一般演題（ポスター）

一般演題（ポスター） Glia・Myelin I

1P-35 Characterization of zinc uptake by mouse primary cultured astrocytes and microglia Koike Akihiro，Segawa Shohei，Tatsumi Nao，Ohishi Akihiro，Nishida Kentaro，Nagasawa Kazuki Department of Environmental Biochemistry, Kyoto Pharmaceutical University Under severe pathophysiological conditions, zinc is released excessively from glutamatergic synaptic boutons, and induces brain injury. Neuronal death is exacerbated by excessive activation of microglia induced by zinc released from astrocytes in addition to that from the neurons. Therefore, regulation of the extracellular zinc is important for maintenance of brain homeostasis, and the extracellular zinc has to be cleared by specific systems. To elucidate the regulatory mechanism for extracellular zinc in the CNS, we examined the zinc uptake characteristics in mouse astrocytes and microglia. Zinc was taken up into mouse astrocytes and microglia time-dependently, and the cell-to-medium concentration（C/M）ratio in the initial uptake phase in astrocytes was significantly smaller than that in microglia, while in the steady state phase, there was no difference in their C/M ratios. In both astrocytes and microglia, the zinc uptake was mediated, at least in part, by high- and low-affinity systems. There were no differences for both in the Km values for zinc uptake between astrocytes and microglia, and those for the low-affinity system in both cell types were the same as that for mouse ZIP1 reported previously. On the other hand, the Vmax values for both systems were greater in microglia than in astrocytes. Among ZIP isoforms, expression of ZIP1 was high in astrocytes and microglia. Nickel, a competitive inhibitor of ZIP1, and ZIP1 knock-down decreased zinc uptake by both types of cells. Overall, it is demonstrated that astrocytes and microglia had a similar uptake system for zinc including ZIP1, and the differences found in their uptake profiles imply that they play different roles in the regulation of extracellular zinc to maintain brain homeostasis.		1P-36 Oxidative stress enhances zinc clearance via upregulation of ZIP1 expression at the plasma membrane in astrocytes Furuta Takahiro，Ohshima Chiaki，Takebayashi Naoto，Matsumura Mayu，Nishida Kentaro，Nagasawa Kazuki Department of Environmental Biochemistry, Kyoto Pharmaceutical University Zinc plays roles as neuro- and glio-transmitters in maintenance of brain homeostasis. Under pathological conditions, zinc is released from glutamatergic boutons and astrocytes, and excessive zinc in extracellular space kills neurons and also induces extensive activation of microglia, resulting in exacerbation of brain injury. Therefore, extracellular zinc levels have to be strictly regulated in narrow physiological ranges. Recently, we demonstrated that astrocytic uptake of zinc has a primary role in zinc clearance, and a zinc transporter ZIP1 expressed by astrocytes is one of the responsible molecules for the uptake. On the other hand, under pathological conditions, it is reported that astrocytic clearance for transmitters such as glutamate and GABA is upregulated, but that for zinc is unknown yet. Here, we examined whether functional expression of zinc clearance system is altered under oxidative stress-loaded cultured astrocytes. Treatment of astrocytes with hydrogen peroxide at 0.4 mM for 24 h treatment caused apparent activation of them with increased expression of GFAP and 4-hydroxynonenal without cell toxicity. The activated astrocytes exhibited increased zinc uptake activity, and the Vmax value for the uptake was significantly greater than that in control group, but there was no change in the Km value, which is comparable wih that of ZIP1. Expression of ZIP1 in the activated astrocytes was increased in whole cell lysates and plasma membrane fraction. Taken together, it is indicated that under oxidative stress-loaded conditions, astrocytes increase the zinc clearance activity and this is due, at least in part, to the increase of ZIP1 expression at their plasma membrane.

1P-37 Involvement of Mlc1 in white matter development and maintenance Sugio Shouta^1,2，Ikenaka Kazuhiro²，Tanaka Kenji³ ¹Department of Molecular and Cellular Neurobiology, Gunma University School of Medicine，²Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences，³Department of Neuropsychiatry, Keio University School of Medicine For many decades, astrocytes have been considered as supporting cells for neuronal and brain functions. However, this notion has been strongly modified by studies on neuron-astrocytes and blood vessel-astrocytes interactions that should modulate synaptic transductions and blood flow. These new findings have been proposed during last decade, while little is known about relationship between astrocytes and white matter development and/or maintenance. Recent studies have suggested that astrocytes are involved in the white matter development and maintenance. Deficiency in several genes that are expressed in astrocyte specific manner results in leukoencephalopathy, and the damaged astrocytes could contribute to the pathological onset of the leukoencephalopathy. To expand the knowledge on the relationship between astrocytes and the white matter development, we focused on astrocyte-specific gene, Mlc1. Mlc1 is a mouse homologue of the human MLC1 that is responsible for a human leukoencephalopathy, Megalencephalic leukoencephalopathy with subcortical cysts（MLC：# 604004 at Online Mendelian Inheritance in Man（OMIM））. MLC is a rare autosomal recessive neurological disorder with an infantile-onset leukoencephalopathy, which is characterized by a chronic white matter edema, macrocephaly, a slowly progressing deterioration of motor function, cerebellar ataxia, spasticity, and mental decline. Although human MLC1 and murine Mlc1 encode an eight transmembrane protein, its precise function has remained unclear. Here, we generated Mlc1 null mouse and Mlc1 over-expressing mouse, and examined their loss-of- and gain-of-functional effects against astrocytes and brain white matter.		1P-38 Activation of P2X7 receptor/HIF-1α signal pathway in astrocytes induces ischemic tolerance Hirayama Yuri¹，Ikeda-Matsuo Yuri³，Koizumi Schuichi² ¹Dept. Liaison Academy, Sch. Med., Univ. Yamanashi，²Dept. Neuropharmacol., Interdisciplinary Grad. Sch. Med., Univ. Yamanashi，³Dept. Pharmacol., Sch. Pharm. Sci., Univ. Kitasato A mild ischemic episode（preconditioning；PC）induces resistance to a subsequent severe ischemic injury. This phenomenon, known as ischemic tolerance, is an endogenous process that provides robust neuroprotection. We previously showed that PC-induced activation of astrocytes and subsequent upregulation of P2X7 receptor are essential for ischemic tolerance（astrocyte-mediated ischemic tolerance）. However, the downstream signals of P2X7 receptors responsible for the ischemic tolerance remain unknown. Here we show that hypoxia inducible factor-1α（HIF-1α）, a master molecule that induces various neuroprotective factors in astrocytes has an indispensable role for this event. Using in vivo middle cerebral artery occlusion（MCAO）model in mice, we found that PC（15 min-MCAO）increased HIF-1α in both neurons and astrocytes. It is well-known that decrease in the oxygen supply is a main mechanism that promotes increase in HIF-1α, and actually, the neuronal HIF-1α increase was dependent on hypoxia/ischemia. In contrast, as for astrocytes, activation of P2X7 receptors, rather than decrease in the oxygen supply, was important. We also confirmed these mechanisms using primary cultures of neurons or astrocytes. Furthermore, PC-induced increase in HIF-1α in neurons was transient, whereas that in astrocytes lasted much longer. Such characteristic features of HIF-1α in astrocytes, but not in neurons, were well correlated with that of ischemic tolerance. Thus, it is strongly suggested that P2X7 receptor/HIF-1α signal pathway plays an indispensable role in astrocyte-mediated ischemic tolerance.

1P-39 Hedgehog signaling modulates the release of gliotransmitters from cultured cerebellar astrocytes. Okuda Hiroaki，Tatsumi Kouko，Morita Shoko，Wanaka Akio Dept Anatomy and Neuroscience, Nara Medical University The hedgehog（Hh）signaling pathway is conserved in diverse species from Drosophila to human and plays a key role in regulating organogenesis. Sonic hedgehog（Shh）, a member of the Hh family, is an essential factor in the development of the central nervous system. Recent studies have implied that the Hh signaling pathway also functions in mature astrocytes under physiological conditions. The present study focused on the functions of the Hh signaling pathway in the adult mouse brain. We first examined the expression of Hh signaling molecules in the adult mouse brain by in situ hybridization and immunohistochemistry. Patched homolog 1（ptch1）, a receptor for Hh family members, was expressed in S100beta positive astrocytes and Shh mRNA was expressed in HuC/D-positive neurons in the adult mouse cerebellum. These results suggested that the Hh signaling pathway is involved in neuro-glial interactions. To test this hypothesis, we next examined the effects of recombinant Shh N-terminal（rShh-N）on the functions of cultured cerebellar astrocytes. While glutamate uptake was not affected by activation or inhibition of Hh signaling, activation of Hh signaling by rShh-N influenced gliotransmitter release such as glutamate, ATP and D-serine from cultured astrocytes. However, cyclopamine pretreatment interfered with the release of glutamate and ATP, but not of D-serine. These results suggest that non-canonical Hh signaling pathways such as the MAPK and AKT pathway are evidently important in the release of D-serine from astrocytes. We conclude from these results that the Hh signaling pathway modulates the release of gliotransmitters and participates in neuro-glial interactions in the adult mouse brain.		1P-40 The mechanism of Denosomin in astrocytes leading to release of axonal growth factors Tanie Yoshitaka，Sigyo Michiko，Tanabe Norio，Kuboyama Tomoharu，Tohda Chihiro Div. of Neuromedical Science, Inst. of Natural Medicine, Univ. of Toyama We previously found that a novel compound Denosomin improved hindlimb motor dysfunction of spinal cord injury（SCI）mice, induced axonal growth and increased astrocytes in the injured site. Denosomin enhanced secretion of the intermediate filament protein, vimentin, from cultured astrocytes. The direct treatment of vimentin to cortical neurons increased axonal density. In addition, ratios of astrocytes expressing vimentin and 5-HT-positive axons co-localizing with vimentin were increased inside of the glial scar in SCI mice administered Denosomin. These results suggest that the functional change to astrocyte secreting vimentin as an axonal growth factor is induced by Denosomin treatment, which may contribute to recovery from motor dysfunction. Generally, astrocytes secreting chondroitin sulfate proteoglycan are considered to inhibit axonal regeneration in SCI. Therefore Denosomin-induced conversion of astrocytic properties into beneficial ones of secreting vimentin would be valuable for therapy of SCI. However, the mechanism of Denosomin in astrocytes has not been elucidated. In this study, we aimed to clarify a direct target protein of Denosomin to know its signal pathway in astrocytes. DARTS analysis was peformed using cultured astrocytes（ddY mice, E14）. As a result, one candidate protein was supposed as a vulnerability changed protein against proteolysis with Denosomin coexistence. Confirming that the candidate is a direct binding protein of Denosomin is ongoing.

1P-41 Acetate attenuates LPS-induced nitric oxide production in cultured astrocytes Moriyama Mitsuaki，Kurebayashi Ryosuke，Kawabe Kenji，Hashimoto Ayano，Takano Katsura，Nakamura Yoichi Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University The biomolecule acetate can be utilized for energy production, lipid synthesis, and several metabolic processes. Recently, acetate supplementation reduced neuroglial activation in the model of neuroinflammation induced by intraventricular injection of lipopolysaccharide（LPS）. To understand the mechanisms underlying the anti-inflammatory effect of acetate on glial cells, we examined the effect of acetate on nitric oxide（NO）production in cultured astrocytes, which is experimentally stimulated by LPS. Increasing acetate concentration attenuated the LPS（1 μg/ml）-induced NO production in a dose-dependent manner, significantly more than 5 mM, although cell viability was not affected. The LPS-induced expression of inducible NO synthase protein was significantly decreased by acetate（10 mM）. Acetate also reduced the LPS-induced phosphorylation of p38 MAPK at 24 hr, whereas ERK was not affected. LPS-induced intracellular reactive oxygen species（ROS）productions were decreased at 4-24 hr by the addition of acetate. Furthermore, the addition of acetate significantly reduced hydrogen peroxide（H₂O₂）-induced cytotoxicity by increasing cell viability through the attenuation of intracellular ROS level. These findings suggest that attenuation of NO production by acetate may alleviate glial cell damage during neuroinflammation. Acetate may offer its glioprotection by reducing the oxidative stress.		1P-42 Potent induction of glycogen metabolism by pituitary adenylate cyclase-activating polypeptide on cultured astrocytes Kambe Yuki¹，Nakashima Yu¹，Shintani Norihito²，Hashimoto Hitoshi^2,3，Miyata Atsuro¹ ¹Lab. Pharmacol., Kagoshima Univ., Grad. Sch. Med. Dent. Sci.，²Lab. Mol. Neuropharmacol., Osaka Univ., Grad. Sch. Pharmaceut. Sci.，³Osaka Univ., Uni. Grad. Sch. Child Dev. Glycogen is stored in astrocyte located in regions of high synaptic density, and is important for a variety of brain functions including learning and memory. However, the mechanism how the astrocytic glycogen metabolism is regulated has not been completely elucidated yet. Previously, it was reported that glycogen metabolism was activated by vasoactive intestinal polypeptide（VIP）, which shares common receptors with pituitary adenylate cyclase-activating polypeptide（PACAP）. In addition, gene ontology analysis revealed that carbohydrate metabolism was the closest network induced by PACAP in astrocytes. Therefore, we investigated the effect of PACAP on glycogen metabolism using cultured astrocytes. PACAP or VIP induced glycogenolysis dose-dependently 1 hr after exposure, and these EC₅₀ values were 0.0084 nM or 0.43 nM, respectively. Interestingly, EC₅₀ value of PACAP was at least 50 times less than these of neurotransmitters previously reported to induce glycogenolysis such as VIP, noradrenaline or serotonin. Although PACAP decreased glycogen content 1 hr after exposure, it was over-compensated about 3 times more than control level 7 hr after exposure. This glycogenesis by PACAP or VIP was induced dose-dependently, and these EC₅₀ values were 0.086 nM or 185 nM, respectively. Ratio between EC₅₀ values of glycogenolysis and glycogenesis by PACAP was just 10 times, but that by VIP was about 500 times. In addition, co-application of maxadilan, PAC1 receptor selective agonist, with VIP further improved the VIP mediated glycogenesis by that of PACAP. These results suggested that PACAP and its receptor PAC1 strongly activates glycogen metabolism including glycogenolysis and glycogenesis in cultured astrocytes.

1P-43 Characterization of Olig2-positive astrocytes in the normal adult forebrain Tatsumi Kouko，Okuda Hiroaki，Morita Shoko，Wanaka Akio Department of Anatomy and Neuroscience, Nara Medical University Expression of Olig2, a basic helix-loop-helix（bHLH）transcription factor, persists from embryonic to adult stages in the central nervous system. In the adult stage, most of the Olig2-positive cells co-express NG2 proteoglycan, and constitute a population of oligodendrocyte precursors（OPCs）.“Adult OPCs”have abilities of self-renewal and differentiation. We previously reported that genetically labeled Olig2-positive cells in the adult brain generate NG2 glia, oligodendrocytes and astrocytes（Tatsumi et al., 2008；Islam et al., 2009；Okuda et al., 2009）. We recently found a distinct population of Olig2-positive cells in the gray matter of the adult forebrain. In contrast to the OPCs, these cells are postmitotic and positive for s100β, a marker of mature astrocyte. They are relatively abundant in basal ganglionic nuclei such as the globus pallidus and substantia nigra pars reticulata. Since these nuclei receive inhibitory GABAergic inputs from the striatum and globus pallidus, respectively. Olig2-positive astrocytes are preferentially localized in the vicinity of the inhibitory synapses. Assuming the tripartite synapses theory, Olig2-positive astrocytes may contribute to inhibitory transmission in the adult forebrain.		1P-44 The role of CD38, an Autism Spectrum Disorder（ASD）-associated molecule, in the development of glial cells Hattori Tsuyoshi¹，Takarada Mika¹，Yamamoto Yasuhiko³，Okamoto Hiroshi⁴，Higashida Haruhiro²，Hori Osamu¹ ¹Grad.Sch.Med.KanazawaUniv，²Res Cent Child Mental Dev, KanazawaUniv，³Grad.Sch.Med.KanazawaUniv，⁴Touhoku Univ CD38, a type II transmembrane protein with ADP-ribosyl cyclase activity, is involved in Ca2+ -induced Ca2+ -release in different types of cells, and plays an important role for oxytocin（OXT）secretion in the hypothalamus. Deletion of CD38 gene caused reduction of the central OXT secretion, and caused impaired social behaviors associated with Autism Spectrum Disorder（ASD）. In this study, we investigated the expression and possible role of CD38 in the postnatal development of neurons and glial cells in mice. qPCR and western blot analysis revealed enhanced expression of CD38 from P14 to P28 in wild-type（WT）mice brain. Analysis with WT and CD38 knockout（KO）mice revealed that the expressions of MBP, MAG and CNP, markers of oligodendrocytes, were significantly decreased at mRNA level in the cerebral cortices of CD38 KO mice from P7 to P14. The reduced levels of expression of the MBP and CNP proteins were also observed in CD38 KO mice from P7 to P21 by western blotting and immunohistochemistry. Further analysis using qPCR, western blotting and immunohistochemistry revealed that the expression of GFAP, a marker of astrocytes, was also reduced, and the processes of astrocytes were shorter in CD38 KO mice from P1 to P7. Taken together, CD38 may have some roles not only in the oxytocin neurons but also in the development of glial cells, such as oligodendrocytes and astrocytes.

1P-45 Functional analysis of a Down syndrome-associated gene. Dewa Ken-ichi^1,2，Koizumi Schuichi¹，Hoshino Mikio²，Taya shinichiro²，Arimura Nariko² ¹Department of Pharmacology, University of Yamanashi，²Department of Biochemistry ＆ Cellular Biology, National Institute of Neuroscience Trisomy of chromosome 21 is the major genetic cause of intellectual disability, collectively known as Down syndrome. The neuropathology of Down syndrome suggests that the gross brain pathology is associates with the specific profile of working memory and/or verbal short-term memory. These pathophysiological changes of Down syndrome also include the changes in size of specific brain regions and their connectivity and alternations in the number and/or the morphology of a certain population of neurons. Recently, the candidate genes and their interactions have been explored, however, the whole picture of pathological process of Down syndrome has not been revealed.Previously, we reported that a down syndrome-associated gene regulates the neuronal migration and eventual distribution in the midbrain at embryonic stage. In this study, we find that a down syndrome-associated gene is expressed in the cerebellum at postnatal stage. Moreover, we show the new function of this gene in both neurons and glial cells in the cerebellum.Several studies in Down syndrome fetal brain and in the trisomy mouse, which express this protein at higher level, have reported the reduction in the brain volume and cell number in the hippocampus and cerebellum. Consistently, we found that the number of neurons in mutant mice, which express this protein at lower level, was increased. We will discuss the functional role of this gene in the pathogenesis of Down syndrome.		1P-46 Involvement of Ndrg2 in blood-brain barrier disruption after stroke Takarada-Iemata Mika¹，Yoshikawa Akifumi²，Aida Yasuhiro²，Minh Hieu Ta¹，Hattori Tsuyoshi¹，Manh Thuong Le¹，Kitao Yasuko¹，Nakada Mitsutoshi²，Hori Osamu¹ ¹Dept. of Neuroanat., Kanazawa Univ. Gra. Sch. Med. Sci.，²Dept. of Neurosurg., Kanazawa Univ. Gra. Sch. Med. Sci. Disruption of blood-brain barrier（BBB）is known to be occurred in various pathological conditions including ischemic stroke. However, its regulating mechanism remains elusive. We previously showed that Ndrg2, a gene responding to various stresses in astrocytes, is involved in the regulation of reactive astrogliosis and the protection from infarct damage in a mouse experimental stroke model. This study was aimed to investigate the functional role of Ndrg2 in BBB dysfunction after brain ischemia using a mice middle cerebral artery occlusion（MCAO）model. Vessel permeability, determined by tracer leakage and the extravasation of internal serum proteins, was enhanced in Ndrg2-knockout（KO）mice compared to wild-type（WT）mice after MCAO. Moreover, flow cytometry analysis showed increased infiltration of immune cells in ipsilateral brain hemispheres from Ndrg2-KO mice compared to that from WT mice. Further study revealed that Ndrg2 deficiency results in enhanced expression level of matrix metalloproteases in ipsilateral cortex after MCAO. Similar results were observed in cultured astrocytes isolated from Ndrg2 KO mice. These results suggest that Ndrg2 expressed in astrocytes may play a critical role in the regulation of BBB permeability and immune cell infiltration after ischemic brain stroke.

1P-47 Treatment of hyperbaric oxygenation combined with radiotherapy improves radioresponsiveness of Glioblastoma. Katagiri Chiaki^1,2，Nagamine Hideki¹，Matsushita Masayuki²，Ishiuchi Shogo¹ ¹Dept. of Neurosurgery, Med., Univ., of Ryukyus，²Dept. of Mol. Physiol., Med., Univ., of Ryukyus Glioblastoma multiforme（GBM）is the most aggressive malignancies among primary malignant brain tumors. Certain areas of the tumor tissues are coursed to hypoxic condition because of insufficient blood vessel supply. Such hypoxic condition area is also considered to induce radioresistance. Molecular oxygen has been recognized an enhancer of radiatiosensitivity. Hyperbaric oxygenation（HBO）improves the oxygen supply to hypoxic tumor cells. We examined the effect of radiotherapy just after HBO breathing in experimental tumors using a tumor growth assay. U87-MG cells were transplanted into balb/c nu/nu mice leg. After the subcutaneous xenograft reached approximately 200mm³, mice were started by radiotherapy, 2Gy/day for 10days, with or without HBO, 2.5 atmosphere with 100% oxygen for 40min. A significant growth delay was observed in the xenograft with radiotherapy after HBO, and the tumor size increased 7.0 fold in no-treatment, 4.2 fold only radiation treatment, and 2.4 fold in radiation after HBO treatment, respectively. Next we analyzed the changes of gene expression of tumor cells using mRNA differential display method. Gene expressions induced by HIF-1, a hypoxic response transcription factor, were reduced by HBO. The result indicates that HBO treatment induced oxygenation of hypoxic tumor cells and activated sensitivity of radiation.