role of gap junction hemichannels in protective effect of nad+ against parp-triggered astrocyte...

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e238 Abstracts / Neuroscience Research 68S (2010) e223–e334 els in cultured astrocytes under prolonged exposure to ammonia. Using an enzyme-linked high-performance liquid chromatography assay to detect glutamate, prolonged (48 h) exposure of cultured astrocytes to ammonia resulted in a concentration- and time-dependent increase in extracellu- lar glutamate. Similar increases were observed when ammonia-containing medium (pH 7.8) was adjusted to the pH of control medium (pH 7.4), indi- cating that the effect is not due to pH. Treatment of astrocytes with an antioxidant (l-ascorbic acid), an NADPH oxidase inhibitor (apocynin), a Ca 2+ chelator (BAPTA-AM), an NMDA receptor antagonist (NK801), or a mito- chondrial permeability transition inhibitor (cyclosporine A) suppressed the increase of extracellular glutamate in response to prolonged ammonia expo- sure. Prolonged exposure to ammonia increased extracellular glutamate through the NMDA receptor, increased intracellular Ca 2+ levels, and upreg- ulation of excitatory amino acids. The addition of ATP further increased extracellular glutamate levels in astrocytes subjected to prolonged ammonia treatment (5 mM, 48 h) in a dose-dependent manner. These results indicate that the deregulation of glutamate release from astrocytes may contribute to the dysfunction of glutamatergic neurons in patients with acute liver failure. doi:10.1016/j.neures.2010.07.1051 P2-b38 PLD4 is implicated in phagocytosis in major phago- cytes Yoshinori Ohtani 1 , Yoshihide Yamaguchi 1 , Hiroshi Kitani 2 , Yumi Sato 3 , Teiichi Furuichi 3 , Hiroko Baba 1 1 Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences 2 Nat. Inst. Agrobiol. Sci., Tsukuba, Japan 3 Lab. for Mol. Neuro- genesis, RIKEN Brain Sci Inst, Wako, Japan Phospholipase D (PLD) represented by PLD1 and PLD2 hydrolyzes phos- phatidylcholine to produce phosphatidic acid and choline. These PLDs are known to be involved in various cellular functions including membrane trafficking, secretion and mitogenesis. Previously, we reported that the expression of a novel member of PLD family, PLD4, was specifically upregu- lated in amoeboid microglia in the white matter of mouse cerebellum, both in the developmental stage and the pathological conditions. In the analy- sis using cultured microglial cell line (MG6), we demonstrated that PLD4 was primarily located in the nucleoplasm while accumulated in phagosomes containing BioParticles during phagocytosis. Because, unlike PLD1 and PLD2, PLD4 did not exhibit PLD enzymatic activity, the role of PLD4 in phagocytotic cascade was totally unknown. In this study, to clarify the function of PLD4, we focused on the functional association of PLD4 with other PLDs during phagocytosis. In another major phagocyte, macrophage, PLD1 and PLD2 were reported to be involved in phagocytosis. To determine whether PLD4 was expressed in such activated macrophages in the lesion of the PNS, we per- formed immunohistochemistry for PLD4 and Iba1 on lysolecithin-induced demyelinated area of mouse sciatic nerve. The result indicated that PLD4 was localized in the Iba1 positive lesion where activated macrophages were accumulated. Thus, PLD4 may play a role in the phagocytosis together with PLD1 and/or PLD2. Therefore, to investigate the relationship of PLD4 with other PLDs in the process of phagocytosis, we examined their localizations during phagocytosis using SNAP/CLIP systems. As a result, PLD4 was colocal- ized with PLD1 in the phagosomes of MG6 cells. These results suggest that PLD4 may be involved in the phagocytic process associated with PLD1. doi:10.1016/j.neures.2010.07.1052 P2-b39 Administration of Chinpi, a component of the herbal medicine ninjinyoeito, ameliorates cuprizone- induced demyelination and promotes remyelination. Yumi Misawa , Kenji Watanabe, Hiroaki Asou Center for Kampo Medicine, Keio Univercity School of Medicine, Japan Introduction The myelin sheath in the central nervous system (CNS) forms as an oligodendrocyte wraps layer upon layer of its own membrane around the axon in a tight spiral forming an electrically insulting sheath around the axon. Myelin loss occurs in many demyelinating diseases such as multiple sclerosis. In previous studies, we have demonstrated that the recovery from demyelination in aged mice is achieved after administration of the herbal medicine ninjinyoeito (NYT), and also found a potentially therapeutic effect of NYT against age-induced demyelination, resulting the level of phosphory- lated myelin basic protein (P-MBP) was markedly and inversely correlated with demyelination. Methods and results Feeding cuprizone (bis-cyclohexanone oxalydihydra- zone) to young C57BL/6 mice leads to a reproducible demyelination of the corpus callosum and the superior cerebellar peduncle within several weeks with nearly complete remyelination after withdraw of the cuprizone from the diet. We examined the efficacy of Chinpi, the major active constituent of NYT, to demyelinated animals in a 0.2% cuprizone diet. Immunohistochem- ical studies of cuprizone-exposed mice brain section by using anti-myelin basic protein (MBP) antibodies and anti- P-MBP antibodies showed that in the Chinpi treated mice for 5 weeks was marked increase both T-MBP and P-MBP positive fibers in corpus callosum. We also found that cuprizone- exposed for 5 weeks mice with a 0.5% Chinpi in drinking water for 7 weeks induce a marked increase in myelin deposition compared with that observed in spontaneous recovery. Conclusions In conclusion, our immunohistochemical findings suggest that Chinpi significantly ameliorates cuprizone-induced demyelination and pro- motes remyelination. doi:10.1016/j.neures.2010.07.1053 P2-b40 Role of gap junction hemichannels in protective effect of NAD + against PARP-triggered astrocyte death Hiroto Okuda , Yuka Tokotani, Kentaro Nishida, Kazuki Nagasawa Department of Environ. Biochem., Kyoto Pharm. University, Kyoto, JAPAN Objective Excessive activation of poly(ADP-ribose) polymerase (PARP), fol- lowing ischemia and trauma, etc., depletes cellular NAD + and leads to cell death. PARP activation-triggered astrocyte death has been demonstrated to be prevented by addition of NAD + extracellularly. It has been known that intact form of NAD + acts as a cytoprotective molecule, and that P2X7 recep- tor (P2X7R) and connexin43 hemichannel are candidate molecules for NAD + fluxes in several types of cells, but the detailed molecular mechanism remains still unclear. The aim of this study was to clarify the pathway through which NAD + can enter astrocytes under PARP-activated conditions. Methods Astrocyte primary cultures were prepared from mouse newborn pups and cultured in EMEM supplemented with FBS. DNA damage and PARP activation were induced in astrocyte cultures with a DNA alkylating agent, N- methyl-N?-nitro-N-nitrosoguanidine (MNNG). siRNAs were transfected into cells by a lipofection methods. Cell viability was determined with an LDH assay, and cellular NAD + content was assessed by enzymatic recycling assay. Results and discussion MNNG-triggered astrocyte death was completely pre- vented by extracellular administration of 5 mM NAD + , and this was not affected by P2X7R antagonists, oxidized ATP and KN-62. Furthermore, knock- down of P2X7R and pannexin1, which can be opened following P2X7R activation, had no effect on NAD + -induced increase of cellular NAD + level of MNNG-treated astrocytes. On the other hand, pharmacological treatment known to block gap junction hemichannels, 18-glycyrrhetinic acid, octanol and carbenoxolone resulted in decrease of NAD + -induced increase of cell viability. Taken together, these data suggested that NAD + could be taken up into astrocytes through gap junction hemichannels, but not P2X7R, and this process might play a key role in NAD + -induced prevention against PARP- triggered astrocyte death. doi:10.1016/j.neures.2010.07.1054 P2-c01 Death or survival of Toll-like receptor 4-activated microglia is regulated by extracellular ATP Kana Harada 1 , Izumi Hide 1 , Shunsuke Fujiwara 1 , Takahiro Seki 1 , Shigeru Tanaka 1 , Yoshihiro Nakata 2 , Norio Sakai 1 1 Dept. Mol. and Pharmacol. Neurosci., Grad. Sch. Biomed. Sci., Hiroshima Univ., Hiroshima, Japan 2 Dept. Pharmacol., Grad. Sch. Biomed. Sci., Hiroshima Univ., Hiroshima, Japan Microglia, the CNS resident macrophages, migrate to the injured sites and promote tissue repair. Hyperactivated microglia, however, induce excessive inflammation, which is closely implicated in the exacerbation of neurode- generation. Recently, it has been shown that endogenous ligands of Toll-like receptors (TLRs), which are released from damaged cells, elicit inflammatory responses through the activation of microglia. Moreover, ATP is also released from damaged cells and conveys a danger signal to microglia. In this study, we examined whether lipopolysaccharide (LPS), a TLR4 ligand, and extracel- lular ATP might control death and survival of microglia. Microglia started to undergo spontaneous cell death shortly after being isolated from rat primary mixed glial cultures and all the cells died within 2 days. The high concen- trations of LPS (5–100 ng/mL) accelerated microglial cell death, whereas lower concentration of LPS (1 ng/mL) significantly extended the survival time of microglia without affecting cell death. Interestingly, ATP (1 mM) further enhanced the cell death of microglia activated with the high concentration of LPS (5 ng/mL), while promoting microglial survival induced by lower concen- tration of LPS (1 ng/mL). The stimulatory effect of ATP on microglial survival

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238 Abstracts / Neuroscience R

ls in cultured astrocytes under prolonged exposure to ammonia. Usingn enzyme-linked high-performance liquid chromatography assay to detectlutamate, prolonged (48 h) exposure of cultured astrocytes to ammoniaesulted in a concentration- and time-dependent increase in extracellu-ar glutamate. Similar increases were observed when ammonia-containing

edium (pH 7.8) was adjusted to the pH of control medium (pH 7.4), indi-ating that the effect is not due to pH. Treatment of astrocytes with anntioxidant (l-ascorbic acid), an NADPH oxidase inhibitor (apocynin), a Ca2+

helator (BAPTA-AM), an NMDA receptor antagonist (NK801), or a mito-hondrial permeability transition inhibitor (cyclosporine A) suppressed thencrease of extracellular glutamate in response to prolonged ammonia expo-ure. Prolonged exposure to ammonia increased extracellular glutamatehrough the NMDA receptor, increased intracellular Ca2+ levels, and upreg-lation of excitatory amino acids. The addition of ATP further increasedxtracellular glutamate levels in astrocytes subjected to prolonged ammoniareatment (5 mM, 48 h) in a dose-dependent manner. These results indicatehat the deregulation of glutamate release from astrocytes may contribute tohe dysfunction of glutamatergic neurons in patients with acute liver failure.

oi:10.1016/j.neures.2010.07.1051

2-b38 PLD4 is implicated in phagocytosis in major phago-ytesoshinori Ohtani 1 , Yoshihide Yamaguchi 1, Hiroshi Kitani 2, Yumiato 3, Teiichi Furuichi 3, Hiroko Baba 1

Department of Molecular Neurobiology, Tokyo University of Pharmacy andife Sciences 2 Nat. Inst. Agrobiol. Sci., Tsukuba, Japan 3 Lab. for Mol. Neuro-enesis, RIKEN Brain Sci Inst, Wako, Japan

hospholipase D (PLD) represented by PLD1 and PLD2 hydrolyzes phos-hatidylcholine to produce phosphatidic acid and choline. These PLDs arenown to be involved in various cellular functions including membranerafficking, secretion and mitogenesis. Previously, we reported that thexpression of a novel member of PLD family, PLD4, was specifically upregu-ated in amoeboid microglia in the white matter of mouse cerebellum, bothn the developmental stage and the pathological conditions. In the analy-is using cultured microglial cell line (MG6), we demonstrated that PLD4as primarily located in the nucleoplasm while accumulated in phagosomes

ontaining BioParticles during phagocytosis. Because, unlike PLD1 and PLD2,LD4 did not exhibit PLD enzymatic activity, the role of PLD4 in phagocytoticascade was totally unknown. In this study, to clarify the function of PLD4,e focused on the functional association of PLD4 with other PLDs duringhagocytosis. In another major phagocyte, macrophage, PLD1 and PLD2 wereeported to be involved in phagocytosis. To determine whether PLD4 wasxpressed in such activated macrophages in the lesion of the PNS, we per-ormed immunohistochemistry for PLD4 and Iba1 on lysolecithin-inducedemyelinated area of mouse sciatic nerve. The result indicated that PLD4as localized in the Iba1 positive lesion where activated macrophages were

ccumulated. Thus, PLD4 may play a role in the phagocytosis together withLD1 and/or PLD2. Therefore, to investigate the relationship of PLD4 withther PLDs in the process of phagocytosis, we examined their localizationsuring phagocytosis using SNAP/CLIP systems. As a result, PLD4 was colocal-

zed with PLD1 in the phagosomes of MG6 cells. These results suggest thatLD4 may be involved in the phagocytic process associated with PLD1.

oi:10.1016/j.neures.2010.07.1052

2-b39 Administration of Chinpi, a component of theerbal medicine ninjinyoeito, ameliorates cuprizone-

nduced demyelination and promotes remyelination.umi Misawa , Kenji Watanabe, Hiroaki Asou

Center for Kampo Medicine, Keio Univercity School of Medicine, Japan

ntroduction The myelin sheath in the central nervous system (CNS) formss an oligodendrocyte wraps layer upon layer of its own membrane aroundhe axon in a tight spiral forming an electrically insulting sheath around thexon. Myelin loss occurs in many demyelinating diseases such as multipleclerosis. In previous studies, we have demonstrated that the recovery fromemyelination in aged mice is achieved after administration of the herbaledicine ninjinyoeito (NYT), and also found a potentially therapeutic effect

f NYT against age-induced demyelination, resulting the level of phosphory-

ated myelin basic protein (P-MBP) was markedly and inversely correlated

ith demyelination.ethods and results Feeding cuprizone (bis-cyclohexanone oxalydihydra-

one) to young C57BL/6 mice leads to a reproducible demyelination of theorpus callosum and the superior cerebellar peduncle within several weeks

ch 68S (2010) e223–e334

with nearly complete remyelination after withdraw of the cuprizone fromthe diet. We examined the efficacy of Chinpi, the major active constituent ofNYT, to demyelinated animals in a 0.2% cuprizone diet. Immunohistochem-ical studies of cuprizone-exposed mice brain section by using anti-myelinbasic protein (MBP) antibodies and anti- P-MBP antibodies showed that inthe Chinpi treated mice for 5 weeks was marked increase both T-MBP andP-MBP positive fibers in corpus callosum. We also found that cuprizone-exposed for 5 weeks mice with a 0.5% Chinpi in drinking water for 7 weeksinduce a marked increase in myelin deposition compared with that observedin spontaneous recovery.Conclusions In conclusion, our immunohistochemical findings suggest thatChinpi significantly ameliorates cuprizone-induced demyelination and pro-motes remyelination.

doi:10.1016/j.neures.2010.07.1053

P2-b40 Role of gap junction hemichannels in protectiveeffect of NAD+ against PARP-triggered astrocyte deathHiroto Okuda , Yuka Tokotani, Kentaro Nishida, Kazuki NagasawaDepartment of Environ. Biochem., Kyoto Pharm. University, Kyoto, JAPAN

Objective Excessive activation of poly(ADP-ribose) polymerase (PARP), fol-lowing ischemia and trauma, etc., depletes cellular NAD+ and leads to celldeath. PARP activation-triggered astrocyte death has been demonstrated tobe prevented by addition of NAD+ extracellularly. It has been known thatintact form of NAD+ acts as a cytoprotective molecule, and that P2X7 recep-tor (P2X7R) and connexin43 hemichannel are candidate molecules for NAD+

fluxes in several types of cells, but the detailed molecular mechanism remainsstill unclear. The aim of this study was to clarify the pathway through whichNAD+ can enter astrocytes under PARP-activated conditions.Methods Astrocyte primary cultures were prepared from mouse newbornpups and cultured in EMEM supplemented with FBS. DNA damage and PARPactivation were induced in astrocyte cultures with a DNA alkylating agent, N-methyl-N?-nitro-N-nitrosoguanidine (MNNG). siRNAs were transfected intocells by a lipofection methods. Cell viability was determined with an LDHassay, and cellular NAD+ content was assessed by enzymatic recycling assay.Results and discussion MNNG-triggered astrocyte death was completely pre-vented by extracellular administration of 5 mM NAD+, and this was notaffected by P2X7R antagonists, oxidized ATP and KN-62. Furthermore, knock-down of P2X7R and pannexin1, which can be opened following P2X7Ractivation, had no effect on NAD+-induced increase of cellular NAD+ levelof MNNG-treated astrocytes. On the other hand, pharmacological treatmentknown to block gap junction hemichannels, 18�-glycyrrhetinic acid, octanoland carbenoxolone resulted in decrease of NAD+-induced increase of cellviability. Taken together, these data suggested that NAD+ could be taken upinto astrocytes through gap junction hemichannels, but not P2X7R, and thisprocess might play a key role in NAD+-induced prevention against PARP-triggered astrocyte death.

doi:10.1016/j.neures.2010.07.1054

P2-c01 Death or survival of Toll-like receptor 4-activatedmicroglia is regulated by extracellular ATPKana Harada 1 , Izumi Hide 1, Shunsuke Fujiwara 1, TakahiroSeki 1, Shigeru Tanaka 1, Yoshihiro Nakata 2, Norio Sakai 1

1 Dept. Mol. and Pharmacol. Neurosci., Grad. Sch. Biomed. Sci., HiroshimaUniv., Hiroshima, Japan 2 Dept. Pharmacol., Grad. Sch. Biomed. Sci., HiroshimaUniv., Hiroshima, Japan

Microglia, the CNS resident macrophages, migrate to the injured sites andpromote tissue repair. Hyperactivated microglia, however, induce excessiveinflammation, which is closely implicated in the exacerbation of neurode-generation. Recently, it has been shown that endogenous ligands of Toll-likereceptors (TLRs), which are released from damaged cells, elicit inflammatoryresponses through the activation of microglia. Moreover, ATP is also releasedfrom damaged cells and conveys a danger signal to microglia. In this study,we examined whether lipopolysaccharide (LPS), a TLR4 ligand, and extracel-lular ATP might control death and survival of microglia. Microglia started toundergo spontaneous cell death shortly after being isolated from rat primarymixed glial cultures and all the cells died within 2 days. The high concen-trations of LPS (5–100 ng/mL) accelerated microglial cell death, whereas

lower concentration of LPS (1 ng/mL) significantly extended the survival timeof microglia without affecting cell death. Interestingly, ATP (1 mM) furtherenhanced the cell death of microglia activated with the high concentration ofLPS (5 ng/mL), while promoting microglial survival induced by lower concen-tration of LPS (1 ng/mL). The stimulatory effect of ATP on microglial survival