Poster

Poster 1 Development 1 ポスター 1 発生・分化1

P1-1 Abnormal organization of pinceau in the cerebellum of Kirrel3-deficient mice Kirrel3遺伝子欠損マウスの小脳におけるピンスー構造の異常 Hisaoka Tomoko（久岡朋子）¹，小森忠祐¹，北村俊雄²，森川吉博¹ ¹Dept. of Anatomy and Neurobiology, Wakayama, Japan ²Div. of Cellular Therapy, Advanced Clinical Research Center, The Inst. of Medical Science, The Univ. of Tokyo, Tokyo, Japan Synaptic cell adhesion molecules play important roles in the synapse development, including synapse formation, maturation, and stabilization. Several autism spectrum disorder (ASD)-implicated genes encode synaptic cell adhesion molecules, suggesting that synaptic dysfunction may be involved in the pathogenesis of ASD. Kirrel3 is also one of the synaptic cell adhesion molecules and the disruption of KIRREL3 gene has been reported as being a candidate gene for ASD. At the 59th meeting of Japanese Society for Neurochemistry, we presented that Kirrel3-deficient (Kirrel3^-/-) mice exhibited ASD-like behaviors, including the social and communication deficits, repetitive behaviors, and sensory abnormalities as well as hyperactivity. In the present study, we investigated the molecular mechanism of ASD-like behaviors in Kirrel3^-/- mice. Recent reports suggest that cerebellar abnormalities, such as abnormal development of Purkinje cells (PCs), are involved in ASD-like behaviors. Previously, we reported that Kirrel3 mRNA was highly expressed in the molecular layer of the cerebellum (Neuroscience, 2005). In the present study, we observed that Kirrel3 protein was intensely expressed in the PSD95-positive axonal terminals of basket cells, which form pinceau around the axon initial segment of PCs. To investigate the structure of pinceau, we performed immunofluorescence staining for PSD95 in Kirrel3^-/- mice. We found that the PSD95-positive pinceau areas were increased in Kirrel3^-/- mice compared to those in wild-type mice. This cerebellar abnormality in Kirrel3^-/- mice may cause ASD-like behaviors, such as the social and communication deficits and repetitive behaviors. This work was supported by JSPS KAKENHI Grant Numbers 15K09873 and 18K07610.		P1-2 Multiple functions of Meis1 in cerebellar granule cell development 転写因子Meis1による顆粒細胞の多段階発生制御機構 Owa Tomoo（大輪智雄）¹，田谷真一郎¹，宮下聡¹，西岡朋生²，貝淵弘三²，中村卓郎³，後飯塚僚⁴，星野幹雄¹ ¹Dept of Biochemistry andCellularBiology National Institute of Neurosucience ²Dept. of Cell Pharmacology, School of Medicine, Nagoya Univ ³Department of Carcinogenesis, Japanese Foundation for Cancer Research ⁴Division of Development & Aging, Research Institute for Biological Sciences, Tokyo University of Science Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. We report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in theGClineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors.

P1-3 Deletion of exons encoding carboxypeptidase domain of Nna1 in mice results in Purkinje cell degeneration phenotype マウスNna1遺伝子のcarboxypeptidaseドメインをコードするエクソンを欠失するとプルキニエ細胞変性の表現型を示す Takebayashi Hirohide（竹林浩秀）¹，周麗^1,2，ホサイン MDイブラヒム¹，山崎真弥²，阿部学²，夏目夏目²，今野幸太郎³，渡辺雅彦³，崎村建司² ¹Div of Neurobiol. & Anat., Niigata Univ., Niigata, Japan ²Dept of Cell. Neurobiol., Brain Res. Inst., Niigata Univ., Niigata, Japan ³Dept of Anat., Hokkaido Univ., Sapporo, Japan Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Activation of apoptotic pathway in PCs was also observed at P20 and granule cells at a later stage, which are consistent with phenotypes of other pcd mutant lines. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin, increased polyglutamated tubulin was detected in the mutant cerebellum. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum.		P1-4 Irregular activation of IP₃R1-mediated signaling through MAMs induces axonal swellings and subsequent cell death in Purkinje neurons in CST^-/- mice パラノーダルジャンクション形成不全マウス小脳プルキンエ細胞軸索腫脹におけるMAMを介したIP₃シグナリングの関与 Ishibashi Tomoko（石橋智子）¹，森友希乃¹，八島祐輔¹，御子柴克彦²，馬場広子¹ ¹Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences ²Laboratory for Developmental Neurobiology, RIKEN Brain Science institute Myelin loops attach to the axonal membrane and form paranodal axoglial junctions (PNJs) at paranodes adjacent to nodes of Ranvier. We have reported before that Purkinje axonal swellings appear in cerebroside sulfotransferase knockout (CST^-/-) mice that partially lack PNJs, and that IP₃R1-positive focal accumulations were the earliest findings in those swellings. Following up on these findings, we investigated CST^-/-IP₃R1^+/- mice, which showed a marked decrease of the number of swellings and almost no Purkinje cell loss. This confirms that the accumulation of IP₃R1 triggers Purkinje axonal swellings and Purkinje cell loss in CST^-/- mice. Here we report on a new study focusing on the alterations of IP₃R1-positive axonal swellings, aiming to reveal the significance of IP₃R1 accumulation in PNJs disrupted axons. We found that IP₃R1-positive axonal swellings contain several proteins that localize to mitochondria-associated ER membranes (MAMs) including Sigma 1 receptor, mitofusion2, VDAC and GRP75. A tethering structure between mitochondria and ER was observed at the swollen region. However, there were no similar observations at the non-swollen axons in the mutant mice. Furthermore, CST^-/-IP3R1^+/- mice lost less weight than CST^-/-IP₃R1^+/+ mice, and the neurological phenotype was milder in CST^-/-IP₃R1^+/- mice in comparison to CST^-/-IP₃R1^+/+ mice. These results imply that the regulation of the activation of IP₃R1-mediated signaling through MAMs is necessary to prevent axonal swellings and subsequent cell death in Purkinje neurons in CST^-/- mice. Because axonal swellings are often found in early stages of a number of neurodegenerative diseases, CST^-/-IP₃R1^+/- mice as well as CST^-/- mice will be useful tools to understand the functional role of IP₃R1 and MAMs in these diseases.

P1-5 Reelin controls N-cadherin-dependent neuronal adhesion by a couple of mechanisms in the developing mouse cortex リーリンはN-cadherinを基盤とした神経細胞接着を2つの経路で誘導する Hayashi Kanehiro（林周宏）¹，仲尾信彦^2,3，井上聖香¹，安達泰治^2,3，久保健一郎¹，仲嶋一範¹ ¹Dept. of Anatomy, Keio University School of Medicine ²Institute for Frontier Life and Medical sciences, Kyoto University ³Department of Micro Engineering, Graduate School of Engineering, Kyoto University The mammalian neocortex has six layered structure, which enables highly brain functions. The secretory molecule ‘Reelin' plays a central role to organize the layered structure. Reelin is a glycoprotein secreted from mainly Cajal-Retzius cells in the marginal zone of the developing neocortex and induces the diverse signals in the migrating neurons. Previously, we reported that Reelin induces the formation of neuronal aggregation which is arranged in a process-rich center area and a cell-body-rich peripheral region, and that formation of this aggregation is mediated by N-cadherin (Kubo et al., 2010, J. Neurosci.; Matsunaga, Noda et al., 2017, PNAS). However, it remains to be elucidated how Reelin promote N-cadherin-dependent cellular adhesion. Here, we present the details of N-cadherin-dependent neuronal adhesion promoted by Reelin. Using Atomic force microscopy (AFM), we analyzed N-cadheirn-based cellular adhesion on primary cultured cortical neurons exposed by the Reelin-medium. Consequentially, we found that Reelin induces the increase of the frequency of N-cadherin-mediated bond upon a unit area of the neuron, and the higher strength of the single adhesion force. The results suggest that Reelin induces a couple of mechanisms to promote the neuronal adhesion based on N-cadherin, that is, the increase of the amount of N-cadherin on the plasma membrane of the migrating neurons, and the formation of N-cadherin complex.		P1-6 Analysis of subcellular distribution of Dab1 in cerebral neocortical excitatory neurons using highly sensitive tagging by genome editing ゲノム編集技術によって高感度タグラベルされたDab1の大脳新皮質興奮性神経細胞における細胞内分布の解析 Honda Takao（本田岳夫），仲嶋一範 Dept. of Anat. Keio Univ. Sch. of Med, Reelin-Dab1 signaling pathway is critically important for the formation of layered structure of brains such as cerebral cortex, hippocampus, and cerebellum. Although we previously found that Dab1 is a nucleocytoplasmic shuttling protein using HEK293T, Neuro2A, or primary cultured cells, it remains unknown whether Dab1 actually exists in the nucleus of migrating or postmigratory neurons. So far, we have been trying to examine the existence of Dab1 in nucleus using immunohistochemical analysis, however, we could not detect the signal even within the cytoplasm presumably due to its low expression level. To overcome the difficulty of the detection, we have added immunohistochemically highly sensitive-tag to the C-terminus of the Dab1 protein by genome editing using CRISPR/Cas9 system. The CRISPR/Cas9 genome editing components were introduced into mouse cerebral cortex by in utero electroporation at embryonic stages, and the subcellular localization of the tagged-Dab1 was examined at various developmental stages of excitatory neurons.

P1-7 Cohesin regulates neuronal network formation in the brain 染色体接着因子コヒーシンによる中枢神経回路形成制御機序 Fujita Yuki（藤田幸）^1,2，山下俊英^1,2,3 ¹Dept. of Mol. Neurosci., Grad. Sch. of Med., Osaka Univ. ²iFReC, Osaka Univ. ³Grad. Sch. of Front. Biosci., Osaka Univ. Gene expression regulated by spatial chromatin organization and nuclear architecture plays key roles in the development of central nervous systems. Here we focus on the role of cohesin complex, which is chromosome-associated multi-subunit protein. Cohesin complex is composed of four subunits, Smc3, Smc1, Scc3, and Scc1/Rad21, and has a role in sister chromatid cohesion, which is crucial for accurate chromosome segregation. Cohesin is also known to be involved in chromatin organization by forming chromatin loops at particular loci and regulates gene expression in postmitotic cells. Disruption of cohesin network results in ‘cohesinopathies’ such as Cornelia de Lange syndrome. These diseases cause higher brain dysfunction, suggesting the role of cohesin in gene regulation rather than chromosome segregation in the postmitotic neurons. To investigate the potential role of cohesin in terminally differentiated cells in vivo, we generated conditional Smc3-knockout mice. We observed increased dendritic complexity and decreased spine density in cortical neurons of heterozygous Smc3-knockout mice. Neuron-specific Smc3-knockout mice showed the same phenotype. Heterozygous Smc3-knockout mice exhibited increased anxiety-related behavior, a symptom of Cornelia de Lange syndrome, also caused by disruption of cohesin. Thus, neuronal cohesin contributes to neural network formation, presumably by modulating gene expression, and cohesin deficiency leads to higher brain dysfunction.		P1-8 The upregulated expression mechanism of nur77 gene and its downstream target gene in early stage of the forskolin-induced differentiation in PC12 cells フォルスコリンによって誘導されるPC12細胞の分化初期段階に関与するnur77遺伝子とその下流遺伝子の発現上昇の分子機構 Maruoka Hiroki（丸岡弘規），山添亮輔，谷尾啓介，高橋亮太，星川郁也，下家浩二 Lab. of Neurobiology, Dept. of Life Science and Biotech., Fac. of Chemistry, Materials and Bioengineering, Kansai University Forskolin, one of the natural compounds, is known to have an effect of neuronal differentiation. Forskolin increases intracellular cAMP concentration by activation of adenylate cyclase and promotes neuronal differentiation of several types of neurons via the PKA-CREB-dependent signaling pathway. However, it is still unclear how it controls transcription factors, which bind to CREB, during forskolin-induced neuronal differentiation. In this study, we revealed that one of the immediate early genes, nur77 gene, contributed to early stage of the CREB-dependent neuronal differentiation of forskolin-treated PC12 cells. After the treatment of forskolin, the expression of Nur77 was upregulated within 1 hour. In addition, a knock-down of nur77 gene inhibited the neurite outgrowth induced by forskolin. In order to elucidate the detailed upregulated mechanism of nur77 gene expression, ChIP assay were carried out. The four CRE sites upstream of the transcriptional start site (TSS) of nur77 gene were associated with phosphorylated CREB within 1 hour after the treatment with forskolin. To analyze the role of these four CRE sites concerning nur77 gene expression, reporter assay was performed. As a result, it was found that nur77 gene expression was mainly mediated through -78 that positioned on the nearest CRE site of TSS of nur77 gene. We also analyzed molecules controlled by Nur77 after the treatment with forskolin. The analysis using the neuronal marker proteins revealed that Nur77 regulated the expression of Synapsin1, not β-tubulin III and NeuroD. These results indicate that the PKA-CREB-Nur77-Synapsin1 signaling pathway plays a pivotal role in the differentiation process of the forskolin-induced PC12 cells.