Poster Sessions

Poster Sessions 細胞内・細胞間、情報伝達、回路網形成

1P-11 Splicing variation of Long-IRBIT determines the target selectivity of IRBIT family Katsuhiro Kawaai¹，Hideaki Ando¹，Nobuhiko Satoh²，Hideomi Yamada²，Naoko Ogawa¹，Matsumi Hirose¹，Akihiro Mizutani³，Benjamin Bonneau¹，George Seki²，Katsuhiko Mikoshiba¹ ¹Lab. Develop. Neurobiol., Brain Science Institute, RIKEN，²Dept Internal Medicine, Univ of Tokyo，³Dept Pharmacotherapeutics, Showa Pharmaceutical Univ IRBIT (inositol 1,4,5-trisphosphate receptor (IP₃R) binding protein released with inositol 1,4,5-trisphosphate (IP₃)) is a multifunctional protein that regulates several target molecules such as ion channels, transporters, polyadenylation complex, and kinases. Through its interaction with multiple targets, IRBIT contributes to calcium signaling, electrolyte transport, mRNA processing, cell cycle, apoptosis and neuronal function. However, the regulatory mechanism of IRBIT binding to particular targets is poorly understood. Long-IRBIT is an IRBIT homolog with high homology to IRBIT except for a unique N-terminal appendage. Long-IRBIT splice variants have different N-terminal sequences and a common C-terminal region, which is involved in multimerization of IRBIT and Long-IRBIT. In this study, we characterized IRBIT and Long-IRBIT splice variants (IRBIT family). We determined that the IRBIT family exhibits different mRNA expression patterns in various tissues. The IRBIT family formed homo- and hetero-multimers. In addition, N-terminal splicing of Long-IRBIT changed the protein stability and selectivity to target molecules. These results suggest that N-terminal diversity of the IRBIT family and various combinations of multimer formation contribute to the functional diversity of the IRBIT family.		1P-12 Npas4 mediates pentylenetetrazole-induced Homer1a expression in the hippocampus Taku Nagai¹，Wei Shan¹，Motoki Tanaka²，Toshitaka Nabeshima^3,4，Masahiro Shikabe²，Kiyofumi Yamada¹ ¹Dept. Neuropsychopharmacol. Hosp. Pharm., Nagoya Univ. Grad. Sch. Med.，²Mechanobiol. Lab., Nagoya Univ. Grad. Sch. Med.，³Adv. Diag. Sys. Res. Lab., Grad. Sch. Health Sci., Fujita Health Univ.，⁴Aino Univ. Epilepsy is a common and a refractory neurological disorder. The worldwide prevalence is approximately 0.5-1%. Previous studies indicate multiple factors are involved in epileptogenesis by affecting the balance between excitatory and inhibitory, leading to neuronal hyperexcitability and recurrent seizures. Recent findings suggest a role for Npas4, an activity-dependent neuron-specific transcription factor, in the epileptogenesis, but the underlying mechanism that Npas4 regulates intrinsic homeostatic mechanisms remains unknown. Here, we propose that limbic seizure activity up-regulates Npas4-homer1a signaling in the hippocampus which suspects epileptogenesis in mice. C57BL/6 mice were repeatedly administered the sub-convulsive dose of PTZ (25 mg/kg) every 48 h to achieve the fully kindled state. Totally 16 times injections with PTZ were required for the kindling development, whereas no marked change in seizure score was observed in repeated saline and repeated saline plus single treatment with PTZ at the last time of injections. The expression level of Npas4 mRNA was significantly increased after the repeated-PTZ treatment compared with either saline-treated or single PTZ-treated mice A single treatment with PTZ had no effect on the Npas4 mRNA level. When PTZ-induced kindling was compared between wild-type and Npas4 KO mice, Npas4 KO mice developed kindling more rapidly than wild-type littermates. COS7 cells transfected with Npas4 increased the relative luciferase activity of the Homer1a promoter fragments. PTZ-stimulated Homer1a mRNA and protein induction were attenuated in the hippocampus of Npas4 KO mice. These results indicate that Npas4 promotes Homer1a expression in the hippocampus after the convulsive seizure.

1P-13 Phosphorylation of SNAP-25 induced by anesthesia in mice brain. Saori Yamamori¹，Tsukiko Sugaya¹，Masakazu Kataoka² ¹Dept Biochem. Sch med, Kitasato Univ，²Dept Envir Sci and Tech. Engineering, Shinshu Univ Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein, plays an essential role in neurotransmitter release by exocytosis. Protein kinase C phosphorylates SNAP-25 at Ser187 and enhances neurotransmitter release from PC12 cells. Recent study revealed that the phosphorylation of SNAP-25 induced in mouse brain following restraint stress in cold water or in rat brain by kainite administration. However, the mechanisms and physiological roles of the SNAP-25 phosphorylation in brain circuits and higher function remain unknown. Anesthetics hyperpolarize neurons by increasing inhibition or decreasing excitation. Therefore, we investigate whether anesthesia affects the phosphorylation of SNAP-25. By using immunohistochemistry and western blotting, we found isoflurane anesthesia (4% induction, 1.5% maintenance; 5, 30 min) induced the SNAP-25 phosphorylation in part of the mice brain (including the hippocampus, septal, corpus callosum, cortex, etc.) similar to that produced by restraint stress in cold water, in a time-dependent manner. When the mice allow to recover from isoflurane anesthesia, mice awoke in 4 min, and the SNAP-25 phosphorylation immediately decreased. It is known that isoflurane potentiates many receptors (GABA_A, glycine, 5-HT₃ and kainite) and channels, and inhibits some receptors (AMPA and nicotinic acetylcholine) and channels. Next, we also investigated the effects of a combination anesthetic (0.75 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol), that enhances the effect of the neurotransmitter GABA on the GABA_A receptors, and that potentiates alpha2-adrenoreceptors and cannabinoid receptors. Consequently we also found the combination anesthetic induced the SNAP-25 phosphorylation in mice brain.		1P-14 Physiological Analysis of Lipid Raft Molecules in the Brain Norihiro Kotani¹，Takanari Nakano¹，Yui Ida¹，Rina Ito¹，Miki Hashizume¹，Arisa Yamaguchi²，Makoto Seo³，Tomoyuki Araki¹，Yasutsugu Hojo¹，Koichi Honke²，Takayuki Murakoshi¹ ¹Dept Biochem. Saitama Med. Uni.，²Dept Biochem. Kochi Uni. Med. School，³Facul Pharmacy and Pharmaceutical Sci., Fukuyama Univ. Due to the nonphysiological conditions used in typical lipid raft analyses, their results cannot be utilized directly in the common neuroscience fields. Hence the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the “Enzyme-Mediated Activation of Radical Sources (EMARS)” method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, which recognizes ganglioside GM1 as a lipid raft-resident glycolipid. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called “EMARS products” distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several GM1-enriched lipid raft molecules were confirmed by the EMARS method in living hippocampal slices.

1P-15 Involvement of the CaM kinase family in signal transduction that stimulates the tyrosine kinase pathway in response to gonadotropin-releasing hormone Hideyuki Yamamoto，Sayomi Higa-Nakamine，Shiho Okitsu，Hidetsugu Torihara Dept Biochem. Grad Sch Med, Ryukyu Univ Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons) and stimulates GnRH receptors belonging to the G-protein-coupled receptors in anterior pituitary gonadotrophs. GnRH neurons also have GnRH receptors, and the stimulation of the receptors regulates the functions of GnRH neurons. In the previous study, we found that CaM kinase IIδ2 was involved in GnRH-induced ERK activation in cultured GnRH neurons (GT1-7 cells)¹⁾. Recently, we found that protein kinase D (PKD), belonging to the CaM kinase family, is activated by GnRH receptor stimulation²⁾. Our study suggested that PKD was activated by novel PKC isoforms and involved in ERK activation. It has been reported that proline-rich tyrosine kinase 2 (PYK2) was activated by activation of PKC, as well as by the increase in the intracellular Ca²⁺. It was also reported that PYK2 was involved in GnRH-induced ERK activation in GT1-7 cells through activation of the ErbB family. In the present study, we examined the possibility that CaM kinase IIδ2 and PKD activated PYK2. 1) Fyn existed in the activated form in GT1-7 cells, and dasatinib, a Src family inhibitor, completely inhibited GnRH-induced PYK2 activation. 2) Among PKD isoforms, PKD1 was expressed in GT1-7 cells. 3) A PKD inhibitor and knockdown of PKD1 inhibited GnRH-induced PYK2 activation, while they had no effects on Fyn activation. 4) Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited GnRH-induced PYK2 activation. These results strongly suggested that PKD1 and CaM kinase IIδ2 were involved in Fyn-induced PYK2 activation. PYK2 may activate the ErbB family, resulting in the activation of the ERK pathway. 1) Arch. Biochem. Biophys. 2007, 234-241. 2) J. Biol. Chem. 290, 25974-25985.		1P-16 The Role of Nck as a downstream effector of the Reelin signaling Kanehiro Hayashi，Seika Inoue，Ken-ichiro Kubo，Kazunori Nakajima Dept Anatomy, Keio University School of Medicine Mammalian neocortex has a 6-layered structure, which is essential for higher brain functions. The neurons in the layers are aligned in a birth-date dependent "inside-out" manner. A glycoprotein, Reelin is secreted from Cajal-Retzius neurons in the marginal zone of the cortex during development. Reelin deficient mice, reeler, exhibit a roughly inverted laminar structure in the cortex, indicating Reelin is indispensable for the formation of the cortical architecture. Although Reelin transduces the signal to the several downstream molecules such as phosphatidylinositol 3-kinase, Crk family, Lis1, SOCS family, the cellular and molecular mechanisms of Reelin function on the layer formation of the mammalian neocortex are not yet fully understood. In this study, we investigated the role of Nck as a downstream molecule of the Reelin signaling. Nck directly interacts with Dab1, which is a key downstream molecule of the Reelin signaling. In the migrating neuron, Nck is localized at the base of the leading process when a neuron reachs the primitive cortical zone beneath the marginal zone. Knockdown of Nck in migrating neurons of the mouse embryonic neocortex by the in utero electroporation technique caused delayed migration of neurons and poor dendritic arborization in the migrated neurons. Furthermore, introduction of a Nck knockdown vector with a Reelin-expressing vector resulted in abnormality of the neuronal aggregation caused by ectopic Reelin expression. These results indicate multiple roles of a Reelin-Nck signaling during mouse development.

1P-17 The importance of ion dynamics for low temperature-induced tyrosine phosphorylation of SIRPα in neurons Hiroshi Ohnishi¹，Shinya Kusakari¹，Miho Hashimoto¹，Kaori Imai¹，Eriko Urano¹，Takashi Matozaki² ¹Dept Lab Sciences. Grad Sch Health Sci, Gunma Univ，²Dept Biochem Mol Biol. Grad Sch Med, Kobe Univ Signal regulatory proteinα (SIRPα) is a neuronal membrane protein that has tyrosine phosphorylation sites in the cytoplasmic region. Tyrosine phosphorylated SIRPα binds and activates protein tyrosine phosphatase Shp2. Previously, we have shown that SIRPα undergoes tyrosine phosphorylation in the brain of mice in response to forced swim stress in cold water, and this signaling mechanism is implicated in regulation of immobility of mice during the swimming. The forced swim-induced tyrosine phosphorylation of SIRPα strongly depends on lowered body temperature in cold water, and tyrosine phosphorylation of SIRPα was also increased in cultured neurons by lowering the temperature of culture medium. Here we found that Na⁺ or Cl^- depletion also induced tyrosine phosphorylation of SIRPα in cultured neurons. Cl^- channel blockers suppressed the effect of Na⁺ or Cl^- depletion. Furthermore, these blockers also suppressed low temperature-induced tyrosine phosphorylation of SIRPα in neurons. Neuronal ion dynamics is thus an important factor for low temperature-induced tyrosine phosphorylation of SIRPα that contribute to the control of brain functions under hypothermic conditions.		1P-18 Dab1 haploinsufficiency causes reduction of layer I thickness and splitting of CA1 pyramidal cell layer Takao Honda，Kazunori Nakajima Dept of Anat. Keio Univ Sch of Med Reelin signaling pathway is essential for the formation of the cerebral cortex, hippocampus, and other regions of the mammalian brain. Reelin is a large secreted protein mainly produced from Cajal-Retzius cells in the marginal zone. Excitatory radially migrating neurons express Reelin receptors ApoER2 and/or VLDLR, and the signal is transmitted to the cytoplasmic adaptor protein Dab1. Although Dab1 has been thought to be a cytoplasmic protein, we have shown that Dab1 contains two nuclear localization and export signals (NLSs/NESs) and shuttles between cytoplasm and nucleus. Overexpression study showed that although wild-type Dab1 caused migration defect, Dab1 NESs mutant, which is mainly localized in nucleus, did not cause severe migration defect. In addition, migration rescue experiment of dab1 mutant yotari mouse using wild-type Dab1 and Dab1 NLS1 mutant resulted in more superficial positioning of the Dab1 NLS1 mutant-transfected cells compared with wild-type Dab1-expressing cells. These results raised a possibility that the level of the cytoplasmic Dab1 protein might be important for the function of Dab1 to regulate the positioning of excitatory neurons. To examine the effect of reduced amount of Dab1 proteins on its function, we compared the structures of cerebral cortex and hippocampus between wild type and heterozygous yotari mice. We found that heterozygous yotari mice at P7 had a thinner layer I in the cerebral cortex compared with that of wild-type mice. Furthermore, we found that layer II-IV neurons in yotari heterozygous mice had a tendency to locate more superficially than those in wild-type mice. As to hippocampus, we found that the CA1 pyramidal cell layer is abnormally splitted in heterozygous yotari mice.

1P-19 Netrin-5 is upregulated in the peri-infarct region after middle cerebral artery occlusion. Satoru Yamagishi¹，Mika Takarada²，Masato Sawada³，Kazunobu Sawamoto^3,4，Osamu Hori²，Kohji Sato¹ ¹Dept Anatomy, Hamamatsu Univ Sch Med，²Dept Neuroanatomy, Faculty of Medicine, Kanazawa Univ，³Dept Develop and Regeneration, Nagoya City Univ Sch Med，⁴Division Neural Develop Regeneration, NIPS Mammalian netrin family proteins are involved in targeting of axons, neuronal migration, and angiogenesis and act as repulsive and attractive guidance molecules. Netrin-5 is a new member of the netrin family with homology to the C345C domain of netrin-1. Strong netrin-5 expression was observed in the olfactory bulb (OB), rostral migrate stream (RMS), the subventricular zone (SVZ), and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus, where neurogenesis occurs in the adult brain. In the SVZ and RMS, netrin-5 expression was observed in Mash1-positive transit-amplifying cells and in Doublecortin (DCX)-positive neuroblasts, but not in GFAP-positive astrocytes. In the OB, netrin-5 expression was maintained in neuroblasts, but its level was decreased in NeuN-positive mature neurons. In the hippocampal SGZ, netrin-5 was observed in Mash1-positive cells and in DCX-positive neuroblasts, but not in GFAP-positive astrocytes, suggesting that netrin-5 expression occurs from type 2a to type 3 cells. Next, we generated ischemia mouse model with middle cerebral artery occlusion. After 3 days from the stroke, netrin-5 was highly upregulated in the infarct region. Netrin-5 positive cells were expressing both NG2 and Iba1, suggesting they are BINCS (Brain Iba1+ /NG2+ Cells) derived from bone marrow and beneficial. These data suggest that netrin-5 is involved in neurogenesis and phagocytosis in the peri-infarct region of the cortex.		1P-20 LOTUS, a neural circuit formation factor, promotes neurite outgrowth Yuji Kurihara，Yutaka Kawakami，Kohtaro Takei Mol. Med. Biosci. Lab., Grad. Sch. of Med. Life Sci., Yokohama City Univ. Axons fail to regenerate following damage to the adult central nervous system. This failure is attributed to the binding of axonal growth inhibitors such as Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), B lymphocyte stimulator (BLyS) and chondroitin sulfate proteoglycans (CSPGs), to Nogo receptor-1 (NgR1). We have recently reported that lateral olfactory tract usher substance (LOTUS) binds to NgR1 and suppresses axonal growth inhibition induced by the interaction of these five inhibitors with NgR1. However, another function of LOTUS remains indeterminate. In this study, we found that retinal ganglion cell (RGC) neurons showed drastic neurite outgrowth on LOTUS substrate. The promoting activity of LOTUS on neurite outgrowth was similarly observed in RGC neurons from ngr1-deficient mice. Furthermore, we found that LOTUS bound to retinoic acid (RA)-treated Neuro2A cells and promoted neurite outgrowth in RA-treated Neuro2A cells. On the other hand, we identified neurotrophic tyrosine kinase receptor type 2 (TrkB) as a novel LOTUS binding protein. These findings suggest that LOTUS may promote neurite outgrowth through TrkB and unidentified LOTUS binding protein(s) expressed in RA-treated Neuro2A cells. We are attempting to identify the LOTUS-interacting molecule(s) expressed in RA-treated Neuro2A cells using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis.

1P-21 Super-resolution analysis of the interaction between drebrin and CaMKIIβ in dendritic spines Hiroyuki Yamazaki，Noriko Koganezawa，Tomoaki Shirao Dept. Neurobiol and Behav. Gunma Univ. Sch. Med Drebrin is a major F-actin binding protein in neurons, and is localized in dendritic spines. In this study, we isolated CaMKIIβ as a drebrin-binding protein by yeast two-hybrid screen and investigated drebrin-CaMKIIβ relationship in dendritic spines. CaMKIIβ  is localized in dendritic spines more than in dendritic shaft. However, drebrin knockdown (KD) caused diffuse localization of CaMKIIβ  in dendrites, suggesting that drebrin anchors CaMKIIβ in dendritic spines. To analyze drebrin-dependence of CaMKIIβ stability in dendritic spine more directly, we performed ﬂuorescence recovery after photobleaching (FRAP) experiments on individual dendritic spines. We found that the stable fraction of CaMKIIβ in drebrin-KD neurons was greater than that of control neurons. This result seems to be inconsistent with the results that CaMKIIβ diffused in dendrites of drebrin-KD neurons. We consequently think that drebrin-independent stable pool became dominant in drebrin-KD neurons. To test the hypothesis, we used stochastic optical reconstruction microscopy (STORM) to elucidate the localization of drebrin and CaMKIIβ in a dendritic spine on the nanometer scale. Our super resolution data showed that CaMKIIβ is partially co-localize with drebrin in the inner side of dendritic spines. In addition, the co-localization of CaMKIIβ and drebrin was separated by NMDA receptor activation, suggesting that active form of CaMKIIβ is free from drebrin. Furthermore, FRAP analysis showed that NMDA receptor activation markedly increased the stable fraction of CaMKIIβ. Together, these results suggest that CaMKIIβ is localized in dendritic spines as both drebrin-dependent pool and drebrin-independent more stable pool.