一般演題（ポスター）

一般演題（ポスター） Neurotransmission II

2P-07 A role for BMP4 signaling pathway in mouse neural stem cell survival Yamamoto Hanako^1,2，Kurachi Masashi¹，Naruse Masae¹，Shibasaki Koji¹，Ishizaki Yasuki¹ ¹Dept. Mol. Cell. Biol., Grad. Sch. Med., Gunma Univ.，²Center Med. Educ., Grad. Sch. Med., Gunma Univ. We recently reported that bone morphogenetic protein 4（BMP4）does not promote differentiation of CD44-positive astrocyte precursor cells into mature astrocytes but greatly promotes their survival（1）. Although only few studies have examined the survival-promoting effects of BMPs in the nervous system, it has been shown that BMPs acts as important survival factors in various kinds of cells including those constituting primordial follicles（2）. While the anti-apoptotic effects of BMPs have been described in these reports, the molecular mechanism by which they inhibit apoptotic death remains unclear. We isolated neural stem/progenitor cells（NSCs）from ganglionic eminence of postnatal day 0 mouse brain, and examined the effects of BMP4 on their proliferation and survival. BMP4 did not promote their proliferation but promoted their survival, just as in the case of CD44-positive APCs. Microarray analysis suggested us some candidate molecules in the signaling pathway downstream of BMP4. We will present the data and discuss the mechanism by which BMP4 promotes survival of NSCs. 1）. Cai N, Kurachi M, Shibasaki K, Okano-Uchida T, Ishizaki Y. CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum. Cerebellum. 2012；11：181-193. 2）. Nilsson EE, Skinner MK. Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development. Biol. Reprod. 2003；69：1265-1272.		2P-08 PACAP induces Bdnf expression through selective activation of NMDA receptor/calcineurin pathway in neurons. Watanabe Baku，Fukuchi Mamoru，Ichimura Mina，Ochi Yuki，Tabuchi Akiko，Tsuda Masaaki Dept. of biol. Chem., Grad. Soh. of med. ＆ Pharm. Sci., Univ.of Toyama It has been reported that the activation of GPCR（G protein-coupled receptor）, which is a major receptor for modulatory neurotransmitters such as monoamines and neuropeptides, modulates the function of NMDA（N-methyl-D-aspartate）, one of ionotropic glutamate receptors. We here focused on the regulation of gene expression under the activation of GPCR in cultured cortical cells. However, intracellular signaling pathways evoked by the activation of these two receptors are still unclear. We found that the activation of PAC1, a Gs/q-coupled GPCR, with PACAP（Pituitary adenylate cyclase-activating polypeptide）induced the expression of a group of genes including Bdnf（brain-derived neurotrophic factor）through the NMDA receptor and calcineurin pathway. Interestingly, co-activation of NMDA receptor and PAC1 selectively enhanced the NMDA receptor/calcineurin pathway, resulting in the synergic induction of Bdnf expression. We demonstrated that the activation of the NMDA receptor/calcineurin pathway induced CREB（cAMP response element-binding protein）-dependent transcription mediated by nuclear localization of CRTC1（CREB-regulated transcriptional coactivator 1）. Taken together, it is suggested that glutamatergic and modulatory neurotransmissions activate CRTC1/CREB-dependent gene expression via the selective activation of the NMDA receptor/calcineurin pathway in neurons. To further elucidate the molecular mechanisms underlying the selective activation of this pathway, we are now focusing on intracellular anchoring molecules like AKAP（A kinase anchoring protein）, which can coordinate multiple kinases and phosphatases such as PKA（protein kinase A）, PKC（protein kinase C）, and calcineurin, and modulate the function of NMDA receptor.

2P-09 Dopamine phosphorylates GEF-H1 through PKA to regulate GEF-H1 activity in striatum Zhang Xinjian¹，Kuroda Keisuke¹，Takenaka Hiroyuki¹，Kondo Reon¹，Oda Kaishu¹，Nishioka Tomoki¹，Nakamuta Shinichi¹，Nagai Taku²，Kaibuchi Kozo¹ ¹Department of Cell Pharmacology, Nagoya University Graduate School of Medicine，²Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University Graduate School of Medicine In the brain, dopamine functions as a neurotransmitter and associates with emotion regulation. The functions of dopamine are mediated by Protein kinase A（PKA）-dependent signaling cascades in the neurons. Phosphorylation of PKA substrates regulates the structural and functional plasticity of neurons. However, many important PKA substrates are still unknown, so further investigation is required to determine the substrate for PKA and its function. Here, we treated mouse striatum with D1 agonist（SKF81297）or cyclic AMP（cAMP）inducer（forskolin）and then concentrated phosphoprotein with GST-14-3-3z pull-down assay followed by LC-MS/MS analysis. We identified GEF-H1, a Rho guanine nucleotide exchange factor, as a novel PKA substrate. Although present throughout the brain, GEF-H1 is enriched in striatum and is a major RhoGEF in striatum. GEF-H1 is phosphorylated by PKA at Ser-885 in response to activation of D1 dopamine receptors. Phosphorylation of GEF-H1 is associated with inhibition of GEF activity as demonstrated by GST-RhoA-G17A pull down assay. These results suggest that dopamine can decrease RhoA activity through phosphorylation of GEF-H1 in striatum so as to exert its control over behavior.		2P-10 Transduction from the protein kinase C pathway to the tyrosine kinase pathway in cultured hypothalamic neurons Yamamoto Hideyuki，Nakamine Sayomi，Maeda Noriko，Toku Seikichi Dept. of Biochem. Med., Univ. of the Ryukyus The receptor for gonadotropin-releasing hormone（GnRH）belongs to the G-protein-coupled receptors, and its stimulation activates extracellular signal-regulated protein kinase（ERK）. We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized hypothalamic GnRH neurons（GT1-7 cells）^1）. In the present study, we examined signal transduction comprising the activation of ERK after GnRH treatment. Experiments with two types of PKC inhibitors, Go 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms were involved in ERK activation. Our inhibitor experiments indicated that the novel PKC isoforms activated protein kinase D（PKD）after its translocation by treatment with GnRH. Knockdown experiments suggested that PKD stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn. We found that Src, Fyn and PYK2 were involved in ERK activation. Taken together, it is highly possible that PKD plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway for ERK activation.1）J. Cell. Physiol., 2012, 2492-2501.

2P-11 Behavioral phenotypes of neuron-specific Shp2 conditional knockout mice Ohnishi Hiroshi¹，Kusakari Shinya²，Hashimoto Miho¹，Ishikawa Shuya¹，Urano Eriko¹，Kotani Takenori³，Murata Yoji³，Matozaki Takashi³ ¹Dept. of Lab. Sci., Gunma Univ. Grad. Schl. of Health Sci.，²Lab. of Biosig. Sci., IMCR, Gunma Univ.，³Div. Mol. Cell. Signal., Dept. Biochem. Mol. Biol., Kobe Univ. Grad. Schl. Med. A protein tyrosine phosphatase, Shp2（Src homology 2-containg protein tyrosine phosphatase 2）, acts as a positive regulator of Ras-MAPK cascade downstream of growth factor receptors. Forebrain neuron-specific genetic ablation of Shp2 resulted in the reduction of synaptic transmission and of post-tetanic potentiation, a form of short-term synaptic plasticity, in hippocampal slices. To further address the functional roles of Shp2 in the brain, forebrain neuron-specific Shp2 conditional KO（cKO）mice were subjected to a battery of behavioral tests. The mutant mice showed normal behavior in the rotarod test or the thermal preference test, suggesting normal motor coordination, thermosensation, and nociception in the Shp2 cKO mice. In contrast, the mutant animals exhibited reduced stay time in the dark place, as well as increased number of transition and distance traveled, in the light-dark transition test. The mutant mice also exhibited reduced immobility in the forced swim test and an increased acoustic startle response, whereas prepulse inhibition was normal. In addition, Shp2 cKO mice exhibited reduced freezing behavior during re-presentation to the context in the fear conditioning test, while the fear response to an auditory conditioned stimulus（CS）was not affected. The mutant mice also showed temporary impaired memory formation in Morris water maze. Some of these abnormal behaviors are likely due to the hyperactivity in Shp2 cKO mice, because the mutant mice exhibited markedly increased locomotor activity in the open field test and in the home cage as shown in our previous report. In contrast, other abnormal behaviors, such as reduced stay time in the dark place or impaired memory formation, may be independent from the hyperactive phenotype of the mutant mice.		2P-12 Two Gq-coupled octopamine receptors function non-redundantly to mediate food deprivation signaling of C. elegans Suo Satoshi¹，Yoshida Midori²，Ishiura Shoichi² ¹Department of Pharmacology, Faculty of Medicine, Saitama Medical University，²Graduate School of Arts ＆ Sciences, the University of Tokyo Amine neurotransmitters act primarily through G protein-coupled receptors（GPCRs）. In many cases, there are multiple receptors that bind to the same neurotransmitter and activate the same intracellular signaling cascades. In a model animal Caenorhabditis elegans, there are two Gq-coupled receptors for octopamine, the biological equivalent of norepinephrine in invertebrate. It has been previously shown that octopamine induces activation of CREB（cAMP response element-binding protein）in the cholinergic SIA neurons during food deprivation and that this activation is mediated through activation of the Gq-coupled octopamine receptor SER-3 that is expressed in these neurons. We also analyzed the other Gq-coupled octopamine receptor, SER-6, which is highly homologous to SER-3. As seen for ser-3 deletion mutants, CREB activation induced by exogenous octopamine and food deprivation was decreased in ser-6 deletion mutants compared to wild-type animals, suggesting that SER-6 is also required for this signal transduction. Expression of SER-6 in the SIA neurons was sufficient to restore CREB activation in the ser-6 mutants, indicating that SER-6 functions in these neurons as does SER-3. Furthermore, overexpression of one receptor subtype did not fully restore CREB activation in the absence of the other receptor. These results demonstrate that two types of similar GPCRs, SER-3 and SER-6, are required for normal signaling and function in the same cells in a non-redundant manner.

2P-13 The Strip-Hippo pathway regulates synaptic terminal formation by modulating actin organization at the Drosophila neuromuscular junction Chihara Takahiro^1,2，Sakuma Chisako¹，Saito Yoshie¹，Umehara Tomoki¹，Kamimura Keisuke³，Mosca Timothy⁴，Maeda Nobuaki³，Miura Masayuki^1,2 ¹Dept Genetics, Pharm Sci, Univ of Tokyo，²CREST, JST，³Dept of Brain Dev and Neural Regen, Tokyo Met Inst of Med Sci，⁴Dept Biology, Stanford Univ The Hippo（Hpo）pathway is well known for its role in growth control in both flies and mammals. Although postmitotic roles of Hpo have also been uncovered such as regulation of dendrite tiling and photoreceptor specification, there has been no report of Hpo function in synapse development. Here, we show the role of Hpo and its negative regulator Strip in synapse formation of Drosophila larval neuromuscular junction（NMJ）. Strip, an evolutionarily conserved protein functions as a platform for endosome maturation that is required for axon elongation（Nat Commun 5, 5180, 2014）. In addition, we found that endogenous Strip is predominantly localized at presynaptic sites of NMJ. strip knockdown in motor neurons resulted in the significant increase in the number of small synaptic bouton called satellite bouton. Furthermore, strip knockdown larvae exhibited the defects in synapse transmission. As Strip was also reported as a component of STRIPAK（PP2A）complex, a negative regulator of Hpo in growth control, we examined the relationship between Strip and Hpo. First, we found that strip knockdown in S2 cells significantly increased the phosphorylation level of Hpo. Consistent with this, the satellite bouton phenotype by strip knockdown was significantly suppressed in hpo heterozygous background, suggesting that Strip negatively regulates Hpo activity in synapse development. We also observed overexpression of Hpo caused the satellite bouton phenotype, suggesting that Hpo positively regulates satellite bouton formation. Here we would like to present the molecular mechanism how Hpo and Strip regulate synapse development.		2P-14 Investigation of novel CREB interacting proteins using DNA affinity beads. Kondo Reon，Kuroda Keisuke，Kaibuchi Kozo Department of Cell Pharmacology, Nagoya University Graduate School of Medicine Dopamine is a neurotransmitter that encodes emotional information such as reward, pleasure, attention and anxiety. It acts as a modulator of synaptic plasticity by altering the resting potential of the post-synaptic membrane and changing gene expression. In brain, most of the dopaminergic neuron project to striatum. Dopamine receptors comprise 7-transmembrane domain receptors and are associated with guanosine triphosphate-binding proteins（or G proteins）that mediate their effects. One major effect of D1 receptors is to raise cAMP levels and thereby activate a cyclic AMP dependent protein kinase（PKA）. PKA phosphorylates various substrates including cyclic AMP response element binding protein（CREB）and regulate their functions. CREB is a transcription factor that binds to cyclic AMP response element（CRE）sequence and activate transcription phosphorylation-dependent manner. Although we know that CREB activity is regulated by phosphorylation at Ser133 and interaction with cofactors such as CBP/p300, the precise mechanism of transcription induced by CREB is yet to be revealed. Therefore, we tried to obtain comprehensive understanding of protein complex that is responsible for CREB-induced transcription. We prepared the DNA affinity beads coupled to oligonucleotides containing CRE sequence in promoter region of c-fos gene. And the ability of the beads to enrich CREB and the specificity of the beads were confirmed both in vitro and in vivo. By LC/MS/MS we identified several proteins that bound to the beads with sequence specificity. Using DNA affinity beads enables us to elucidate the regulatory mechanism of transcriptional activation and to obtain comprehensive understanding of protein complex that acts in that process.

2P-15 Physiological analysis of lipid raft molecules on mouse brain slices Kotani Norihiro¹，Ida Yui¹，Shinozaki Rina¹，Seo Makoto^1,2，Nakano Takanari¹，Hashizume Miki¹，Yamaguchi Arisa³，Honke Koichi³，Murakoshi Takayuki¹ ¹Department of Biochemistry, Saitama Medical University，²Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University，³Department of Biochemistry, Kochi University Medical School The brain consists of numerous neural cells which give rise to the voluntary action potential resulting in the transmission of complex and intermittent signals to the succeeding cells. The constitution of such neural network is quite essential to maintain higher brain function in vertebrate. Also, the signal transduction system simultaneously contributes to not only neural cell functions but also higher brain function. It has been known that lipid raft domain serve as cell surface platforms of signal transduction especially plays the crucial roles of neural cell functions, such as the release and internalization of neurotransmitter in presynaptic nerve terminal. Although lipid raft in the brain has been intensely investigated, the neural culture cells or brain lysates that do not retain native neural network were used for experimental materials because of the limitation in biochemical strategies. The physiological living brain tissue（brain slice）is, however, considered to be appropriate to explore the relationship between higher brain functions and actual lipid raft domains. Herein, we focused on lipid raft domains on living brain slices（200μm）derived from several portions of mouse brain processed by using brain slicer. To identify the lipid raft molecules, Enzyme-Mediated Activation of Radical Sources（EMARS）method previously developed（Kotani N. et al. Proc Natl Acad Sci U S A. 105：7405-7409（2008））was applied to label specific lipid raft molecules on living brain slices. The physiological lipid raft molecules in mouse brain were significantly different among brain region, suggesting that there needs to use living brain slice for understanding the contribution of lipid raft domain to neural network and higher brain functions.		2P-16 VAMP7 regulates autophagy to maintain mitochondrial homeostasis and to control second phase insulin secretion in pancreatic β-cells. Aoyagi Kyota，Ohara-Imaizumi Mica，Nishiwaki Chiyono，Nakamichi Yoko，Kishimoto Takuma，Nagamatsu Shinya Dept of Biochem, Kyorin Univ Sch of Med VAMP7 is a member of VAMP family proteins required for membrane trafficking and the fusion, and recently reported to be involved in autophagy. VAMP7 is expressed in pancreatic β-cells but its physiological function have not been elucidated. In this study, we investigated the role of VAMP7 in the β-cell function using the β-cell specific VAMP7 deletion（VAMP7 βKO）mice. In isolated VAMP7 βKO islets, the glucose-induced second phase but not the first phase insulin secretion was decreased. The deletion of VAMP7 did not affect the distribution of insulin granules and the glucose-induced cytosolic Ca2+ dynamics during the second phase. On the other hand, p62 was slightly accumulated and the starvation-induced LC3-II accumulation was disturbed in VAMP7 βKO islets, indicating a defect in autophagy. Because damaged mitochondria are eliminated by autophagy to maintain mitochondrial homeostasis, we focused on the mitochondrial function in VAMP7 null β-cells. The glucose-induced ATP production was significantly reduced during the second phase in VAMP7 βKO islets. Both the hyperpolarization of mitochondrial membrane and increase in mitochondrial Ca2+, which was essential for the second phase insulin secretion, was blunted in VAMP7 null β-cells. In addition, morphologically abnormal mitochondria were found in VAMP7 null β-cells. Finally, we found that the expression of VAMP7 in VAMP7 null β-cells restored the glucose-induced secretion and the starvation-induced LC3-II accumulation. These results suggest that VAMP7 plays an important role in the maintenance of mitochondrial homeostasis which is critical for the regulation of the second phase insulin secretion.

2P-17 A novel effect of tributyltin on mitochondrial quality control Yamada Shigeru¹，Kotake Yaichiro²，Sekino Yuko¹，Kanda Yasunari¹ ¹Division of Pharmacology, National Institute of Health Sciences，²Graduate School of Biomedical and Health Sciences, Hiroshima University Organotin compounds, such as tributyltin（TBT）, are well-known endocrine disruptors. TBT is known to cause neurodevelopmental defects such as behavioral abnormalities and teratogenicity. Several reports have shown that micromolar TBT levels induce neuronal degeneration via mitochondria-mediated ROS generation in neurons. We have recently reported that nanomolar TBT levels reduce cell growth and ATP content via mitochondrial NAD⁺-dependent isocitrate dehydrogenase（NAD-IDH）, which metabolizes isocitrate into α-ketoglutarate, in human embryonic carcinoma cells NT2/D1. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we evaluated the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar TBT levels induced mitochondrial fragmentation. TBT also degraded the mitochondrial fusion proteins, mitofusin 1 and 2. Interestingly, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. Incubation with an α-ketoglutarate analogue partially recovered TBT-induced mitochondrial dysfunction, supporting the involvement of NAD-IDH. Our data suggest that nanomolar TBT levels impair mitochondrial quality control via NAD-IDH in NT2/D1 cells. Thus, mitochondrial function in embryonic cells could be used to assess metal toxicity.