e ポスター

e ポスター 8. シナプス、細胞シグナリング e Poster 8. Synapse, Cell signaling

2020/9/11　14:00～15:00　オンデマンドB-1 P2-15 ASDリスク遺伝子MyosinId mRNAの脳内発現と翻訳産物の樹状突起棘への局在 Localization of Myosin Id, an ASD Risk Gene Product in Dendritic Spines 佐々木哲也¹、鮑培毅¹、武井陽介¹ 1. 国立大学法人筑波大学 Tetsuya Sasaki¹, Peiyi Bao¹, Yosuke Takei¹ 1. University of Tsukuba The majority of excitatory synapses in the mammalian cerebral cortex occur at small protrusions, or spines, on the dendrites. Dendritic spines are capable of changing their shape and size to modulate synaptic transmission. The actin cytoskeleton and a variety of actin-binding proteins play a critical role in the dynamics of dendritic spines. Abnormalities of spine dynamics are implicated in several psychiatric disorders, such as autistic spectrum disorder (ASD), schizophrenia, and Rett's syndrome. Class I myosins are monomeric motor proteins that move along actin filaments using the energy of ATP hydrolysis. Of these class I myosins, myosin Id has been reported to be expressed in not only oligodendrocytes but also neurons, whereas its subcellular localization in neurons remained unknown. The linkage analysis suggests that myosin Id is a potential risk gene for ASD. In this study, we investigated the subcellular localization of myosin Id using EGFP-fusion myosin Id, and determined the domain responsible for it. We found that myosin Id is enriched in the dendritic spines of primary hippocampal neurons. The mutant form lacking the TH1 domain is less distributed in dendritic spines than is the full-length form. Immunohistochemical analysis revealed myosin Id is colocalized with several postsynaptic markers. Taken together, our findings reveal that myosin Id localizes in dendritic spines through the TH1 domain. These results provide the first clues to understand the role of this molecule in the development and pathophysiology of ASD.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-16 脳幹のミクログリア活性は末梢神経損傷に伴う視床回路の再構築を促進する Microglia in the brainstem promote peripheral nerve injury-induced circuit reorganization in the thalamus 植田禎史¹、宮田麻理子¹ 1. 東京女子医科大学医学部生理学講座（神経生理学分野） Yoshifumi Ueta¹, Mariko Miyata¹ 1. Dept Physiol, Div Neurophysiol, Sch Med, Tokyo Women's Med Univ Peripheral nerve injury rapidly reorganizes somatotopic representation in the brain. We have previously reported that the mouse whisker deafferentation, which is induced by transection of the infraorbital nerve, disrupts topographic projections from the brainstem to the thalamic ventral posteromedial nucleus (VPM) by recruiting non-whisker-information carrying ‘ectopic' axons in VPM. However a mechanism underlying this central plasticity is still largely unknown. Here, we show the role of pathway- and region-specific microglial activation in the principal trigeminal nucleus (Pr5), a whisker sensory-recipient brainstem region. Systemic or local manipulation of microglia revealed that reactive microglia in Pr5 were necessary and sufficient for recruiting ectopic axons in VPM. Microglia associated with deafferentation-induced Pr5 neuronal hyperexcitability. Inactivation of Pr5 neurons was capable of suppressing deafferentation-induced recruitment of ectopic axons in VPM in spite of microglial activation in Pr5, suggesting the important function of neuron-microglia interaction in Pr5. Whisker deafferentation-induced reorganization of thalamic circuits underlies ectopic mechanical hypersensitivity in response to lower jaw tactile stimulation. We found that inhibition of microglial activity abolished this ectopic mechanical hypersensitivity. Our results emphasize the critical role of microglia in the brainstem for promoting peripheral nerve injury-induced reorganization of thalamic circuits, which underlies abnormal behavior.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-17 CaMKIIの液液相分離によるグルタミン酸受容体のシナプス内区画化 Subsynaptic segregation of glutamate receptor subtypes by CaMKII-mediated phase separation 細川智永¹、劉品吾、林康紀 1. 京都大学医学部 Tomohisa Hosokawa¹, PinWu Liu, Yasunori Hayashi 1. Department of Pharmacology, Graduate School of Medicine, Kyoto University At synaptic contact, the constituent proteins segregate into distinct nanodomains both within each side of the synapse and across the cleft. Such segregation is modulated by neuronal activity thereby potentially serving as a mechanism to regulate the efficacy of synaptic transmission. However, how such segregation is accomplished largely remain elusive. Here we report that Ca2+/calmodulin-dependent protein kinase II (CaMKII), an abundant postsynaptic kinase involved in synaptic plasticity, undergoes liquid-liquid phase separation (LLPS) and forms protein condensate in Ca2+-dependent manner. Once formed, the condensate withstand removal of Ca2+ via autophosphorylation-mediated mechanism. This segregates two subtypes of glutamate receptors, NMDAR and AMPAR into distinct condensate. Super resolution imaging of the hippocampal neurons revealed that CaMKII segregates of AMPAR from NMDAR in synapse as well. Furthermore, a postsynaptic cell adhesion molecule neuroligin, that can interact with presynaptic neurexin and eventually with the active zone components, partitioned together with AMPAR while NMDAR is segregated. We propose a novel CaMKII-mediated mechanism of synaptic transmission by subsynaptically segregating AMPARs from NMDARs and aligning AMPAR with presynaptic active zone, through LLPS.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-18 【誌上発表】ポストシナプスに局在するSRFコアクチベーターMRTFBの局在変化を介した神経活動依存的な遺伝子発現誘導機構の解析 Activity-dependent gene expression mediated by nuclear translocation of SRF coactivator MRTFB from postsynapses 伊原大輔¹、田邉広樹¹、今西詩織¹、小坂彩¹、佐野友香里¹、阪上洋行²、加藤真之佑¹、田渕明子¹ 1. 富山大学学術研究部薬学・和漢系分子神経生物学、2. 北里大学医学部解剖学 Daisuke Ihara¹, Hiroki Tanabe¹, Shiori Imanishi¹, Aya Kosaka¹, Yukari Sano¹, Hiroyuki Sakagami², Shinnosuke Katoh¹, Akiko Tabuchi¹ 1. Laboratory of Molecular Neurobiology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2. Department of Anatomy, Kitasato University School of Medicine Neuronal activity-dependent gene expression plays crucial roles in neuronal plasticity. It has been reported that CREB-regulated transcription cofactor 1 (CRTC1), a CREB coactivator, contributes to the activity-dependent gene expression through its translocation from synapses into a nucleus. Myocardin-related transcription factors (MRTFs), highly expressed in the brain, have actin-binding motifs and function as transcriptional cofactors of serum response factor. Recently, we reported that MRTFs are localized in postsynapses and involved in synaptic maturation. However, it remains unclear how MRTFs are involved in neuronal activity-dependent gene expression. Therefore, we analyzed the molecular mechanism underlying the nuclear translocation of MRTFs and the following immediate early gene expression in primary cultured rat cortical neurons. Immunostaining revealed that MRTFB transiently translocated into nucleus after stimulation by bicuculline/4-aminopyridine (Bic/4AP), which causes synaptic activation. Next, we found that pretreatment with APV or FK506, inhibitors of NMDA receptor or calcineurin, respectively, blocked the MRTFB translocation into a nucleus caused by Bic/4AP. As the results of qPCR, we preliminarily found that APV, FK506 decreased the expression of JunB mRNA induced by Bic/4AP. Moreover, using ChIP assay, we preliminarily found that accumulation of not only MRTFB but MRTFA on the enhancer of JunB gene increased by Bic/4AP. Taken together, these results indicate that MRTFB, as well as CRTC1, is a key molecule which contributes to the synapse-to-nuclear signaling for neuronal plasticity.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-19 【誌上発表】生薬による樹状突起スパインからのNMDA受容体依存的、非依存的なドレブリン減少～ハイコンテントイメージング解析による検討～ NMDA receptor dependent and independent loss of drebrin from dendritic spines induced by crude drugs in Japanese Kampo 小金澤紀子¹、関野祐子²、白尾智明^1,3 1. 群馬大学大学院医学系研究科薬理学、2. 東京大学大学院薬学系研究科ヒト細胞創薬寄付講座、3. アルメッド（株） Noriko Koganezawa¹, Yuko Sekino², Tomoaki Shirao^1,3 1. Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, 2. Endowed Laboratory of Human Cell-Based Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3. AlzMed, Inc. An assay to detect drug effects on cognitive function in vitro is required to develop, especially among drug discovery fields. To this end, in-vitro evaluation system of synaptic function which underlies learning and memory is important. We have recently developed in-vitro high-throughput assay for evaluation of synaptic function focusing of drebrin. Drebrin is an actin binding protein and stabilizes actin filaments. The drebrin-decorated stable actin filaments accumulate in dendritic spines and the stable actin filaments are thought to play an important role in synaptic plasticity. Once synaptic inputs arrive at synapses and Ca²⁺ influx occurs through NMDA receptors (NMDAR), drebrin accumulation decrease. Therefore, aberrant localization of drebrin at synapses can be a surrogate maker for detecting changes of synaptic states. In this study, we used drebrin-based evaluation of synaptic functions using high-content imaging analysis to examine effects of natural compounds and crude drugs in Japanese Kampo. Here we used 3 natural compounds (nobiletin, diosgenin and tenuifolin) and 5 crude drugs (Uncaria Hook, UH; Bezoar Bovis, BB; Coptis Rhizome, CR; Phellodendron Bark, PB; Polygala Root, PR). These compounds are well studied especially in relation with Alzheimer's disease or cognitive dysfunction. Using 96-well plates, the hippocampal neurons were cultured for 3 weeks and then fixed. After implementation of immunocytochemistry, automated image acquisition and automated quantification were performed. We found that high concentration treatments of diosgenin, tenuifolin, CR, PB and PR decreased drebrin cluster density. Because reduction of drebrin cluster density is known to be caused by NMDAR activation, we next investigated if the drebrin cluster reduction we observed was also NMDAR dependent. Our data showed that reduction of drebrin cluster density by PB and PR was caused by NMDAR activation. Using drebrin-based evaluation of synaptic functional change in 96-well palates, some of crude drugs are shown to affect synaptic function which is underlain by NMDAR activation.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-20 GnRHによるPyk2の活性化のシグナル伝達へのCaMキナーゼグループの関与 Involvement of the CaM kinase group in signal transduction that activates Pyk2 in response to gonadotropin-releasing hormone. 山本秀幸¹、仲嶺三代美¹、澳津志帆¹、鳥原英嗣¹ 1. 琉球大学 Hideyuki Yamamoto¹, Sayomi Higa-Nakamine¹, Shiho Okitsu-Sakurayama¹, Hidetsugu Torihara¹ 1. University of the Ryukyus It has been reported that proline-rich tyrosine kinase 2 (Pyk2) was activated by the tyrosine phosphorylation after the activation of protein kinase C (PKC), as well as after the increase in the intracellular Ca²⁺. However, the molecular mechanisms for the activation are not clear at present. 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 and GnRH neurons. In the previous study, we found that PKD1, belonging to the CaM kinase group, was activated by the novel PKC isoforms after GnRH receptor stimulation in cultured GnRH neurons (GT1-7 cells)¹⁾. Our data suggested that PKD1 activated Pyk2. In addition, we found that CaM kinase IIδ2 was involved in GnRH-induced Pyk2 activation²⁾. In the present study, we examined the molecular mechanisms by which PKD1 and CaM kinase IIδ2 activated Pyk2. 1) Fyn existed in the activated form in GT1-7 cells, and dasatinib, an inhibitor of Fyn, completely inhibited GnRH-induced Pyk2 activation. 2) A PKD inhibitor, CRT0066101, and knockdown of PKD1 inhibited GnRH-induced Pyk2 activation, while they had no effects on Fyn activation. 3) Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited GnRH-induced Pyk2 activation. 4) Intermolecular autophosphorylation of Pyk2 after GnRH treatment augmented the binding of Pyk2 and Fyn. 5) Fyn phosphorylated Pyk2, leading to the full activation of Pyk2. These results strongly suggested that PKD1 and CaM kinase IIδ2 triggered the Pyk2 and Fyn interaction through the stimulation of Pyk2 oligomerization³⁾. 1) J. Biol. Chem. 2015, 25974-25985. 2) J. Cell. Physiol. 2019, 6865-6875. 3)FEBS J. 2020, in press

2020/9/11　14:00～15:00　オンデマンドB-1 P2-21 Characterization of Oxysterol-binding protein (OSBP)-related protein (ORP) 6. 望月信弥¹、三木玄方¹、周如贇¹、野田泰子¹ 1. 自治医科大学 Shinya Mochizuki¹, Harukata Miki¹, Ruyun Zhou¹, Yasuko Noda¹ 1. Jichi medical university Oxysterol-binding protein (OSBP)-related proteins (ORPs) are lipid-binding proteins that share a conserved OSBP-related ligand binding domain (ORD) in their C-terminal region. Twelve ORPs have been identified and classified into 6 groups in mammals. Increasing evidence indicates that they are localized at membrane contact site (MCS) of two different membrane organelles. ORP6, are member of ORP family Ⅲ, is one of the least examined molecules because of the lower yield by exogenous expression. I had previously reported that ORP6 is expressed in the adult brain, cerebellum, spinal cord, embryonic brain and its increased during development. ORP6 localizes at ER and ER-PM membrane contact site in cultured cerebellar neuron. I also identified PI4P, PI(4,5)P2, PI(3,4,5)P3 and phosphatidic acid as binding lipid of the PH domain of ORP6. I showed the interaction of ORP6 with ORP3 and ORP6 itself but not ORP5. This time, I have further examined the knockdown effect of ORP6 on the neuron-derived cells and analyzed the mechanism of the lipid transport of ORP6 at ER-PM membrane contact site.