K Inoue, T Mizuno, K Wada, M Hagiwara, JOURNAL OF BIOLOGICAL CHEMISTRY, 275, (42) 32793 - 32799,   2000年10月, Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in the mRNA processing and export and are post-translationally modified by methylation at arginine residues in their arginine-glycine-rich (RGG) domains. We screened the factors that can interact with the RGG domain of Np13p only in the presence of Hmt1p with the two-hybrid system in Saccharomyces cerevisiae, An isolated clone, YIL079, encodes a novel RING finger protein that was not directly bound to Np13p but associated with the N terminus of Hmt1p. Thus, we designated the gene product Air1p (arginine methyltransferase-interacting RING finger protein). Air1p inhibited the Hmt1p-mediated methylation of Np13p in vitro. Overexpression of Air1p repressed the Hmt1p-dependent growth of cells. Since homology searches indicate that the YDL175 gene product has significant identity (45%) with Air1p, we designated the gene AIR2. Air2p also has a RING finger domain and was bound to Hmt1p. Although single disruption of either gene gave no effect on the cell growth, cells lacking Air1p and Air2p grew at an extremely slow rate with accumulated poly(A)(+) RNA in the nucleus. Thus, Air1p and Air2p may affect mRNA transport by regulating the arginine methylation state of heterogeneous nuclear ribonucleoproteins.

競争的資金

  • カハール・レチウス細胞等のGABAの積極的興奮性とその生理的意義の解明, 文部科学省, 科学研究費補助金(新学術領域研究(研究領域提案型)),   2011年 - 2012年
  • タウリンによるKCC2活性制御の分子基盤とその意義の解明, 文部科学省, 科学研究費補助金(基盤研究(C)),   2011年 - 2011年
  • グリオーマの移動・浸潤時に起こる細胞内イオン変化の可視化とその応用性の検討, 文部科学省, 科学研究費補助金(基盤研究(C)),   2008年 - 2010年, 細胞内イオンイメージング法を用いて、グリオーマ細胞の移動時における自発的な細胞内Ca^<2+>とCl^-濃度の変化を可視化してその役割を検討した。グリオーマ細胞では細胞内Ca^<2+>濃度の頻度の変化は著しい加速を示す移動細胞の指標と成ったが、移動速度との関連性は無かった。細胞内Cl^-イオン濃度をFRET型インディケーターで測定し、細胞内におけるCl^-の局在性について検討した。また、細胞内Cl^-イオン濃度を変化させるとグリオーマ細胞の移動速度を変化させることが分かった。
  • 分子標的発見による肝臓強化新療法の基礎的研究, 文部科学省, 科学研究費補助金(萌芽研究),   2005年 - 2006年, 2系統のRbトランスジェニックマウスを得た。A系統のマウスはトランスジーンを1倍体当たり11コピーもち、B系統のマウスは1倍体当たりトランスジーンを4コピーもっている。今年度は、このトランスジェニックマウスが化学発癌抵抗性を示すかどうかを検討した。化学発癌にはRbトランスジェニックマウス雌とC3H雄のF1(遺伝的背景:B6C3F1)を用いた。生後6週齢のマウスにジエチルニトロソアミンを腹腔内投与し、1週後からフェノバルビタール含有水を35週継続飲水させる。コントロールマウス、Rbトランスジェニックマウス肝臓の病理標本を作製し、腫瘍性病変の性質、悪性度、頻度を検討した。まず、コントロールマウスでは雄、雌とも肝細胞癌が誘導された。雄の発癌性は雌の2-3倍であった。Rbトランスジェニックマウスでは雄雌とも肝細胞癌はまったく認められなかった。次に、結節病変について検討した。コントロールマウスでは雄は平均3.85個の結節を生じるに対し、雌は平均1.70個の結節を生じた。RbトランスジェニックマウスA系統では、雄は平均0.82個の結節を生じ、雌は平均0.33個の結節を生じた。A系統のマウスでは雄雌とも、トランスジェニックマウスは癌抑制性に働いていた。一方、B系統のマウスの雄では、平均0.60個の結節を生じ、雌では平均1.20個の結節を生じた。有意検定をしたところ、B系統では雄のみに有意に...
  • 発達期大脳皮質神経細胞の垂直移動・分化関連遺伝子の網羅的同定及び解析, 文部科学省, 科学研究費補助金(基盤研究(C)),   2005年 - 2006年, 子宮内電気穿孔法を用いて胎生13日齢のICRマウス胎仔脳室周囲の神経前駆細胞にEGFPを導入した。それらの細胞は徐々に脳表に移動(radial migration)していくが、遺伝子導入3日後ではEGFP陽性細胞は脳室帯、中間帯、辺縁帯にほぼ均等に分散している。そこで、胎生15日の脳室帯、胎生16日の中間帯、胎生18日の辺縁帯を脳スライスから切り出し、細胞を分離させた後、cell sortingによりEGFP陽性細胞のみを回収した。細胞数が不十分であったため、in vitro転写法により、2段階でRNAを増幅し後マイクロアレイを行った。細胞の垂直方向への移動と関係が示唆される遺伝子であり、シグナル強度が十分で、かつ、胎生16日中間帯で3倍以上発現が上昇しその後減少していたSema6cとSyntaphifilinについて、免疫組織学的手法により解析をおこなった。胎生17日齢の胎仔大脳皮質におけるSema6CとSyntaphilinの局在を免疫染色法により観察した結果、中間帯に強く発現していることが認められた。この結果から、マイクロアレイにより得られたSema6CとSyntaphilinの遺伝子レベルでの解析結果は、タンパクの局在とほぼ一致することが確認できた。したがって、脳室帯において子宮内穿孔法により標識された神経細胞では、radial migration中にSema6CやS...
  • 環境因子がC1^-ホメオスタシス変化を介して皮質神経回路の発達と再生に与える影響, 文部科学省, 科学研究費補助金(基盤研究(B)),   2004年 - 2006年, 発達期のプログラムされたCl^-ホメオスタシスがGABAを興奮性に作用させ細胞移動による回路構築を促し、環境刺激によるCl^-ホメオスタシス変化が、神経回路形成や再生にも影響するという仮説の検証を目的とした。まず、GAD67-GFP knock-inマウス胎仔脳へHcRed遺伝子をインビボで導入しradial移動中のglutamate細胞とtangential移動中のGABA細胞を生きた状態で識別する方法を確立した。胎仔脳に豊富に存在するタウリンがGABA_A受容体の内因性アゴニストとして作用して、autocrine的にradial移動細胞に働いたり、paracrine的にtangential移動細胞へのシグナルとなっている可能性が示唆された。細胞移動中に強制発現させたKCC2はin vivoでは機能しないことが示唆された。そこで、Cl^-トランスポーターの転写過程を阻害するshRNAに最適な塩基配列をつデザイン・合成して、shRNA発現プラスミドベクターを構築した。またradial移動に影響を与えるCajal-Retzius細胞を含む辺縁帯の興奮伝播では、GABAがGABA_A受容体の、そしてタウリンがglycine受容体の内在性アゴニストとして働き、しかもNKCC1によって[Cl^-]_iが高く維持されることにより、本来抑制性であるGABA_Aやglycine受容体が興奮...
  • 大脳皮質回路形成過程の細胞間クロストークにおけるGABAとタウリンの役割, 文部科学省, 科学研究費補助金(特定領域研究),   2005年 - 2005年, 1.radial及びtangential移動細胞におけるCl^-トランスポーター遺伝子発現変化の解析[福田・井上・熊田]:GAD67-GFP knock-inマウス脳室帯の新生ニューロンへのHcRed遺伝子の導入を行い、GABA含有量が異なるホモ、ヘテロ、野生型間でradial及びtangential移動の状態を比較したが全く差は見られなかった。さらに、パッチクランプ法でradial移動中のglutamate細胞におけるGABAのdose-response curveを測定したが全く差は無かった。HPLC法を用いてGABAの細胞外濃度を測定したところ、予想通り野生型に比べてヘテロタイプ65%、ホモタイプ7%であった。以上からGABA以外の内因性アミノ酸が代償作用を持つと考え、細胞外タウリン濃度を調べたところGABAの1000-10000倍であり、GABAの1%程度の親和性でGABA_A受容体を介した脱分極を惹起した。すなわち、タウリンがGABA以上にGABA_A受容体の内因性アゴニストとして作用しているため、細胞外GABA濃度が異なるどのgenotypeでもGABA_A受容体やCl^-トランスポーター発現に差が出なかったと考えられた。2.辺縁帯におけるCajal-Retzius細胞/non Cajal-Retzius細胞間のクロストークの解析[福田・Luhmann]:辺縁帯の...
  • 会話言語遺伝子FOXP2の高次脳機能における役割の解明, 文部科学省, 科学研究費補助金(特定領域研究),   2005年 - 2005年, マウスFoxp2染色体遺伝子をまず単離した。この遺伝子は19エキソンからなる全長が300kbp以上の大きい遺伝子であることが判明した。エキソン14をloxPで挟み込んだターゲティングベクターを作製し、マウスES細胞にエレクトロポレーションにて遺伝子導入した。相同的に組み換えを起こしたクローンをサザンブロット法にて判定し、5クローンを得た。現在、キメラマウスを作製中である。メダカFoxP2遺伝子は、マウスFoxp2遺伝子との相同性を用いてクローニングし、全長のcDNAを得ることに成功した。マウスFoxp2蛋白が715のアミノ酸からなるに比し、メダカFoxP2蛋白は766アミノ酸からなることが判明した。構造的特徴であるジンクフィンガー、ロイシンジッパー、フォークヘッドドメインは保存されていた。フォークヘッドドメインのN末側に16個のアミノ酸からなる領域が、C末側に54個のアミノ酸からなる領域が挿入されていた。また、ヒトやマウスに保存されていたポリグルタミン配列が欠失していた。ヒトとマウス間では3アミノ酸が異なるのみであったが、マウスとメダカ間では、20%のアミノ酸が置換していた。メダカにおけるFoxP2遺伝子の発現を抗メダカFoxP2抗体によるホールマウント免疫染色法で観察すると、前脳の一部、中脳の広範囲に強い発現が認められたが、小脳における発現は弱い発現であることが判明した。マ...
  • フォークヘッド遺伝子Foxc2による乳癌細胞転移促進作用とその分子機構の解明, 文部科学省, 科学研究費補助金(特定領域研究),   2005年 - 2005年, 中胚葉発生制御遺伝子Twistが上皮-間葉転換を介して乳癌細胞の転移能を増加させる報告を見て、我々が研究してきたFoxc2遺伝子についても同様な機能があるのではないかと考えた。Foxc2遺伝子は、多くの中胚葉由来組織の発生を制御していることが以前の我々のノックアウトマウスを用いた研究からわかっていた。例えば、体節は最初上皮様構造をとっているが、その後硬節を経て中胚葉細胞に転換し骨格系の前駆細胞になる。まず、マウス乳癌EpRas細胞にFoxc2発現ベクターを遺伝子導入し高発現永久株を樹立した。親株のEpRas細胞やFoxc2高発現株細胞をマウス乳腺皮下の脂肪組織に移植した。約6週後に、原発巣および肺転移の癌を検討した。原発巣の腫瘍サイズや数は親株と高発現株の間に差はなかった。しかし、肺への転移巣の数は親株を移植した場合10個であるのに対し、Foxc2高発現株では40個と4倍になっていた。また、転移巣の癌細胞はFoxc2高発現が持続していた。そこで、Foxc2遺伝子による転移能亢進の分子メカニズムとして上皮-間葉転換がおこっている可能性を明らかにするために、上皮細胞MDCKにFoxc2発現ベクターを遺伝子導入し、永久株を得た。Foxc2高発現細胞における上皮マーカー、間葉マーカーの変化を検討した結果、Foxc2高発現細胞株では、上皮マーカーのE-cadherin, catenin...
  • 三叉神経痛モデル動物の作成と痛み情報伝達におけるATP受容体の役割に関する研究, 文部科学省, 科学研究費補助金(基盤研究(C)),   2004年 - 2005年, ATP受容体は神経因性疼痛の形成に重要な役割を果たしていることが,明らかとてきた。さらに,中枢神経における主要な抑制性伝達物質であるGABAが,ATP受容体活性化を通じて興奮性に働くことも明らかとなってきた。正常神経細胞においては,細胞内外のClイオン濃度はそれぞれ,10mM以下及び120-130mMを示し,この条件下において,GABAはGABAタイプA受容体活性化によりClイオン流入を引き起こし,膜電位を過分極させる。一方,発達段階や,末梢神経障害により細胞内Clイオン濃度が上昇すると,GABAによりClイオン流出がおこり結果として,膜電位脱分極と細胞興奮が引き起こされる。このようなGABA機能の逆転が神経因性疼痛時におこり,この細胞内Clイオン濃度を調節するトランスポータの役割を電気生理学的およびyeast-two hybridシステムを用い解析した。yeast-two hybridシステムにより神経特異的なKCC2トランスポータが脳型creatine kinaseと相互作用する可能性が資された。そこでHEK293細胞にKCC2を導入し,stable lineを作成し,kinaseをさらに強制発現させ機能解析を行った。電気生理学的手法により,細胞内のCl濃度を測定し,細胞内でKCC2がこのkinaseにより,活性化され,細胞内Clイオン濃度上昇を来すことを明らかとした。ま...
  • 細胞外Cl^-を調節して神経回路の興奮性を制御するグリアのCl^-トランスポーター, 文部科学省, 科学研究費補助金(特定領域研究),   2004年 - 2005年, 1.脳スライスのアストロサイトからの[Cl^-]_i測定とグリアによる神経回路機能調節の証明[窪田・福田]:まずアストロサイト親和性色素のsulforhodamine 101を用いて、海馬スライス中のastrocytesを同定した。次にパッチ電極内液にCl^-感受性蛍光色素のMEQを加えておき、同定した多数のastrocytesにsingle-cell electroporation法で次々とMEQを注入し、同時に5-6個のastrocytesでのCl^-イメージングを可能にした。シェーファー側枝をテタヌス刺激した時のastrocytesの[Cl^-]_i変化をこれら複数個のastrocytesで同時に観察した(astrocyte回路網のCl^-イメージング)。シェーファー側枝の高頻度刺激で記録したすべてのastrocyteでMEQ蛍光が低下、すなわち[Cl^-]_i上昇が見られた。これらの[Cl^-]_i上昇は刺激の頻度や強度に応じて変化した。しかしこの変化は、以前我々が報告した錐体細胞でのGABA作用を抑制性から興奮性に逆転させるCl^-蓄積(J.Neurophysiol. 2003)とは時間経過が異なり、静止電位におけるastrocyteのCl^-の電気化学勾配もニューロンと逆向きとの報告もあるので、このastrocyteでの[Cl^-]_i上昇はニューロンとは全く別の...
  • 大脳皮質形成期の接線方向移動と放射状移動の細胞間クロストークにおけるCl^‐の役割, 文部科学省, 科学研究費補助金(特定領域研究),   2004年 - 2004年, 1.GFPとHcRed標識による接線方向移動細胞と放射状移動細胞の識別[井上・山田・福田]:GAD67-GFP knock-inマウス(C57BL/6j)胎仔にHcRed遺伝子を胎生14日に電気穿孔法で脳室帯の新生ニューロンに導入し、発達の各段階で脳スライスを作成した。接線方向移動中のGABA細胞(GFP蛍光)と放射状移動中のglutamate細胞(HcRed蛍光)が識別できた。放射状移動細胞は大脳皮質II/III層を形成し、接線方向移動細胞は主に中間帯と辺縁帯を移動し、中間帯から皮質版に進入していた。2.radial移動細胞における細胞内Cl^-濃度とCl^-トランスポーター遺伝子発現変化の解析[福田・窪田・山田・Luhmann]:グラミシジン穿孔パッチクランプ法で、放射状移動中のglutamate細胞のGABA/グリシン反応とCl^-平衡電位を測定して、Cl^-トランスポーターとGABA_A/グリシン受容体mRNAのsingle-cell RT-PCRを行った。その発現パターンを各々の細胞の発生時期や移動状態のちがいで比較し、放射状移動細胞の[Cl^-]_i高値と、定着後の[Cl^-]_i低下に伴うGABA_A/グリシン作用の興奮→抑制転換の分子的要因を明らかにした。また、辺縁帯のCajal-Retzius細胞でもグリシン(タウリン)反応と[Cl^-]_iを解析し、グリシ...
  • 内分泌攪乱物質によるCl^-ホメオスタシス破綻と神経細胞移動障害による神経回路異常, 文部科学省, 科学研究費補助金(基盤研究(C)),   2003年 - 2004年, 移動細胞と[Cl^-]_iの測定胎生14日齢のマウス胎仔に電気穿孔法によりGFP遺伝子を導入し、その胎仔の羊水中にトリブチルスズを注入した。トリブチルスズ1μlを注入し、2-3日後の胎仔の様子を観察した。また、生存していた胎仔の脳スライスを作成し、細胞移動の様子を解析した。・1M:胎仔は萎縮して死亡しており、両側のコントロール(電気穿孔とDMSO1μlのみの胎仔、電気穿孔のみの胎仔、何も処置しない胎仔)も死亡していた。・500mM-100mM:胎仔は萎縮して死亡していたが、両側のコントロールは生存していた。・10mM:胎仔は萎縮はしていなかったが死亡しており、両側のコントロールは生存していた。・1mM:胎仔は生存しており、細胞移動も正常であった。トリブチルスズ暴露によるCl^-ホメオスタシスへの影響・[Cl^-]_iへの影響トリブチルスズ暴露による[Cl^-]_iへの影響をみるため、トリブチルスズ30nM3日、処置したグループとコントロールグループの細胞にグラミシジン穿孔パッチクランプ法を適用し、[Cl^-]_iを測定したが、両グループ間で有意な差はみられなかった。・GABA受容体への影響トリブチルスズ暴露によるシナプス電流に変化が見られたことから、GABA_A受容体が変化していることが考えられる。トリブチルスズ30nM3日間暴露した細胞群とコントロール群の細胞のmRNAを抽...
  • てんかん原性皮質形成異常におけるCl^-ホメオスタシスとGABA機能異常の分子機構, 文部科学省, 科学研究費補助金(萌芽研究),   2002年 - 2004年, 1.ヒト皮質異形成組織標本におけるreelin遺伝子発現変化の解析(福田、井上):Reelinは大脳皮質第一層に存在するCajal-Retzius細胞から分泌され、大脳皮質の神経細胞の配置決定に重要な役割を果たしていると考えられる。大脳皮質形成異常は発生異常に基づく神経細胞の配列や形態異常などの組織像を示すので、難治の部分てんかんに対する外科治療により摘出された皮質形成異常標本を用いてreelin遺伝子の発現変化をin situ hybridization組織化学法を用いて検討した。コントロールとして摘出標本内の組織学的な形成異常がない部位を用い、比較定量的な解析を行ったところ、reelin mRNAが形成異常部位において有意に減少していた。2.皮質形成異常動物モデル標本を用いた異常層構造形成過程の解析(福田、井上、窪田、Luhmann):異常な皮質層構造の形成過程における興奮性GABA/グリシン作用の要因について検討するため、ラットの皮質凍結損傷モデルを用いて、異常皮質形成過程で損傷部位へ移動中の細胞におけるGABA_A受容体、グリシン受容体、Cl^-トランスポーターのmRNAの発現量変化をin situ hybridization法を用いて解析した。凍結損傷4日後の移入細胞でNKCC1が増加、逆にKCC2が減少しており、[Cl^-]_i上昇の原因と考えられた。これらの変化...
  • 大脳新皮質形成過程におけるカハール・レチウス細胞と皮質板細胞のクロストーク, 文部科学省, 科学研究費補助金(特定領域研究),   2003年 - 2003年, 1.マウス胎仔脳への電気穿孔法を用いたEGFP遺伝子導入による移動細胞の可視化(井上、山田、福田):分化直後の神経細胞に特異的にEGFPを発現させて移動過程を可視化する為、子宮内胎仔へのin vivo電気穿孔法により遺伝子導入を行った。すなわち、胎齢13日のマウス胎仔の側脳室にCAGプロモーターにEGFPあるいはDsRed2遺伝子を繋いだプラスミドを注入し、パルス電圧を加え脳室帯で分化直後の神経細胞に遺伝子導入した。これらの細胞がEGFP/DsRed2の蛍光を発しながら脳室帯から皮質板へ移動してinside-outの細胞配置により皮質を形成することを経時的に確認できた。これにより、細胞の発生時期を蛍光で識別することが可能になった。2.皮質板細胞でのCl^-ホメオスタシス調節遺伝子群発現バランスと細胞移動の解析(山田、岡部、福田):子宮内胎仔へのin vivo電気穿孔法により、分化直後の細胞(EGFP蛍光)にKCC2遺伝子を強制発現させ、[Cl^-]_iを低下させた細胞で細胞移動に変化があるかを調べた。まだ例数が少ないため、結論を得るために今後例数を増やす。グラミシジン穿孔パッチクランプ法でCl^-平衡電位を測定し、実際に[Cl^-]_iが低下していることを確認して、移動や[Cl^-]_iとの関係を明らかにする。3.辺緑帯におけるCajal-Retzius細胞間のクロストークの...
  • てんかん原性大脳皮質形成異常モデル動物の確立とアンチセンス治療法への展開, 文部科学省, 科学研究費補助金(基盤研究(B)),   2000年 - 2003年, 部分てんかんの原因として注目されている皮質形成異常は、神経細胞の発生・分化・移動の過程で生じた異常であり、神経細胞の配列や形態異常などの組織像を示すとともに病変自体がてんかん原性を有している。本研究課題では、皮質形成異常の成因やそのてんかん原性にCl^-ホメオスタシス異常によるGABA作用の過分極/脱分極の逆転が関わっている可能性を検討することを目的とした。まず、focal freeze-lesionによる局所的細胞移動障害モデルを作成し、Cl^-トランスポーター、GABA及びグリシン受容体サブユニット、細胞移動関連分子などのmRNAの発現変化をin situ hybridization法を用いて検討し、機能的変化をCa^<2+>イメージングやグラミシジン穿孔パッチクランプ法で解析した。その結果を、正常発達過程のラットやキンドリングモデルと比較して、細胞移動や層構造の異常及び発作波様異常興奮におけるCl^-ホメオスタシス、GABA/グリシン作用や電気生理学的特性の役割を解析した。次いでヒトてんかん原性皮質形成異常摘除組織でのCl^-トランスポーター(NKCC1=Cl^-取込,KCC2=Cl^-排出)と細胞移動関連分子(reelin)のmRNAの発現変化の解析を行い、モデル動物と比較した。その結果、モデル動物の異常皮質形成過程ではNKCC1発現上昇とKCC2発現低下、それに伴う...
  • 大脳新皮質形成過程におけるカハール・レチウス細胞と皮質板細胞のクロストーク, 文部科学省, 科学研究費補助金(特定領域研究),   2002年 - 2002年, 1.ラット胎仔脳への電気穿孔法を用いたEGFP遺伝子導入による移動細胞の可視化(井上、山田、福田):移動中の細胞に特異的にEGFPを発現させて移動後の細胞と識別する為、ラット胎仔を用いて、CMVプロモーターによるEGFP発現プラスミドを用いた遺伝子導入を電気穿孔法により行った。しかし、有効な遺伝子発現が得られず、プロモーターをEF1に変更しても同様であった。そこで、マウス胎仔を用いてCAGプロモーターによる遺伝子導入を行ったところ、脳室帯の細胞にEGFPの蛍光を確認できた。今後は移動中の細胞のみを識別することを可能にするため、各種プロモーターによる発現時期の違いを検討する。2.Cajal-Retzius細胞の生理学的特性と辺縁帯におけるシグナル伝達の解析(福田、岡部、Luhmann):辺縁帯のtanential sliceを作成し、グラミシジン穿孔パッチクランプ法でCaial-Retzius細胞のグリシン応答と[Cl^-]_iを記録した後、Cr^-トランスポーターのKCC2(Cl^-排出)とNKCC1(Cl^-取込)及びglycine受容体subunit(alpha1,alpha2,beta)mRNAのsingle-cell RT-PCRを行って単一細胞でのそれぞれの発現パターンを解析した。Cajal-Retzius細胞では皮質板細胞に比べてNKCC1の発現が優位で、[Cl^...


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