博志 香月

Publications (11)0 Total impact

• Article: Depolarizing stimuli cause persistent and selective loss of orexin in rat hypothalamic slice culture

  Katsuki Hiroshi, Kurosu Shinsuke, Michinaga Shotaro, Hisatsune Akinori, Isohama Yoichiro, Izumi Yasuhiko, Kume Toshiaki, Akaike Akinori, ヒロシ カツキ, シンスケ クロス, [......], トシアキ クメ, アキノリ アカイケ, 博志 香月, 真介 黒須, 昌太郎 道永, 昭哲 久恒, 洋一郎 礒濱, 安彦 泉, 利明 久米, 昭紀 赤池
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  ABSTRACT: A hypothalamic neuropeptide orexin (hypocretin) is a critical regulator of physiological processes including sleep/wakefulness and feeding. Using organotypic slice culture of rat hypothalamus, we found that exposure to elevated extracellular concentration of K+ (+10-30 mM) for 24-72 h led to a substantial decrease in the number of neurons immunoreactive for orexin and a co-existing neuropeptide dynorphin-A. In contrast, the same treatment affected neither the number of melanin-concentrating hormone-immunoreactive neurons nor the number of total neurons. A substantial decrease of orexin-immunoreactive neurons was also induced by 72 h treatment with 1-10 μM veratridine, a Na+ channel activator. The effect of elevated K+ was only partially reversible, and that of veratridine was virtually irreversible, although the decrease in orexin immunoreactivity was not associated with signs of cell damage assessed by propidium iodide uptake and Hoechst 33342 nuclear staining. In addition, the level of preproorexin mRNA did not decrease during treatment with elevated K+ or veratridine. After treatment with elevated K+ and veratridine, c-Fos immunoreactivity appeared in orexin-immunoreactive neurons but not in melanin-concentrating hormone-immunoreactive neurons, suggesting selective excitation of orexin neurons. However, the amount of orexin released extracellularly was paradoxically decreased by treatment with elevated K+ and veratridine. Overall, these characteristics of orexin neurons may be taken into consideration to understand the behaviors of these neurons under physiological and pathophysiological conditions.
  01969781.
• Article: Aminoglutethimide prevents excitotoxic and ischemic injuries in cortical neurons

  Hisashi Shirakawa, Hiroshi Katsuki, Toshiaki Kume, Shuji Kaneko, Akinori Akaike, ヒサシ シワカワ, ヒロシ カツキ, トシアキ クメ, シュウジ カネコ, アキノリ アカイケ, 久志 白川, 博志 香月, 利明 久米, 周司 金子, 昭紀 赤池
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  ABSTRACT: Aminoglutethimide is a clinically available drug that suppresses steroid biosynthesis by inhibiting enzymes such as cytochrome P450scc and aromatase. Because several members of neurosteroids regulate glutamate receptors, we investigated the effect of aminoglutethimide on cell death induced by overactivation of glutamate receptors in CNS neurons.
• Article: ミクログリアの活性化を介する黒質線条体系ニューロン変性の機序とその防御

  博志 香月, ヒロシ カツキ, Hiroshi Katsuki
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  ABSTRACT: ミクログリア活性化の制御を念頭に置き、培養中脳組織切片を用いて諸種化合物のドパミンニューロン保護作用について検討した。1.ミクログリアの活性化を抑制することが報告されているレチノイド受容体リガンドの作用を検討した。RARアゴニストのAm80はIFN-γ/LPSの誘導するドパミンニューロン死を抑制したが、RXRアゴニストのHX630は無効であった。HX630はAm80と併用するとAm80のドパミンニューロン保護作用を遮断した。 Am80はIFN-γ/LPSによるNO産生の増大やミクログリアの活性化型形態への移行に影響を与えなかったが、その保護作用はTrkBのキナーゼ活性を阻害するK252aなどによって抑制されたことから、神経栄養因子類の産生増大が関与する可能性が示唆された。2.ポリフェノール系化合物の resveratrolは、MPP+、アジ化ナトリウムあるいはトロンビンの誘発するドパミンニューロン死を濃度依存的に抑制した。 Resveratrolはsirtuinファミリーのヒストン脱アセチル化酵素を活性化することが知られているが、ヒストン脱アセチル化酵素阻害薬は MPP+毒性に対するresveratrolの保護作用に影響を与えなかった。一方、resveratrolはMPP+による活性酸素種産生増大やグルタチオンの枯渇を顕著に抑制したことから、抗酸化作用がresveratrolの保護作用に関わることが示唆された。これに対して、DNAアルキル化薬の誘発するドパミンニューロン死およびp53アセチル化の亢進は、resveratrolやNADによって抑制され、sirtuin活性化作用がドパミンニューロン保護作用に関与する場合もあることが示された。
• Article: Thrombin-induced delayed injury involves multiple and distinct signaling pathways in the cerebral cortex and the striatum in organotypic slice cultures

  Shinji Fujimoto, Hiroshi Katsuki, Toshiaki Kume, Akinori Akaike, シンジ フジモト, ヒロシ カツキ, トシアキ クメ, アキノリ アカイケ, 真二 藤本, 博志 香月, 利明 久米, 昭紀 赤池
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  ABSTRACT: Thrombin, a serine protease essential for blood coagulation, also plays an important role in cellular injury associated with intracerebral hemorrhage. Here, we show that, in organotypic cortico-striatal slice cultures, thrombin evoked delayed neuronal injury in the cerebral cortex and shrinkage of the striatum. These effects were prevented by cycloheximide and actinomycin D but not by a caspase-3 inhibitor. Thrombin-induced shrinkage of the striatum was abolished by a thrombin inhibitor argatroban or prior heat inactivation of thrombin, and significantly attenuated by a protease-activated receptor-1 antagonist FR171113. However, thrombin-induced cortical injury was not prevented either by heat inactivation or by FR171113, and was only partially inhibited by argatroban. In addition, inhibition of extracelluar signal-regulated kinase (ERK), Src tyrosine kinase and protein kinase C prevented both neuronal injury in the cortex and shrinkage of the striatum, whereas inhibition of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase prevented shrinkage of the striatum only. Thrombin treatment promptly induced phosphorylation of ERK, which was not prevented by inhibition of Src and protein kinase C. Thus, thrombin induces cellular injury in the cerebral cortex and the striatum, by recruiting multiple and distinct signaling pathways in protease activity-independent as well as dependent manner.
• Article: Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice culture

  Hiroshi Katsuki, Mitsugu Okawara, Haruki Shibata, Toshiaki Kume, Akinori Akaike, ヒロシ カツキ, ミツグ オオカワラ, ハルキ シバタ, トシアキ クメ, アキノリ アカイケ, 博志 香月, 賦 大川原, 治樹 柴田, 利明 久米, 昭紀 赤池
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  ABSTRACT: Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent.
• Article: Contribution of endogenous glycine site NMDA agonists to excitotoxic retinal damage in vivo

  Yasuhiro Hama, Hiroshi Katsuki, Yoshinaga Tochikawa, Chihiro Suminaka, Toshiaki Kume, Akinori Akaike, ヤスヒロ ハマ, ヒロシ カツキ, ヨシナガ トチカワ, チヒロ スミナカ, トシアキ クメ, アキノリ アカイケ, 康博 浜, 博志 香月, 宣永 栃川, ちひろ 角中, 利明 久米, 昭紀 赤池
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  ABSTRACT: N-Methyl-d-aspartate (NMDA) receptors, which play an important role in neuronal excitotoxicity, require not only agonists at the glutamate-binding site but also co-agonists at the glycine site for their activation. Here we examined the role of endogenous agonists at the glycine site of NMDA receptors in excitotoxic retinal damage in vivo. To quantify the number of surviving retinal ganglion cells (RGCs), we injected a retrograde tracer, fluoro-gold, into the superior colliculus bilaterally and subsequently counted RGCs on whole-mounted retinas. Co-injection of 5,7-dichlorokynurenic acid (300 nmol), a competitive antagonist at the glycine site of NMDA receptors, rescued RGCs from damage induced by 200 nmol NMDA. On the other hand, RGC death induced by 20 nmol NMDA was enhanced by addition of glycine (10 nmol), d-serine (10 nmol) or a competitive glycine transporter-1 inhibitor, sarcosine (0.3 or 3 nmol). Moreover, application of d-serine-degrading enzyme, d-amino acid oxidase (30 mU), partially suppressed RGC death induced by 20 nmol NMDA. These results suggest that the severity of excitotoxic retinal damage in vivo depends on the levels of both glycine and d-serine.
• Article: Thrombin induces striatal neurotoxicity depending on mitogen-activated protein kinase pathways in vivo

  Shinji Fujimoto, Hiroshi Katsuki, Masatoshi Onishi, Mikako Takagi, Toshiaki Kume, Akinori Akaike, シンジ フジモト, ヒロシ カツキ, マサトシ オオニシ, ミカコ タカギ, トシアキ クメ, アキノリ アカイケ, 真二 藤本, 博志 香月, 正俊 大西, 美佳子 高木, 利明 久米, 昭紀 赤池
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  ABSTRACT: Intracerebral hemorrhage represents stroke characterized by formation and expansion of hematoma within brain parenchyma. Blood-derived factors released from hematoma are considered to be involved in poor prognosis of this disorder. We previously reported that thrombin, a blood-derived serine protease, induced cytotoxicity in the cerebral cortex and the striatum in organotypic slice cultures, which depended on mitogen-activated protein kinase (MAPK) pathways. Here we investigated the mechanisms of thrombin cytotoxicity in the striatum in vivo. Thrombin microinjected into the striatum of adult rats induced neuronal death and microglial activation around the injection site. Neuronal loss without any sign of nuclear fragmentation was observed as early as 4 h after thrombin injection, which was followed by gradual neuronal death exhibiting nuclear fragmentation. Thrombin-induced damage assessed at 72 h after injection was partially but significantly reduced by concomitant administration of inhibitors of MAPK pathways. Activation of extracellular signal-regulated kinase (ERK) and p38 MAPK in response to thrombin was verified by Western blot analysis. Moreover, phosphorylated ERK and p38 MAPK were localized prominently in reactive microglia, and inhibition of microglial activation by minocycline attenuated thrombin-induced damage, suggesting that reactive microglia were responsible for thrombin-induced neuronal death. Thus, MAPK pathways and microglial activation may serve as therapeutic targets of pathogenic conditions associated with hemorrhagic stroke.
• Article: Resveratrol protects dopaminergic neurons in midbrain slice culture from multiple insults

  Mitsugu Okawara, Hiroshi Katsuki, Emi Kurimoto, Haruki Shibata, Toshiaki Kume, Akinori Akaike, ミツグ オオカワラ, ヒロシ カツキ, エミ クリモト, ハルキ シバタ, トシアキ クメ, アキノリ アカイケ, 賦 大川原, 博志 香月, 恵実 栗本, 治樹 柴田, 利明 久米, 昭紀 赤池
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  ABSTRACT: Increasing lines of evidence show that resveratrol, a polyphenol compound contained in several dietary products, exhibits cytoprotective actions. Notably, resveratrol activates sirtuin family of NAD-dependent histone deacetylases implicated in regulation of various cellular processes including gene transcription, DNA repair and apoptosis. Here we examined neuroprotective effect of resveratrol on dopaminergic neurons in organotypic midbrain slice culture. Resveratrol and quercetin, another sirtuin-activating polyphenol, prevented the decrease of dopaminergic neurons and the increase of propidium iodide uptake into slices induced by a dopaminergic neurotoxin 1-methyl-4-phenyl pyridinium (MPP+). Resveratrol also provided concentration-dependent neuroprotective effects against sodium azide, a mitochondrial complex IV inhibitor, and thrombin (EC number 3.4.21.5), a microglia-activating agent. Sirtuin inhibitors such as nicotinamide and sirtinol did not attenuate the protective effect of resveratrol against MPP+ cytotoxicity. Instead, we found that resveratrol prevented accumulation of reactive oxygen species, depletion of cellular glutathione, and cellular oxidative damage induced by MPP+, suggesting involvement of antioxidative properties in the neuroprotective action of resveratrol. On the other hand, resveratrol as well as a sirtuin activator NAD inhibited dopaminergic neurotoxicity of a DNA alkylating agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Moreover, MNNG-induced increase in acetylation of p53, a representative target of sirtuin deacetylase activity, was suppressed by resveratrol. These results indicate that resveratrol can exert neuroprotective actions in dopaminergic neurons. Either antioxidative activity or sirtuin-activating potential may play an important role in the neuroprotectice actions of resveratrol against different kinds of insults.
• Article: Involvement of thrombin and mitogen-activated protein kinase pathways in hemorrhagic brain injury

  Masatoshi Ohnishi, Hiroshi Katsuki, Shinji Fujimoto, Mikako Takagi, Toshiaki Kume, Akinori Akaike, マサトシ オオニシ, ヒロシ カツキ, シンジ フジモト, ミカコ タカギ, トシアキ クメ, アキノリ アカイケ, 正俊 大西, 博志 香月, 真二 藤本, 美佳子 高木, 利明 久米, 昭紀 赤池
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  ABSTRACT: Thrombin is thought to play an important role in brain damage associated with intracerebral hemorrhage (ICH). We previously showed that activation of mitogen-activated protein (MAP) kinases and recruitment of microglia are crucial for thrombin-induced shrinkage of the striatal tissue in vitro and thrombin-induced striatal damage in vivo. Here we investigated whether the same mechanisms are involved in ICH-induced brain injury. A substantial loss of neurons was observed in the center and the peripheral region of hematoma at 3 days after ICH induced by intrastriatal injection of collagenase in adult rats. Intracerebroventricular injection of argatroban or cycloheximide, both of which prevent thrombin cytotoxicity in vitro, exhibited a significant neuroprotective effect against ICH-induced injury. ICH-induced neuron loss was also prevented by a MAP kinase kinase inhibitor (PD98059) and a c-Jun N-terminal kinase inhibitor (SP600125). These drugs had no effect on hematoma size or ICH-induced brain edema. Activation of extracellular signal-regulated kinase in response to ICH was observed in both neurons and microglia. Despite their neuroprotective effects, MAP kinase inhibitors did not decrease the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appearing after ICH. Identification of cell types revealed that TUNEL staining occurred prominently in neurons but not in microglia, whereas inhibition of MAP kinases resulted in appearance of TUNEL staining in microglia. These results suggest that thrombin and the activation of MAP kinases are involved in ICH-induced neuronal injury, and that neuroprotective effects of MAP kinases are in part mediated by arrestment of microglial activities.
• Article: Contribution of endogenous glycine and d-serine to excitotoxic and ischemic cell death in rat cerebrocortical slice cultures

  Hiroshi Katsuki, Yoshinori Watanabe, Shinji Fujimoto, Toshiaki Kume, Akinori Akaike, ヒロシ カツキ, ヨシノリ ワタナベ, シンジ フジモト, トシアキ クメ, アキノリ アカイケ, 博志 香月, 佳典 渡辺, 真二 藤本, 利明 久米, 昭紀 赤池
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  ABSTRACT: N-methyl-d-aspartate (NMDA) receptors, whose activation requires glycine site stimulation, play crucial roles in various physiological and pathological conditions in the brain. We investigated the regulatory roles of potential endogenous glycine site agonists, glycine and d-serine, in excitotoxic and ischemic cell death in the cerebral cortex. Cytotoxicity of NMDA on rat cerebrocortical slice cultures was potentiated by addition of glycine or d-serine. In contrast, cell death induced by oxygen/glucose deprivation (OGD) was not affected by exogenous glycine or d-serine, although blockade of NMDA receptors by MK-801 abolished cell death. In addition, higher concentrations of 2,7-dichlorokynurenic acid (DCKA), a competitive glycine site antagonist, were required to suppress OGD-induced cell death than those to suppress NMDA cytotoxicity. We also found that OGD triggered a robust increase in extracellular glycine. A glycine transporter blocker ALX 5407 increased the extracellular level of glycine, and the protective effect of DCKA against NMDA cytotoxicity was diminished in the presence of ALX 5407. Sensitivity of NMDA cytotoxicity to DCKA was also diminished by l-serine that increased the extracellular level of d-serine. These results indicate that both glycine and d-serine can act as endogenous ligands for NMDA receptor glycine site in the cerebral cortex, and that endogenous glycine may saturate the glycine site under ischemic conditions. The present findings are important for the interpretation of the mechanisms of NMDA and OGD cytotoxicity.
• Article: Plasminogen potentiates thrombin cytotoxicity and contributes to pathology of intracerebral hemorrhage in rats

  Shinji Fujimoto, Hiroshi Katsuki, Masatoshi Onishi, Mikako Takagi, Toshiaki Kume, Akinori Akaike, シンジ フジモト, ヒロシ カツキ, マサトシ オオニシ, ミカコ タカギ, トシアキ クメ, アキノリ アカイケ, 真二 藤本, 博志 香月, 正俊 大西, 美佳子 高木, 利明 久米, 昭紀 赤池
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  ABSTRACT: Thrombin and plasmin are serine proteases involved in blood coagulation and fibrinolysis, whose precursors are circulating in blood stream. These blood-derived proteases might play important roles in the pathogenesis of intracerebral hemorrhage by acting on brain parenchymal cells. We previously reported that thrombin induced delayed neuronal injury through extracellular signal-regulated kinase (ERK)-dependent pathways. Here, we investigated potential cytotoxic actions of plasminogen, a precursor protein of plasmin, using slice cultures prepared from neonatal rat brain and intracortical microinjection model in adult rats. Although plasminogen alone did not evoke prominent neuronal injury, plasminogen caused significant neuronal injury when combined with a moderate concentration of thrombin (30 U/mL) in the cerebral cortex of slice cultures. The cortical injury was prevented by tranexamic acid and aprotinin. The combined neurotoxicity of thrombin and plasminogen was also prevented by PD98059, an inhibitor of ERK pathway, as well as by other agents that have been shown to prevent cortical injury induced by a higher concentration (100 U/mL) of thrombin alone. Extracellular signal-regulated kinase phosphorylation after plasminogen exposure was localized in cortical astrocytes. Moreover, microinjection of plasminogen in vivo potentiated thrombin-induced cortical injury, and inhibition of plasmin ameliorated hemorrhage-induced neuronal loss in the cerebral cortex. These results suggest that plasminogen/plasmin system augmenting thrombin neurotoxicity participates in hemorrhagic cortical injury.