ポスター発表 (Poster Sessions)

受容体、輸送体 Receptors and Transporters
P2-2-13 ドコサヘキサエン酸によるアストロサイトグルタミン酸トランスポーター EAAT2 機能増強 The function of glial excitatory amino-acid transporter EAAT2 is enhanced by docosahexanoic acid ○高橋華奈子¹, 入江智彦¹, 関野祐子¹, 佐藤薫¹ ○Kanako Takahashi¹, Tomohiko Irie¹, Yuko Sekino¹, Kaoru Sato¹ 国立医薬品食品衛生研究所薬理部¹ Division of Pharmacology, National Institute of Health Sciences,¹ Astrocytic EAAT2 is important for removal of L-Glutamate (L-Glu) from synaptic cleft and maintenance of efficient synaptic transmission. Docosahexaenoic acid (DHA; C22:6), one of polyunsaturated fatty acids (PUFAs), is major constituents of membrane phospholipids and highly concentrated in the brain. Although astrocytes synthesize DHA mainly in the brain and release DHA after stimulation with L-Glu and ATP, its acute effects on astrocytic EAAT2 is almost unknown. In this study, we investigated the effects of DHA on EAAT2 currents using two-electrode voltage clamp technique in Xenopus oocytes expressing EAAT2. Exogenously-applied DHA (100 μM) increased the amplitude of L-Glu-induced EAAT2 currents and this effects were reversible. PUFAs are known to regulate membrane protein function by affecting the elasticity of the lipid bilayer. Triton X-100, which increases membrane fluidity, little affected EAAT2 currents, indicating that the DHA-induced enhancement of EAAT2 currents is independent of membrane elasticity. Moreover, the injection of the fatty acid scavenger, bovine serum albumin (BSA), into oocytes expressing EAAT2 largely prevented the enhancement, suggesting that DHA regulates EAAT2 from cytoplasmic space. Currently, we are investigating the characteristics and the mechanisms of the effects of DHA on EAAT2 in more detail.	P2-2-14 Sequence and distribution of vesicular glutamate transporter3 mRNA in the brain of pigeons and chickens Sequence and distribution of vesicular glutamate transporter3 mRNA in the brain of pigeons and chickens 齋藤正一郎¹, 阿閉泰郎¹ ○Mohammad R. Karim¹, Shouichiro Saito¹, Yasuro Atoji¹ 岐阜大・応用生物・獣医解剖¹ Lab Vet Anat, Gifu Univ, Gifu¹ Two types of vesicular glutamate transporters (VGLUTs) have been identified in birds: VGLUT2 and VGLUT3. VGLUT2 mRNA is cloned in the pigeon and chicken, and mRNA and protein of pigeon VGLUT2 are widely distributed in the central nervous system. Sequence of chicken VGLUT3 mRNA has been registered in a gene data base, but its distribution in the brain is unknown. In the present study, in order to compare structural characteristics of avian VGLUTs, we analyzed cDNA sequences of pigeon and chicken VGLUT3, and mapped distribution of VGLUT3 mRNA in brains of the two species. We identified a 1,761 base pair of VGLUT3 cDNA in each species. The VGLUT3 cDNA sequences were deduced to encode 586 amino acids. Comparison of the deduced amino acid sequences of the pigeon and chicken VGLUT3 showed 98% identity with each other, 86% identity with human VGLUT3, and 74% identity with pigeon and chicken VGLUT2. RT-PCR detected a low level of VGLUT3 mRNA in the telencephalon and a moderate level in the cerebellum of the pigeon and chicken. In situ hybridization using oligonucleotide probes labeled with [³⁵S] dATP showed moderate expression of VGLUT3 mRNA in the granular layer of the cerebellum but no expression was detected in the molecular layer and Purkinje cell layer of the pigeon and chicken. In the telencephalon of both species VGLUT3 mRNA signal was not found. The present study revealed that the cDNA sequences of VGLUT3 are well conserved in the pigeon and chicken, and VGLUT3 mRNA distribution in brains of the two species is restricted to the cerebellum in comparison with wide distribution of VGLUT2 mRNA in the pigeon
P2-2-15 アストロサイトに発現するP2X7受容体によるengulfment活性の制御 P2X7 receptors expressed by mouse astrocytes play a role in their engulfment activity ○山本美菜¹, 鎌塚洋祐¹, 西田健太朗¹, 長澤一樹¹ ○Mina Yamamoto¹, Yosuke Kamatsuka¹, Kentaro Nishida¹, Kazuki Nagasawa¹ 京都薬大衛生化学¹ Dept. of Environ. Biochem., Kyoto Pharm. Univ. Kyoto¹ [Objective] P2X7 receptor (P2X7R) has roles in the CNS homeostasis. It is known that activation of P2X7Rs expressed by neurons kills them, and that by microglia regulates their migration. Astrocytes also express P2X7Rs and their activation causes release of pro-inflammatory cytokines and glio-transmitters such as ATP. We previously indicated that P2X7Rs expressed by cultured mouse astrocytes were activated without any exogenous stimuli, but the physiological roles were unknown yet. It is reported that astrocytes have engulfment activity, and basal activity of P2X7Rs regulates phagocytotic activity of macrophages. In this study, therefore, we investigated the possibility that P2X7Rs might regulate engulfment activity of mouse astrocytes.[Materials & Methods] Mouse astrocyte cultures were obtained from ddY-strain mouse cortices as reported previously. Activity of engulfment and P2X7R in astrocytes was assessed by measuring their uptake of yellow-green carboxylate latex beads (1 μm) and YO-PRO-1, respectively. Acute slice cultures of mouse cortex were prepared using a microslicer.[Results & Discussion] Bead uptake by astrocytes was time-dependently increased, and the beads taken up by them were detected in the intracellular space by GFAP- and CellTracker-staining. The bead uptake was inhibited by cytochalasin D, an F-actin polymerization inhibitor, and the agonists (ATP and BzATP) and antagonists (KN-62 and A438079) for P2X7R apparently decreased the uptake. Basal activity of astrocytic P2X7Rs were observed in both primary and acute slice cultures, and YO-PRO-1 uptake by cultured astrocytes was diminished by the agonists and antagonists. These findings suggested that basal activity of P2X7Rs expressed by astrocytes might be involved in regulation of their engulfment activity.	P2-2-16 神経細胞における恒常的Gs活性化受容体GPR3の局在と機能 The subcellular localization and function of Gs-linked receptor GPR3 in neuronal cells ○宮城達博¹, 田中茂¹, 秀和泉¹, 関貴弘¹, 酒井規雄¹ ○Tatsuhiro Miyagi¹, Shigeru Tanaka¹, Izumi Hide¹, Takahiro Seki¹, Norio Sakai¹ 広島大学大学院　医歯薬保健学総合研究科　神経薬理学¹ Department of Molecular and Pharmacological Neuroscience, Hiroshima University Graduate school of Biomedical and Health Sciences, Hiroshima, Japan¹ G-protein coupled receptor (GPR) 3 is a member of GPRs that constitutively activates the alpha subunit of the adenylate cyclase-stimulating G protein (Gs). We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) enhances neurite outgrowth and modulates proliferation of CGNs. However, the distributions and functions of GPR3 in the CNS have not been fully elucidated. In the present study, we revealed that GPR3 is distributed in the cortex, striatum, hippocampus, medial habenular nucleus, and cerebellum in mice. Further, we applied monomeric green fluorescent protein tagged GPR3 to neuronal cells for visualizing time-dependent distributions of GPR3. GFP fluorescence was distributed along the plasma membrane and in the cytoplasm, such as endoplasmic reticulum and goldi body. On the other hand, fluorescent punctae of GFP-GPR3 were translocated along the neurite in both directions. Translocation of GFP-GPR3 punctae was completely abrogate by administration of blebbistatin, which is the potential myosin II inhibitor. We further asked if migration of GFP-GPR3 punctae is associated with local activation of PKA. Application of PKA FRET indictor (AKAR3EV) to the GPR3 transfected cells revealed that the activity of PKA was relatively higher in neurite than in cell body. These results thus indicated that GPR3 may be related with local PKA activation, thereby affect neuronal functions.
P2-2-17 Bidirectional phosphorylation of glucocorticoid receptors in the hippocampus for antidepressant-like effects of Cortex Mori Radicis extract ○Song Her¹, Young Han Kim¹, Wan-Soon Park¹, Mi-Sook Lee², Insop Shim², Kyoji Morita³ Korea Basic Science Institute¹, Kyung Hee University², Shikoku University³ Excessive and prolonged secretion of adrenal glucocorticoids leads to a wide range of pathophysiological processes, including depression. Glucocorticoids, which act at glucocorticoid receptors (GR), are key regulators of the limbic-HPA axis. In the present study, the antidepressant-like effects of the alcohol extract Cortex Mori Radicis (CMR) and its role in GR signalling were investigated. Male Wistar rats were administered CMR extract (50, 100, 200 mg/kg, p.o.) daily for 5 days and then exposed to the forced swim test (FST). Behavioural analyses showed that CMR extract dose-dependently decreased immobility time during the forced swim test. CMR extract also decreased the LHPA axis response to the FST, as indicated by an attenuated corticosterone response and decreased c-fos immunoreactivity in the dentate gyrus. Reduced hippocampal GR expression following exposure to the FST was reversed by CMR treatment. Moreover, a prominent increase in GR phosphorylation at S232 and a decrease at S246 were noted following treatment with CMR. This resulted in a high pGR(S232)/(S246) ratio. CMR treatment also produced a downregulation of serine/threonine phosphoprotein phosphatase 5 levels, producing a strong negative relationship with pGR(S232). Taken together, our findings suggest that the alcohol extract CMR promotes antidepressant-like behaviour through bidirectional phosphorylation of GR at S232 and S246.	P2-2-18 膜輸送体OCTN1によるマウス脳神経の発達制御 Neuronal development regulated by solute carrier OCTN1/SLC22A4 in mouse brain ○中道範隆¹, 細谷拓史¹, 石本尚大¹, 杉浦智子¹, 加藤将夫¹ ○Noritaka Nakamichi¹, Hiroshi Hosotani¹, Takahiro Ishimoto¹, Tomoko Sugiura¹, Yukio Kato¹ 金沢大学医薬保健研究域薬学系　分子薬物治療学研究室¹ Faculty of Pharmacy, Kanazawa University, Kanazawa¹ Carnitine/organic cation transporter OCTN1/SLC22A4 was recently clarified to be functionally expressed in brain neurons (Nakamichi et al., Neurochem Int 61, 1121, 2012). OCTN1 accepts naturally occurring antioxidant ergothioneine (ERGO) as a typical substrate. Identification of physiological function of this antioxidant-related transporter in brain neurons may lead to elucidation of mechanisms underlying the development of neurodegenerative diseases. The aim of the present study was to clarify it using octn1 gene knockout (octn1^-/-) mice and primary cultured neurons from mouse neocortex. Real-time PCR analysis revealed that expression of neuronal marker βIII-tubulin and synapse formation marker synapsin I of octn1^-/- mice was significantly lower than that in wild-type mice at the age of 4-weeks-old. Immunocytochemical analysis showed colocalization of OCTN1 protein with βIII-tubulin in cultured cortical neurons, which exhibited time-dependent and saturable uptake of [³H]ERGO. The expression of OCTN1 mRNA was largely decreased in cortical neurons transfected with small interfering RNA for OCTN1 by electroporation. Consistent with the results obtained using octn1^-/- mice, this knockdown of OCTN1 significantly attenuated expression of βIII-tubulin and synapsin I. In addition, OCTN1 knockdown induced expression of Sox2, which is required to maintain the properties of undifferentiated neural stem cells, in cortical neurons. Interestingly, the addition of ERGO to culture medium including serum of cortical neurons also resulted in down-regulation of βIII-tubulin and synapsin I, and up-regulation of Sox2, at the mRNA levels as well as the protein levels, suggesting that ERGO may act as an inhibitor of OCTN1 in this case and inhibit uptake of an unidentified substrate of OCTN1 from serum. These results suggest that OCTN1 may regulate neuronal development possibly via transporting an unidentified substrate in mouse brain.
P2-2-19 ドパミン神経細胞における CaMKIIdelta3 核内移行制御機構 The mechanisms of nuclear translocation of CaMKIIdelta3 in dopaminergic neurons ○澤井優広¹, 塩田倫史¹, 福永浩司¹ ○Masahiro Sawai¹, Norifumi Shioda¹, Kohji Fukunaga¹ 東北大院・薬・薬理¹ Dept Pharmacol, Univ of Tohoku, Sendai,Japan¹ We previously reported that stimulation of dopamine D2R activated the calcium/calmodulin dependent protein kinase II (CaMKII) nuclear isoform, CaMKIIdelta3, thereby increasing BDNF gene expression in NG-108 cells. In the present study, we found that CaMKIIdelta3 was directly dephosphorylated by protein phosphatase-1 (PP1) at Ser332 site, thereby translocating into nucleus. CaMKIIdelta3 was expressed in both cytoplasmic and nuclear compartments in Neuro-2a cells, while it translocated into nucleus following co-expression with PP1. In addition, the activity of CaMKIIdelta3 co-expressed with PP1 significantly increased in nuclear fractions as compared to CaMKIIdelta3 alone expression. Most importantly, co-expression of CaMKIIdelta3 and PP1 or expression of CaMKS332A in Neuro-2a cells significantly increased BDNF mRNA compared to that in CaMKIIdelta3-expressed cells. In addition, dopamine D2R stimulation decreased CaMKII phosphorylation at Ser332 in cultured dopaminergic neurons. These results suggest that CaMKIIdelta3 is dephosphorylated by PP1 at Ser332, resulting in translocation into nucleus. The nuclear translocated CaMKIIdelta3 likely mediates BDNF expression in neurons.	P2-2-20 グリシントランスポーター2-Creノックインマウス系統の解析 Characterization of a glycine transporter 2-Cre knock-in mouse line ○柿崎利和¹, 西丸広史², 崎村建司³, 柳川右千夫¹ ○Toshikazu Kakizaki¹, Hiroshi Nishimaru², Kenji Sakimura³, Yuchio Yanagawa¹ 群馬大院・医・遺伝発達行動¹, 筑波大・医学医療系², 新潟大・脳研・細胞神経生物³ Dept Genetic and Behavioral Neurosci, Univ of Gunma, Maebashi, Japan¹, Fuculty of Medicine, Univ of Tsukuba, Ibaraki, Japan², Dept Cellular Neurobiol, Brain Res Inst, Niigata Univ, Niigata, Japan³ Glycine transporter 2 (GLYT2) is an important molecule for glycinergic neurotransmission, and is a reliable marker of glycinergic neurons. The BAC-based GLYT2-Cre transgenic mouse line has been a useful tool for studies on glycinergic neurotransmission. Another transgenic mouse line capable of both the superior expression pattern reproducibility of Cre and gene manipulations in GLYT2-dificient glycinergic neurons will be useful for these studies. With this strategy, we introduced the Cre gene into the GLYT2 locus and generated a GLYT2-Cre knock-in mouse line. The homozygous GLYT2-Cre knock-in mice displayed motor disturbance and died before weaning. These phenotypes are consistent with those of GLYT2-deficient mice (Gomeza et al., 2003), suggesting that GLYT2 is lacked in the homozygous GLYT2-Cre knock-in mice. The spatial activities of Cre were examined by crossing GLYT2-Cre mice with two kinds of Cre reporter mouse lines, ROSA26R and CAG-CAT-EGFP. Histological analyses showed that GLYT2-Cre activities were primarily distributed in the brainstem, cerebellum and spinal cord, in which GLYT2 mRNA was expressed. In order to confirm that GLYT2-Cre activities were specifically detected in glycinergic neuron, we performed double immunohistochemical staining and/or the double in situ hybridization analyses.
P2-2-21 Disruption of metabotropic glutamate receptor signaling in Purkinje cells expressing a lentivirally transduced mutant SCA1 gene ○Anton Nikolaevich Shuvaev¹, Yamato Sato², Hanna Gennawan¹, Dai Yanagihara², Hirokazu Hirai¹ Gunma University, Graduate School of Medicine, Dep. of Neurophysiology¹, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan² Cerebellar Purkinje cells (PCs) are excited by glutamate released from the presynaptic terminals of parallel fibers (PFs) and climbing fibers (CFs), through the activation of postsynaptic AMPA-type glutamate receptors. High-frequency stimulation of PFs causes spillover of glutamate from the synaptic cleft, leading to the activation of perisynaptically localized metabotropic glutamate receptor subtype 1 (mGluR1). mGluR1 is involved in the generation of slow EPSCs and induction of long-term depression (LTD) at PF-PC synapses. Defects in mGluR1 function are known to result in severe ataxia. Hereditary spinocerebellar ataxia type 1 (SCA1), which is caused by the abnormal expansion of CAG repeats in the ATXN1 gene, primarily affects PCs in the cerebellum. To clarify the pathology of SCA1, we used lentiviral vectors to express GFP-tagged ATXN1 with normal (30) or abnormally expanded (76) CAG repeats in the PCs of mature wild-type mice. Behavioral, morphological and electrophysiological examination showed that the mice injected with the lentiviral vectors were almost indistinguishable from the non-injected mice 4 weeks after viral injection. However, at 8 weeks post-injection, the mice treated with lentiviral vectors expressing ATXN1 with abnormally expanded CAG repeats showed significantly poorer rotarod performance, which was accompanied by significant alterations in mGluR-mediated synaptic events, but not by defects in morphology and AMPA receptor-mediated fast synaptic transmission. These results suggest that disruption of mGluR signaling in PCs induced by mutant ATXN1 could provide an underlying explanation for the onset of ataxia during the early phases of SCA1 prior to the manifestation of morphological defects of the cerebellum.	P2-2-22 培養神経細胞におけるNMDA誘発性ミトコンドリア内Ca2+濃度上昇に対するアルコールの抑制作用 Suppression by alcohol of NMDA-induced mitochondrial Ca2+ increase in cultured neurons ○宝田剛志¹, 福森良¹, 藤川晃一¹, 米田幸雄¹ ○Takeshi Takarada¹, Ryo Fukumori¹, Koichi Fujikawa¹, Yukio Yoneda¹ 金沢大学医薬保健研究域薬学系¹ Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School¹ We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity mediated by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to increased mitochondrial Ca2+ levels in HEK293 cells with acquired NMDAR channels. In this study, we evaluated pharmacological profiles of the central depressant alcohol on the NMDA-induced increase in mitochondrial Ca2+ levels in relation to cell death in cultured murine neocortical neurons. Exposure to NMDA led to a significant increase in mitochondrial Ca2+ levels determined by Rhod-2 fluorescence image, while further addition of alcohol inhibited the NMDA-induced increase at concentrations over 50 mM. Lentiviral overexpression of UCP2 accelerated the NMDA-induced increase in Rhod-2 fluorescence, without affecting Fluo-3 fluorescence for intracellular free Ca2+ levels. In these neurons with overexpressed UCP2, alcohol was more effective in inhibiting the NMDA-induced increase in Rhod-2 fluorescence, without altering the increased Fluo-3 fluorescence. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by alcohol in cultured neurons exposed to glutamate. In HEK293 cells with acquired NMDAR channels, moreover, efficient inhibition was similarly induced by alcohol on the NMDA-induced increase in mitochondrial, but not intracellular, Ca2+ levels with concomitant protection of cell death after UCP2 overexpression. These results suggest that alcohol could inhibit mitochondrial Ca2+ incorporation and subsequent cell death after NMDAR activation in a UCP2-dependent manner in cultured neurons.
P2-2-23 ドパミンによるエンドサイト―シスはゴルジ体において細胞内D2L受容体を活性化する Dopamine-induced endocytosis activates the intracellular D2L receptor at golgi apparatus ○塩田倫史¹, 笹原正清², 森寿³, 福永浩司¹ ○Norifumi Shioda¹, Masakiyo Sasahara², Hisashi Mori³, Kohji Fukunaga¹ 東北大院・薬・薬理学¹, 富山大・医・病理学², 富山大・医・分子神経³ Dept. Pharmacol., Grad. Sch. Pharm. Sci., Tohoku Univ, Sendai¹, Dept. Pathol, Grad. Sch. Med and Pharm. Sci., Univ of Toyama, Toyama², Dept. Mol Neurosci, Grad. Sch. Med and Pharm. Sci., Univ of Toyama, Toyama³ The dopamine D2 receptor (D2R) is target for antipsychotic drugs and its abnormality is associated with several neuropsychiatric disorders. It exists as two alternatively spliced isoforms, termed D2LR and D2SR. In D2LR, but not D2SR, its activation involves in the transactivation of receptor tyrosine kinase pathways. We previously demonstrated that D2SR is predominantly expressed in the plasma membrane, whereas D2LR is mostly retained in the Golgi apparatus. However, the molecular mechanisms and physiological roles of the intracellular D2LR remain unclear. When D2LR or D2SR is transfected with PDGFRβ in HEK293T cells, D2SR stimulation with dopamine produced a transient increase in ERK phosphorylation, whereas D2LR stimulation with dopamine caused significant and persistent ERK activation. Interestingly, D2R protein synthesis was significantly enhanced by dopamine within 30min in D2LR transfected cells with PDGFRβ. The persistent kinase activation and protein synthesis were completely blocked by pertussis-toxin, PDGFR inhibitor tyrphostin A9 and dynamin inhibitor Dynasore, suggesting the dopamine-induced receptor internalization was involved in the D2LR signaling. In addition, we analyzed interactions of D2LR with Gαi isoforms using BRET method. Dopamine caused a significant BRET change in cells expressing D2LR-RLuc8 with Gαi3-mVenus compared with the effect of Gαi1 and Gαi2. Gαi3 was localized at the plasma membrane and Golgi apparatus, in where colocalized with D2LR. Taken together, the intracellular dopamine D2LR is predominantly associated with Gαi3, and the dopamine-induced internalization of D2LR leads to making D2LR-PDGFRβ complex in the golgi apparatus, thereby eliciting persist activation of ERK pathways to enhance D2R protein synthesis.	P2-2-24 アデノシン受容体の活性型構造に対するGi1蛋白質の安定化作用についてのFRET解析 FRET analyses of the stabilizing effect of Gi1 protein on the agonist-induced activated conformation of the A1aR ○立山充博¹, 久保義弘¹ ○Michihiro Tateyama¹, Yoshihiro Kubo¹ 生理学研究所神経機能素子研究部門¹ Div. Biophysics & Neurobiology. NIPS¹ The neurotransmitters, neuropeptides and hormones modulate the neuronal excitability and transmission through binding to G protein coupled receptor (GPCR). The agonist-binding induces conformational changes in GPCR, resulting in the G protein coupling and initiation of the downstream signaling. Binding studies have demonstrated that the binding of G protein stabilizes the agonist-binding to GPCR. The stabilizing effect of G protein were shown in the Gq-coupled receptors as the Gq-induced enhancements of the agonist-induced decreases in FRET between the fluorescent proteins tethered at the intracellular third loop (i3) and the C-tail. Here we aimed at examining the effect of Gi1 on the active state of the Gi/o-coupled adenosine receptor type 1 receptor (A1aR) by using the FRET technique. First, YFP was inserted at the i3 of the A1aR with or without junctional linkers. Both of the YFP-fused receptors with or without linkers elicited the current amplitude through the G protein coupled inwardly rectifying K channels upon the agonist applications, indicating that they were able to interact with the Gi1 protein. Then CFP was tethered at the C-terminus of the YFP-fused receptors and the agonist-induced changes in FRET were monitored, to investigate the effects of the Gi1 protein on the active state of the receptors. Application of the agonist induced FRET decreases, reflecting the agonist-induced conformational changes of the receptors. Interestingly, co-expression of the Gi1 protein clearly enhanced the agonist-induced FRET decreases of the A1aR constructs with or without linkers. The enhanced FRET decreases were also observed when the A1aR constructs were co-expressed with the chimeric Gqi5 protein which is reported to interact with Gi-coupled receptor and to stimulate Gq signaling pathway. These results indicate that the binding of Gi protein stabilizes the activated conformation of the Gi-coupled A1aR.
P2-2-25 摂食受容体MCHR1の1次繊毛局在機構 Characterization of ciliary targeting sequence of rat melanin-concentrating hormone receptor 1 ○斎藤祐見子¹, 永田麻実¹, 濱本明恵¹, 堀川学², 吉村健太郎³, 竹田扇³ ○Yumiko Saito¹, Asami Nagata¹, Akie Hamamoto¹, Manabu Horikawa², Kentarou Yoshimura³, Sen Takeda³ 広島大学大学院　総合科学研究科　生命科学領域¹, サントリー生命科学財団　生物有機科学研究所², 山梨大学大学院医学工学総合研究部解剖学講座細胞生物学³ Grad Sch Integrated Arts and Sci, Hiroshima Univ, Hiroshima¹, Bioorganic Research Institute, Suntory Foundation for Life Sciences, Osaka², Dept Anatomy and Cell Biol, Interdisc Grad Sch Med and Engineer, Univ of Yamanashi, Yamanashi³ Mammalian melanin-concentrating hormone (MCH), a cyclic nonadecapeptide produced by neurons of the lateral hypothalamus, is involved in the regulation of feeding, energy homeostasis and emotional processing in rodents. MCH acts via two G-protein-coupled receptors (GPCRs), MCHR1 and MCHR2. Recently, MCHR1 expression was discovered in neuronal immotile primary cilia of the central nervous system in mice. The cilium has an important chemosensory function in many types of cell and ciliary dysfunction is illustrated by the gamut of diseases (ciliopathies) such as polycystic kidney disease, retinal dystrophy, and obesity. The functions of cilia are defined by the signaling proteins expressed on their membranes, so specific targeting of these membrane receptors and channels is essential for ciliary function. Although these sequences have been predicted in the cytoplasmic third loop and/or C-terminus of GPCRs, little is known about the characteristics of MCHR1. We therefore examined the molecular mechanisms of MCHR1 targeting by transiently expressing a series of MCHR1 mutants into ciliated hRPE1 cells and evaluated the effects of these mutations on the ciliary localization of the heterologous receptor. Screening from over 20 substitution mutants, we conformed that the point mutation of third residue Ala to Gly (A242G) in the i3 loop significantly and selectively affected the ciliary localization of MCHR1. In contrast, the C-terminal tail of MCHR1 did not affect the ciliary localization, unlike that in rhodopsin. This study of MCHR1 as a ciliary GPCR provides a potential molecular mechanistic link between defective cilia ciliary and obesity.	P2-2-26 The role of Capsaicin receptors into Locus Coeruleus nucleus in morphine analgesia in rats affected with neuropathic diabetes ○Mohammad Zarei¹, Azam Naderifarjam², Abdolrahman Sarihi¹, Masoomeh Taheri², Shirafkan Talieh², Esmaili Rasul¹, Komaki Alireza¹, Shahidi Siamak¹ Department of Physiology & Neurophysiology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran¹, Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Hamadan Branch, Hamadan, Iran² Morphine inhibits activity of Locus Coeruleus (LC) nucleus neurons which are involved in pain modulation. Capsaicin is an agonist for vanilloid type 1 receptors (TRPV1) which can centrally induce analgesic effects via descending analgesic system into midbrain. Peripheral neuropathy may happen in all diabetic patients' types which have hyperalgesia. This study was performed in male rats affected by peripheral neuropathy (PN) using tail flick (TF) reflex to investigate the effects of intra-PAG injection of Capsaicin (10 nmol) on morphine analgesic test. To induce PN, Streptozotocin was intraperitonealy injected as single dose (55 mg/kg). PN induction was confirmed by weight loose, plasma glucose elevation and allodynia to tactile and thermal stimuli. Activation of TRPV1 receptors into LC, 15, 30 and 45 min after injection increased morphine analgesia in rats affected with PN, but had no effect at 5 and 60 min after injection. In normal rats Capsaicin increased tail flick latency 15, 30, 45 and 60 min after injection during morphine analgesia test. The results of present study can be use in pharmacological therapy for patients affected with peripheral neuropathy by activation of Capsaiciniod system activation.
P2-2-27 バレル皮質錐体細胞においてPRIP遺伝子欠損によりシナプス外GABAA受容体の発現が増加する Deletion of PRIP1/2 causes an increased expression of extrasynaptic GABA_A receptors in pyramidal cells of the barrel cortex ○豊田博紀¹, 齋藤充¹, 佐藤元¹, 兼松隆², 平田雅人², 姜英男¹ ○Hiroki Toyoda¹, Mitsuru Saito¹, Hajime Sato¹, Takashi Kanematsu², Masato Hirata², Youngnam Kang¹ 大阪大院・歯・高次脳口腔機能¹, 九州大学院・歯・口腔細胞工学² Dept Oral Physiol, Osaka Univ Grad Sch Dent, Suita, Japan¹, Lab Mol Cell Biochem, Facul Dent, Kyushu Univ, Fukuoka, Japan² Phospholipase C-related inactive proteins (PRIP1/2) are involved in trafficking of GABA_A receptors (GABA_ARs) containing γ2 subunit to synapses. We studied kinetic properties of IPSCs and GAB_AA currents evoked by a GABA puff in layer 2/3 pyramidal cells (PCs) of the barrel cortex in PRIP1/2 double knockout (PRIP-DKO) mice to elucidate functional expression of GABA_ARs. PRIP deletion resulted in acceleration of desensitization and subsequent resensitization of GABA_ARs at the offset of a GABA puff, yielding a hump-like tail current (tail-I). These kinetic changes were abolished by Ca²⁺ chelators and calcineurin inhibitor. Pentobarbital enhanced the hump-like tail-I in PRIP-DKO PCs while it prolonged the decay of GABA_A currents in wild-type (WT) PCs, generating larger net charges in PRIP-DKO PCs compared to WT. Because BAPTA can prevent only extrasynaptic GABA_ARs from being modulated by Ca²⁺, these data suggest an increased expression of extrasynaptic GABA_ARs paradoxically enhances phasic inhibition in PRIP-DKO PCs.	P2-2-28 ニューロピリン-1（軸索忌避ガイダンス分子受容体）は、血管平滑筋細胞と混合培養すると神経系細胞のラフト領域に局在する Neuropilin-1, a repulsive axon guidance molecule receptor, is localized on raft domains in neuronal cells cocultured with vascular smooth muscle cells ○吉村亮一¹, 神野美和¹, 西田倫希², 遠藤泰久¹ ○Ryoichi Yoshimura¹, Miwa Jinno¹, Tomoki Nishida², Yasuhisa Endo¹ 京都工繊大院・工芸科・応用生物・細胞機能¹, 大阪大学超高圧電子顕微鏡センター² Div Appl Biol, Kyoto Inst Tech Grad Sch Sci Tech, Kyoto, JAPAN¹, Res Ctr UHVEM, Osaka Univ, Ibaraki, Osaka, JAPAN² Axon guidance is mediated by extracellular attractive or repulsive cues through the membrane microdomains, so-called lipid rafts, on the growth cones; that are enriched with cholesterol and sphingolipids, and resistant to cold detergent extraction. The repulsion of neurites may involve Semaphorin 3A and Neuropilin-1 (Npn-1), one of its receptors; therefore, the raft manipulation modulates Semaphorin 3A-induced growth cone repulsion, inhibition, and collapse (Guirland et al., 2004). Previously, we found that neuronal cells NG108-15 (NG) extended their neurites when the cells were cocultured with vascular smooth muscle cells SM-3, but the extended neurites were repelled by the targets. Here, we examined the localization of Npn-1 on the cell membrane including growth cones of the NG cultured solely or cocultured with SM-3, to clarify the mechanism of repulsions of neurites from target cells. Cultures were treated with cold TritonX-100 (TX100) or methyl-beta-cyclodextrin (MCD, a water-soluble cyclic oligomer that extracts cholesterol from the plasma membrane) (Simons and Toomre, 2000). In the monocultured NG, intensity of Npn-1 immuneoreactivity at the membrane decreased after the TX100 treatment, but not MCD. Whereas, in NG cocultured with SM-3, the Npn-1 immunoreactivity level at the membrane did not significantly change after the TX100 treatment, but lowered by the MCD treatment. The results suggest that most Npn-1 in monocultured NG cell surface exists on the membrane except the raft domains and, in the NG cocultured with SM-3, a part of Npn-1 shift the localization onto rafts. Thus, the manipulation of membrane integrity or individual lipid raft components could shed light on the axon guidance receptor dynamics, such as sorting and endocytosis, in the neuronal cells interacting with target cells.
P2-2-29 シナプトタグミン3：ポストシナプスタンパク質の表面発現制御因子 Synaptotagmin3: Potential molecule to regulate surface expression level of synaptic proteins ○篠田陽^1,2, , 阿部麗実¹古市貞一^1,2 ○Yo Shinoda^1,2, Saheeb Ahmed³, Reimi Abe¹, Ankit Awasthi³, Teiichi Furuichi^1,2, Camin Dean³ 東京理科大・理工・応用生物科学¹ Dept. of Appl. Biol. Sci., Fac. of Sci. and Technol., Tokyo Univ. of Sci., Chiba, Japan¹, JST/CREST, Saitama, Japan², Eur. Neurosci. Inst., Goettingen, Germany³ Synaptotagmin3 (SYT3) is one of the synaptotagmin family proteins that are known to be involved in calcium dependent exo- and endocytosis of vesicles. Although SYT1, the most investigated SYT family protein, is well understood as a regulating protein which is associated with synaptic vesicle exocytosis by calcium dependent manner, the function and localization of SYT3 is still unknown. Here we show that the localization and function of SYT3 in hippocampal neuron, which is potential regulative molecule to regulate surface expression level of postsynaptic proteins. Using immunocytochemistry and western blotting, we revealed that SYT3 is localized in postsynaptic site. To study the physiological movement, translocation and their kinetics of SYT3, we made the fused SYT3 protein construct which has pH sensitive green fluorescent protein (pHluorin) in its N-terminal (pHluorin-SYT3). Time-lapse imaging of pHluorin-SYT3 transfected into rat hippocampal dissociated culture showed that 1) SYT3 is highly located in postsynaptic plasma membrane, 2) strong depolarization of membrane potential induces SYT3 endocytosis in calcium dependent manner, 3) SYT3 switches its exo- and endocytosys that depend on the level of neural activity (the amount of calcium influx). These data suggest that SYT3 has a potential to regulate surface expression level of postsynaptic proteins by neural activity level. We will show the potential candidate proteins regulated by SYT3.	P2-2-30 マウス脳における自閉性障害に関係するCadm1とCntnap2の分子複合体の解析 Molecular complexes of cell adhesion molecules Cadm1 and Cntnap2 related to autism spectrum disorder in the mouse brain ○田辺裕子¹, 藤田恵理子^1,2, 桃井隆¹ ○Yuko Tanabe¹, Eriko Fujita^1,2, Takashi Momoi¹ 国際医療福祉大学　基礎医学研究センター¹, 自治医科大学　小児科² Center for Medical Science, International University of Health and Welfare, Kitakanamaru, Otawara, Tochigi, Japan¹, Depatment of Pediatrics, Jichi Medical University, Yakushiji, Shimotsuke, Tochigi, Japan² RA175/SynCAM1/Cadm1 (Cadm1), a member of the immunoglobulin superfamily. It promotes the formation of presynaptic terminals and induces functional synapses in the central nervous system. Mutations in CADM1 and another synaptic adhesion molecule, Cntnap2, are associated with autism spectrum disorder (ASD). Cadm1-knock out (KO) mice exhibits some ASD-like symptoms, including impaired ultrasonic vocalization. Cadm1 has an extracellular region that includes three immunoglobulin domains, a C-terminal transmembrane domain, and a cytoplasmic region with an intracellular type II PDZ-binding domain. The C-terminal regions of Cadm1 interact with CASK at the pre-synapse. But little is known about Cadm1 and Cntnap2 molecular complex at the post-synapse.In this study, we examined the molecular complexes of Cadm1 and Cntnap2 in the mouse brain by GST pull-down assays. The C-terminal peptides of Cadm1 and Cntnap2 associated with Mupp1, which has 13 PDZ domains, but not with PSD-95. Cadm1 and Cntnap2 mainly associated with PDZ2 and PDZ3 of Mupp1, respectively. Mupp1 interacts with SynGAP and GABBR2 at PDZ13 and 5-HTR2C at PDZ10. Cadm1 and Cntnap2 may form Mupp1-receptor complexes at the post-synaptic membrane.
P2-2-31 新規serotonin transporter制御因子としてのN-ethylmaleimide-sensitive factor Modulation of the serotonin transporter by interaction with N-ethylmaleimide-sensitive factor ○岩田圭子¹, 松崎秀夫^1,2, 立花太郎³, 片山泰一⁴, 森則夫^2,5 ○Keiko Iwata¹, Hideo Matsuzaki^1,2, Taro Tachibana³, Taiichi Katayama⁴, Norio Mori^2,5 福井大学　子どものこころの発達研究センター¹, 浜松医科大学子どものこころの発達研究センター², 大阪市立大学大学院工学研究科細胞工学研究室³, 連合小児発達研究科大阪校分子生物遺伝学研究領域⁴, 浜松医科大学精神医学講座⁵ RCCMD, University of Fukui, Fukui, Japan¹, RCCMD, Hamamatsu University School of Medicine, Hamamatsu, Japan², Department of Bioengineering, Graduate School of Engineering, Osaka City University, Osaka, Japan³, UGSCD, Division of Developmental Neuroscience, Suita, Japan⁴, Department of Psychiatry, Hamamatsu University School of Medicine, Hamamatsu, Japan⁵ Many studies on the pathophysiologic mechanism of autism have focused on the serotonergic system. Prior studies consistently found elevated serotonin levels in the whole blood cells and platelets of patients with autism. Importantly, our colleagues reported that serotonin transporter (SERT) and its radioligand binding was significantly lower throughout the brain in autistic individuals compared with controls (Nakamura et al., 2010). These clinical evidences suggest that SERT function may be disturbed in autism. The functions of neurotransmitter transporters are influenced both by the number of transporter molecules present at the cell surface and by the intrinsic activity of individual transporter molecules. It is known that the number and activity are regulated by proteins which interact with transporter. Several SERT-interacting proteins, such as SCAMP2, nNOS, Hic-5 and syntaxin-1A, have been identified mainly by yeast two-hybrid screens. However, it is little known whether some of these proteins bind SERT in the mammalian brain. Thus, in this study, we sought to identify novel SERT-interacting proteins. We identified the N-ethylmaleimide-sensitive factor (NSF) as a candidate SERT-interacting protein by pull-down system using mouse brain. Using biochemical, cellular imaging and knockdown approaches, we determined that NSF interacts with SERT and was required for SERT membrane trafficking and its function. In addition, NSF co-localized with SERT in mouse raphe neurons. It has been reported that NSF interacts with some neurotransmitter receptors, such as glutamate, adrenaline, and dopamine receptors, and regulate membrane trafficking of these receptors. Since it has been reported that many of these receptors are abnormal in autism, it is possible that NSF plays a key role in the pathophysiology of the disorder.	P2-2-32 Withdrawn
P2-2-33 Atp1a3^+/-マウス小脳皮質において登上線維伝達物質のシナプス外拡散は強く抑制されている Suppressed extrasynaptic diffusion of climbing fiber neurotransmitter in the cerebellar cortex of the Atp1a3-deficient heterozygous mice ○佐竹伸一郎^1,2, 池田啓子³, 川上潔⁴, 井本敬二^1,2 ○Shin'Ichiro Satake^1,2, Keiko Ikeda³, Kiyoshi Kawakami⁴, Keiji Imoto^1,2 生理研・生体情報¹, 総研大・生命科学², 兵庫医大・生物³, 自治医大・分子病態治療研究センター⁴ National Institute for Physiological Sciences (NIPS), Okazaki, Japan¹, The Graduate University for Advanced Studies (SOKENDAI), Okazaki, Japan², Hyogo College of Medicine, Nishinomiya, Japan³, Jichi Medical University, Shimotsuke, Japan⁴ Neurotransmitters diffuse out of the synaptic cleft and act on adjacent synapses to exert concerted control of the synaptic strength within neural pathways that converge on single target neurons. Excitatory amino acid transporters (EAATs) underlie the uptake mechanisms surrounding central excitatory synapses to control clearance of glutamate released during neurotransmission. In the cerebellar cortex, two distinct glutamate transporters, GLAST and EAAT4, are expressed most abundantly with different distribution patterns. GLAST is expressed in Bergmann glial processes surrounding excitatory synapses on dendritic spines of Purkinje cells (PCs), while EAAT4 is alternatively accumulated on perisynaptic regions of the PC spines. We previously reported that the excitatory neurotransmitter released from climbing fibers (CFs) depresses GABA release at cerebellar basket cell (BC)-PC synapses through its extrasynaptic diffusion and activation of AMPA receptors expressed on BC axon terminals. EAAT4 in PCs plays a critical role in the control of extrasynaptic diffusion of CF transmitter. Because (1) EAATs are Na-dependent proteins that rely on Na and K gradients generated principally by Na,K-ATPase and (2) Atp1a3 (Na,K-ATPase α3 subunit gene) is highly expressed in PCs, we assumed that EAAT4 activity of PCs weakens in Atp1a3-deficient mice, resulting in increases in extrasynaptic diffusion of CF transmitter and CF-induced depression of GABA release at BC-PC synapses. However, contrary to this assumption, the magnitude of CF-induced heterosynaptic depression was significantly smaller in Atp1a3^+/- mice when compared to wild-type littermates. Application of the GLAST-selective blocker PMB-TBOA rescued the suppressed CF-induced depression of the Atp1a3-deficient mice. By contrast, the EAAT4-selective blocker T3MG did not show significant effect. These results suggest that the glial transporter GLAST compensates deficient activity of the neuronal transporter EAAT4 in Atp1a3^+/- mice.	P2-2-34 CNT発現と酸不溶性画分への酸化ストレスによるチミジン取り込み機構 Expression of CNT and mechanism of thymidine incorporation into acid insoluble fraction on oxidative stress DNA injury ○田中康一^1,2,3, 北中順惠², 北中純一², 横山翔¹, 花谷淳弥¹三宅照久¹, 大井春菜¹, 佐藤友昭³, 西川殷維³, 竹村基彦², 馬場明道¹, 西山信好¹ ○Koh-ichi Tanaka^1,2,3, Nobue Kitanaka², Junichi Kitanaka², Shyohn Yokoyama¹, Junya Hanatani¹, Minse Pak¹, Teruhisa Miyake¹, Haruna Oh-i¹, Tomoaki Sato³, Takashige Nishikawa³, Motohiko Takemura², Akemichi Baba¹¹, Nobuyoshi Nishiyama¹ 兵庫医療大・薬・薬理¹, 兵庫医大・薬理², 鹿児島大院・医歯・歯科応用薬理³ Div Pharmacol, Dept Pharm, Sch Pharm, Hyogo Univ Health Sci, Hyogo¹, Dept Pharmacol, Hyogo Col Med, Hyogo², Dept Applied Pharmacol, Kagoshima Univ Grad Sch Med & Dent Sci, Kagoshima³ We have found that cultured astrocytes pretreated with N⁶, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP), a permeable analogue of cAMP, but not astrocytes pretreated without DBcAMP and neurons, have the ability to incorporate thymidine into acid insoluble fraction via equilibrative nucleoside transporter 2 (ENT2) and concentrative nucleoside transporter 3 (CNT3) at an early time for repair on hydrogen peroxide (H₂O₂)-induced DNA injury. We studied expression of CNT and the relation between thymidine incorporation into intracellular spaces (membrane transport) and acid insoluble fraction (DNA repair) on cultured astrocytes pretreated with DBcAMP in the presence and absence of H₂O₂. We concerned that astrocytes express CNT2 and CNT3, but not CNT1, by immunocytochemistry and RT-PCR, and H₂O₂ caused decrease in membrane transport of thymidine from extracellular spaces to intracellular spaces and increase in incorporation of thymidine into acid insoluble fraction to cultured astrocytes pretreated with DBcAMP. These finding indicate that cultured differentiated astrocytes could incorporate thymidine effectively into acid insoluble fraction for repair on H₂O₂-induced DNA injury, although the function of ENT2 and CNT3 might be impaired.
P2-2-35 マウス初代培養大脳皮質アストロサイト及びミクログリアにおける亜鉛取り込み特性の比較 Characterization of zinc uptake by mouse primary cultured cortical astrocytes and microglia ○瀬川将平¹, 辰巳奈穂¹, 西田健太朗¹, 長澤一樹¹ ○Shohei Segawa¹, Nao Tatsumi¹, Kentaro Nishida¹, Kazuki Nagasawa¹ 京都薬大・衛生化学¹ Dept Environ Biochem, Kyoto Pharm Univ, Kyoto¹ [Objective] Zinc is an essential trace element and acts as a neurotransmitter in the brain. Under severe ischemic conditions, zinc is excessively released from synaptic vesicles of glutamatergic neurons into extracellular space, and contributes to brain injury. Previously, we have demonstrated that zinc uptake via ZIP1 is involved in zinc-triggered microglial activation, by which neuronal death is deteriorated. Astrocytes play roles in maintenance of the homeostasis by clean up extracellular neuro- and glio-transmitters. Astrocytes are reported to uptake zinc, but its characteristics have not been clarified yet. In this study, therefore, we examined zinc transport system in mouse primary cultured cortical astrocytes, and compared it with the case of microglia. [Material and Methods] Mouse cultured astrocytes and microglia were prepared from 1-day-old ddY mice. Relative mRNA expression of the ZIP isoforms in astrocytes was determined by real-time quantitative PCR. Zinc uptake was characterized by measuring cellular ⁶⁵Zn uptake. [Results and Discussion] In both astrocytes and microglia, the zinc uptake was time- and concentration-dependent, and was mediated, at least in part, by two saturable systems. There were no differences in the Km values for zinc uptake between astrocytes and microglia, but the cell-to-medium ratios and Vmaxs for zinc uptake were greater in the latters than in formers. Of ZIP isoforms, ZIP1 was found to be abundant one in both type of cells, and nickel, a highly selective substrate/inhibitor of ZIP1, exhibited cis- and trans-inhibitory effects on their zinc uptakes. On the other hand, zinc uptake was not affected by an L-type calcium channel blocker and a glutamate receptor antagonist. These findings indicate that zinc is taken up into astrocytes mainly via ZIP1 as the case of microglia, while the zinc uptake activity in astrocytes is less than that in microglia, and this might be explained by the difference in expression of zinc transporters.	P2-2-36 An antidepressant, imipramine, has both acute inhibitory effect and long-lasting excitatory effect on NMDA receptor ○Nghia Nguyen¹, Takae Hirasawa^1,3, Shuichi Koizumi², Takeo Kubota¹ Dept. Epigenetic medicine, Univ.Yamanashi¹, Dept. Pharmacology, Univ. of Yamanashi², Japan Science and Technology Agency, CREST³ Imipramine, a major anti-depressant, is known to inhibit the re-uptake of serotonin and noradrenalin, which is thought to contribute to the recovery from depression. It has recently been reported that imipramine has a role of inhibition of NMDA receptor activity. However, it is not known whether the inhibitory effect to NMDA receptor is associated with treatment of depression. Therefore, we investigated the acute and long-lasting effects of imipramine on NMDA receptor in primary cortex neurons obtained from mice at embryonic Day 15 (E15) using fluorescence calcium imaging. As a result, the acute (30 sec) imipramine treatment dose-dependently decreased Ca²⁺ influx mediated by NMDA receptors as previously reported. This inhibition was recovered to normal condition after washing out of imipramine. However, the long-lasting treatments of imipramine (24 hrs and 48 hrs) unexpectedly increased Ca²⁺ influx mediated by NMDA receptors. Furthermore, the long-lasting treatment induced the increase of the number of nestin-positive cells, suggesting that imipramine may urge neurogenesis, which possibly contribute to the treatment of depression. In order to clarify the mechanism of neurogenesis induced by long-lasting imipramine treatment, we are currently performing BrdU-cell proliferation assay and epigenetic analyses.

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