シナプス後性機構 Postsynaptic mechanisms
O1-7-3-1 精神疾患原因遺伝子IL1RAPL1による下流RhoAシグナル系を介したグルタミン酸作動性シナプス制御 IL1RAPL1 regulates the formation and stabilization of glutamatergic synapses of cortical neurons through RhoA signaling pathway ○林崇1, 吉田知之1, 三品昌美1,2 ○Takashi Hayashi1, Tomoyuki Yoshida1, Masayoshi Mishina1,2 東大院・医・薬理・分子神経生物1, 立命館大・総研・脳科学2 Dept Mol Neurobiol and Pharmacol, Grad Sch Med, Univ of Tokyo, Tokyo1, Brain Sci Lab, Res Org Sci and Tech, Ritsumeikan Univ, Shiga.2 Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is associated with X-linked mental retardation (XLMR) and autism spectrum disorder (ASD). We previously found that IL1RAPL1 regulates excitatory synapse formation of cortical neurons. To investigate how IL1RAPL1 controls excitatory synapse formation, we here screened IL1RAPL1-interacting proteins by affinity chromatography and mass spectroscopy. IL1RAPL1 interacted with Mcf2-like (Mcf2l), a Rho guanine nucleotide exchange factor (RhoGEF), through the cytoplasmic Toll/IL-1 receptor (TIR) domain. Knockdown of endogenous Mcf2l and treatment with an inhibitor of ROCK, the downstream kinase of RhoA, suppressed IL1RAPL1-induced excitatory synapse formation of cortical neurons. The AMPA-type glutamate receptor (AMPA receptor) mediates the fast component of excitatory postsynaptic currents. The AMPA receptor consists of four subunits, GluA1-4. Initially, synaptic delivery of GluA1-containing AMPA receptor occurs in activity- or experience-dependent manner, followed by constitutive replacement of synaptic GluA1 by GluA2/3 during excitatory synapse stabilization. We then employed a total internal reflection fluorescence (TIRF) microscopy to directly visualize IL1RAPL1 effects on the AMPA receptor trafficking on excitatory synapses, such as pHluorin-tagged GluA1, GluA2 and GluA3 insertion to surface in cultured cortical neurons. Our live imaging results showed that the expression of IL1RAPL1 affected the turnover of AMPA receptor subunits. Namely, insertion of GluA1-containing AMPA receptors to the cell surface was decreased, whereas that of AMPA receptors composed of GluA2/3 was enhanced. Mcf2l knockdown and ROCK inhibitor treatment diminished the IL1RAPL1-induced changes of AMPA receptor subunit insertions. Our data suggest that Mcf2l-RhoA-ROCK signaling pathway mediates IL1RAPL1-dependent formation and stabilization of glutamatergic synapses of cortical neurons.	O1-7-3-2 樹状突起スパインの形成におけるPACAPの役割 Pituitary adenylate cyclase-activating polypeptide (PACAP) plays significant roles in dendritic spine formation ○勢力薫1, 早田敦子1,2, 尾形勝弥1, 新谷紀人1, 笠井淳司3, 馬場明道4, 橋本均1,2 ○Kaoru Seiriki1, Atsuko Hayata1,2, Katsuya Ogata1, Norihito Shintani1, Atsushi Kasai3, Akemichi Baba4, Hitoshi Hashimoto1,2 大阪大院・薬・神経薬理1, 大阪大院・連合小児・子どものこころセンター2, 大阪大・未来戦略3, 兵庫医療大・薬4 Mol. Neuropharmacol., Osaka Univ., Osaka1, Center Child Mental Dev., Osaka Univ., Osaka2, Institute for Academic Initiatives, Osaka Univ., Osaka3, Pharmaceut. Sci., Univ. of Health Sci., Kobe4 PACAP (pituitary adenylate cyclase-activating polypeptide) exerts multiple activities as a neurotransmitter, neuromodulator, and neurotrophic factor. Previously, we demonstrated that PACAP-deficient (PACAP-/-) mice showed notable psychomotor abnormalities, most of which were reversed by atypical antipsychotics, and that PACAP gene SNPs were associated with schizophrenia. These findings suggest that alterations in PACAP signaling might be involved in the pathogenesis of psychiatric disorders including schizophrenia. However, a pathogenic pathway of PACAP signaling remains unknown. Recent studies implicate dendritic spines as important substrates of pathogenesis in psychiatric disorders. There are genes that are associated with both psychiatric disorders and abnormal spine formation. Mutant mice of these genes sometimes show abnormal behavior and dendritic spine loss. Abnormal spine formation has also been reported in some patients with psychiatric disorders.In this study, we therefore focused on the effect of PACAP on dendritic spine formation as a possible mechanism for the abnormalities in PACAP-/- mice, and showed that 1) the number of dendritic spines were decreased in hippocampal CA1 neurons but not in the cortex in PACAP-/- mice, 2) the number of PSD-95-labeled synaptic puncta was decreased in primary cultured hippocampal neurons prepared from PACAP-/- mice while it was increased by PACAP in the neurons from wild-type mice, and 3) PACAP increased miR-132 expression and decreased mRNA and protein expression levels of p250GAP which is involved in dendritic spine formation and targeted by miR-132. These results indicated that PACAP is critically implicated in spine formation and miR-132 might be involved therein. In summary, it is suggested that dysfunction of PACAP signaling may contribute to the pathogenesis of psychiatric disorders at least partly through abnormal spine formation.
O1-7-3-3 マウス脳におけるグルタミン酸受容体GluD1の細胞選択的およびシナプス選択的発現 Distinct cellular, subcellular, and synaptic expressions of Glutamate receptor GluD1 in adult mouse brain ○今野幸太郎1, 中本千尋2, 松田恵子3, 崎村建司2, 柚崎道介3, 渡辺雅彦1 ○Kohtarou Konno1, Chihiro Nakamoto2, Keiko Matsuda3, kenji Sakimura2, Michisuke Yuzaki3, Masahiko Watanabe1 北海道大学大学院　医学研究科　解剖発生学1, 新潟大学　脳研究所　細胞神経生物学2, 慶應大学　医学部　神経生理学3 Department of Anatomy and Embryology, Hokkaido University Graduate School of Medicine, Sapporo1, Niigata University, Department of Cellular Neurobiology, Brain Research Institute, Niigata2, Keio University, School of Medicine, Department of Physiology, Tokyo3 Of two members of the δ family of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell synapses and regulates their connectively. However, little is known to date as to expression and function of GluD1. To address this issue, we produced riboprobe and antibody specific to GluD1. In immunofluorescence, GluD1 immunoreactivity was distributed widely in the adult mouse brain with higher levels in the neocortex, retrosplenial granular cortex, hippocampal formation, lateral septum, caudate-putamen, central nucleus of the amygdala, bed nucleus of the stria terminalis, and cerebellar molecular layer. In particular, GluD1 was enriched in the stratum lacunosum-moleculare of the hippocampus. Double-labeling fluorescent in situ hybridization addressed that GluD1 mRNA was expressed in distinct neuronal populations, e.g., glutamatergic neurons in the hippocampus and GABAergic neurons in the cerebellar molecular layer. Preembedding and postembedding electron microscopy demonstrated that GluD1 was widely distributed on synaptic and extrasynaptic membranes in hippocampal pyramidal cells, while it was concentrated at parallel fiber synapses on the soma, but not dendrites, of molecular layer interneurons. The distinct subcellular distribution between the hippocampus and cerebellum was consistent with immunoblot data showing less marked condensation in the postsynaptic density fraction in the former. Thus, GluD1 displays distinct cellular and synaptic expressions, as is the case for GluD2. However, the types of neurons and synapses expressing the two GluD members are quite distinctive, suggesting their complementary or distinct roles in synaptic function and regulation.	O1-7-3-4 CaMKK-CaMKIVのシナプスから核へのシグナル伝達の動態解析 Resolving CaMKK-CaMKIV signal transfer from synapse to nucleus ○井上昌俊1, 藤井哉1, 奥野浩行1, 竹本-木村さやか1,2, 尾藤晴彦1,3 ○Inoue Masatoshi1, Hajime Fujii1, Hiroyuki Okuno1, Sayaka Takemoto-Kimura1,2, Haruhiko Bito1,3 東京大院・医・神経生化1 Dept. of Neurochem., Grad. Sch. of Med., Univ. of Tokyo, Tokyo, Japan1, PRESTO-JST, Saitama, Japan2, CREST-JST, Tokyo, Japan3 Synaptic activity-regulated gene expression is critically required for the late phase of synaptic plasticity and for maintenance of long term memory. High-frequency synaptic stimulation can rapidly trigger gene expression through CREB phosphorylation in a CaMKK-CaMKIV cascade-dependent manner. However, the biochemical basis of the spatiotemporal transfer of information from the initial synaptic Ca2+ rises to the nuclear transcriptional activation event is not fully understood. Indeed, it remains a technical challenge to perform live, quantitative and comparative activity measurements of each CREB-stimulating player within the extended dendritic space that links the stimulated synapses and the nucleus. To address this question, we first developed a series of sensitive FRET sensors of Ca2+ signaling (or FRESCA probes) that enabled quantitative imaging of each step of the Ca2+-CaMKK-CaMKIV-pCREB pathway. Second, using FRESCA probes, we found in dissociated hippocampal neuron that several delay time to induce nuclear CREB phosphorylation whereas somatic CaMKK and CaMKIV were rapidly activated after UV glutamate uncaging at soma. Third, UV glutamate uncaging showed that synaptic stimulation within one localized dendritic segment was sufficient to trigger Ca2+ responses that led to CREB phosphorylation, which was induced even when Ca2+ rises were restricted in the dendrite, and did not spread to the nucleus. A minimal CREB stimulation protocol triggered spine and dendritic Ca2+ entries within seconds after the beginning of the stimulation train, while we detected significant latencies in the onset of nuclear CREB phosphorylation, which could be delayed for more than 10 minutes. Furthermore, we confirmed that this CREB phosphorylation pathway depended on CaMK pathway. These results suggest that activated CaMKIV information transfer from the stimulated synapses to the nucleus contributes to determining the rate of CREB phosphorylation.

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