Poster Sessions

Poster Sessions 回路網形成、神経系の発生・再生

2P-24 Growth cones in 3D culture have different structural dynamics from those in 2D culture Ryota Negishi^1,2，Shigo Koinuma¹，Naoyuki Wada²，Yuichi Sakumura³，Takeshi Nakamura¹ ¹Research Institute for Biomedical Sciences, Tokyo University of Science，²Department of Applied Biological Science, Tokyo University of Science，³Graduate School of Biological Sciences, Nara Institute of Science and Technology During development of the nervous system, newborn neurons extend neurites their targets. At the neurite tips, growth cones integrate various guidance cues and regulate neurite outgrowth. In a two-dimensional culture, growth cones take a fan-like shape and their peripheries consist of actin-rich structures, filopodia and lamellipodia. In contrast, previous studies have shown that under in vivo (three-dimensional) situations, growth cones appear remarkably different structure from 2D counterparts. Some 3D growth cones are simple and have less filopodia without lamellipodia. This leads to a question whether or not the same molecular mechanism regulates structural dynamics of 2D and 3D growth cones. However, we do not have an enough information about structural dynamics of 3D growth cones; this is mainly due to the technical limitations such as low spatial resolution and S/N ratio. To overcome these difficulties, we tried to image a dynamical behavior of growth cones of primary cultures and neuronal cell lines which grew in a two- or three-dimensional environment using confocal microscope. In PC12 cells, we used GFP harboring the C-terminus of K-Ras to visualized the plasma membrane. In DRG neurons, cells were stained with CellMask. If necessary, Lifeact-mCherry was co-expressed to visualize F-actin. Thereafter we processed raw images and constructed stereoscopic structures. Overall structure of 3D growth cones was dissimilar to that of 2D growth cones. Typical 3D growth cones look like harpoons having several filopodia, while typical 2D growth cones were a fan-shape and have well-developed lamellipodia. Their speed of extension was almost comparable. Now we are starting a morphometrical analysis to perform a more quantitative comparison between 2D and 3D growth cones.		2P-25 Cdk5 regulation of the GRAB-mediated Rab11-Rab8 cascade in axon outgrowth Kotaro Furusawa¹，Akiko Asada¹，Pamela Urrutia³，Christian Gonzalez-Billault³，Mitsunori Fukuda²，Shin-ichi Hisanaga¹ ¹Dept Biol Sci, Grad Sch Science Tokyo Metropolitan Univ，²Dept Dev Biol and Neurosci, Grad Sch Life Sci, Tohoku Univ，³Dept Biol, Univ of Chile Neurons communicate with each other through their axons and dendrites. However, a full characterization of the molecular mechanisms involved in axon and dendrite formation is still incomplete. Neurite outgrowth requires the supply of membrane components for surface expansion. Two membrane sources for axon outgrowth are suggested, Golgi secretary vesicles and endocytic recycling endosomes. In non-neuronal cells, trafficking of secretary vesicles from Golgi is regulated by Rab8, a member of Rab small GTPases, and that of recycling endosomes is by Rab11, another member of Rabs. However, whether these vesicles are coordinately or independently transported in growing axons is unknown. Herein, we find that GRAB, a guanine nucleotide exchange factor for Rab8, is a novel regulator of axon outgrowth. Knockdown of GRAB suppressed axon outgrowth of cultured mouse brain cortical neurons. GRAB mediates the interaction between Rab11A and Rab8A and this activity is regulated by phosphorylation at Ser169 and Ser180 by Cdk5-p35. The non-phosphorylatable GRAB mutant S169/180A promoted axonal outgrowth to a greater extent than did the phospho-mimic GRAB mutant S169/180D. Phosphorylation of GRAB suppressed its guanine nucleotide exchange factor activity and its ability to recruit Rab8A to Rab11A-positive endosomes. In vivo function of GRAB and its Cdk5-phophorylation was shown in migration and process formation of developing neurons in embryonic mouse brains. These results indicate that GRAB regulates axonal outgrowth via activation and recruitment of Rab8A to Rab11A-positive endosomes in a Cdk5-dependent manner.

2P-26 Bisphenol A attenuates neuronal differentiation in cerebral cortical neurons. Kumi Mastuura¹，Ryosuke Yamazoe¹，Hiroki Maruoka¹，Koji Shimoke² ¹Grad Sch Sci.&Tech., Kansai Univ，²Dept Chem. Mat.&Biol., Kansai Univ An endocrine disrupter, bisphenol A (BPA), is widely used in the products of plastics and coating. It has been reported that the effect of BPA linked to obesity, cancer, and childhood neurological disorders such as anxiety or hyperactivity. Furthermore, it has also been reported that BPA induced developmental neurotoxicity in offspring rat exposed with BPA in the prenatal period. However, it is still remains unclear how BPA induces these developmental neurotoxicity. Thus, the main purpose of the study is to elucidate the effect of BPA on neurons, especially, focusing on by embryonic cerebral cortical neurons. Then, we speculate that the effects of BPA exposure is occurred during periods on cellular and molecular alterations in the maturation process. The result is that BPA did not affect the dendrite elongation although 100 μM BPA attenuated the atonal elongation of cultured cerebral cortical neurons in the days in vitro 2 or 3 (DIV 2 or 3). Moreover, we found that branches of dendrite increased by the addition of BPA, suggesting that BPA induces neuronal toxicity via the decreased.		2P-27 Munc18-1 plays an essential role for cortical neuron migration during brain development Nanako Hamada^1,2，Hidenori Tabata¹，Koh-ichi Nagata¹ ¹Dept of Mol Neurobiol, Inst for Developmental Res, Aichi Human Service Cent，²Res Fellow of Japan Society for the Promotion of Sci While Munc18-1 is essential for presynaptic vesicle fusion in developed neurons, this molecule is likely to be involved in brain development since gene abnormalities in MUNC18-1 (STXBP1) cause early infantile epileptic encephalopathy with suppression-burst (Ohtahara syndrome), neonatal epileptic encephalopathy and other neurodevelopmental disorders. We analyzed physiological and pathophysiological relevance of Munc18-1 during the cortical development. Knockdown of Munc18-1 using in utero electroporation caused abnormal migration of cortical neurons during corticogenesis. Time-lapse imaging revealed that multipolar-bipolar transition of Munc18-1-deficient cells occurred normally whereas subsequent radial migration was hampered in the cortical plate. Syntaxin1A was critical for radial migration downstream of Munc18-1. As for the underlying mechanism, Munc18-1-knockdown in cortical neurons hampered post-Golgi vesicle trafficking and subsequent vesicle fusion at the plasma membrane in vivo and in vitro, respectively. Notably, Syntaxin1A-silencing did not affect the post-Golgi vesicle trafficking. Taken together, Munc18-1 was suggested to regulate radial migration by modulating not only vesicle fusion at the plasma membrane to distribute various proteins on the cell surface for interaction with radial fibers, but also preceding vesicle transport from Golgi to the plasma membrane. Although knockdown experiments suggested that Syntaxin1A does not participate in the vesicle trafficking, it was supposed to regulate subsequent vesicle fusion under the control of Munc18-1. Disruption of Munc18-1 function may result in the abnormal corticogenesis, leading to neurodevelopmental disorders with MUNC18-1 gene abnormalities.

2P-28 A positive charge cluster of EC domain in LOTUS is involved in the molecular interaction with PirB Hirokazu Takaya^1,2，Yuji Kurihara¹，Yutaka Kawakami¹，Yasuyuki Kurihara²，Kohtaro Takei¹ ¹Molecular Medical Bioscience Laboratory, Graduate School of Medical Life Science, Yokohama City University, Japan，² Laboratory of Molecular Biology, Graduate School of Engineering, Yokohama National University, Japan Nogo interacts with both Nogo receptor 1 (NgR1) and paired immunoglobulin-like receptor B (PirB) on the cell membrane of neurons, and thereby restricts axonal regeneration after injury in the central nervous system (CNS). We previously identified lateral olfactory tract usher substance (LOTUS), a neural circuit formation factor, as an antagonist for NgR1 and PirB. We also found that the two carboxyl terminal domains of LOTUS, UnbV/ASPIC (UA) domain and epidermal growth factor-like calcium binding (EC) domain, contribute to the molecular interaction between LOTUS and NgR1. However, the binding site of LOTUS to PirB remains unknown. In this study, binding analysis using deletion mutants of LOTUS revealed that UA alone, EC alone, and both (UA/EC) interacted with PirB. To further determine the binding site of LOTUS to PirB based on the 3D structure of UA/EC, we searched for proteins having homologous amino-acid sequences in Protein Data Bank (PDB). While BLAST indicated that no protein structures had a similar sequence to UA domain, cbEGF9-hyb2-cbEGF10 region (PDB ID: 2W86) of fibrillin-1 had a similar sequence to EC. Based on a part of this structure and its sequence, we predicted the 3D structure of EC using homology modeling and molecular dynamics simulation methods. The predicted surface potential map showed that EC was mostly covered with negative charges but had a positive charge cluster. We hypothesized that this cluster plays an important role in the binding of EC to PirB, and generated a triple point mutant of EC (K591E/R592E/R595E) which makes this cluster smaller. Ligand-receptor binding assay showed that this mutant bound to PirB more weakly than wild type. These data suggest that this positive charge cluster in EC is involved in the binding of LOTUS to PirB.		2P-29 Promotion of nerve regeneration and functional recovery after spinal cord injury by an endogenous Nogo receptor antagonist, LOTUS Tomoko Hirokawa，Yuji Kurihara，Kohtaro Takei Mol. Med. Biosci. Lab., Grad. Sch. of Med. Life Sci., Yokohama City Univ. After spinal cord injury (SCI), primates, such as humans, hardly recover the locomotor function, and the basic therapy has not been established. But rodents, such as mice and rats, show a partial spontaneous recovery of the locomotor function. However, the factors associated with this partial improvement of nerve regeneration after SCI in rodents remain completely unknown. It has been considered that limitation of neuronal regeneration is mainly caused by axon growth inhibitors such as Nogo, MAG, OMgp, CSPG and BLyS and a common receptor of these ligands, Nogo receptor-1 (NgR1). We previously identified lateral olfactory tract usher substance (LOTUS) suppressed axonal growth inhibition induced by interaction between these NgR1 ligands and NgR1. Therefore, LOTUS may be useful in future therapeutic approaches as an endogenous potent inhibitor of NgR1 for promoting neuronal regeneration. First, we found that lotus-deficient mice showed delayed locomotor functional recovery of behavioral outcome and LOTUS expression was down-regulated in injured site of wild mice. The down-regulation of LOTUS expression well associated with decrease of locomotor activity after SCI. Then, we hypothesized that supply of LOTUS could promote spontaneous recovery. To examine effects of overexpression of LOTUS on the recovery of the locomotor function after SCI using lotus-transgenic (LOTUS-Tg) mice overexpressing LOTUS in neurons. We clearly found functional recovery in LOTUS-Tg mice when compared with wild type mice. Furthermore, we detected that increase of serotonin-positive regenerating axon and expression of GAP-43, growth associated protein marker in the spinal cord of LOTUS-Tg mice after SCI. These findings suggest that LOTUS may contribute to promotion of functional recovery after SCI.

2P-30 Analyses of Cyclin D2 mRNA transportation in the cortical development using CRISPR/Cas9 genome editing system Takako Kikkawa¹，Yukiko U. Inoue²，Takayoshi Inoue²，Noriko Osumi¹ ¹Dept Dev Neurosci. Grad Sch Med, Tohoku Univ，²Dept Biochem and Cell Biol. NCNP During cortical development in mammals, radial glial (RG) cells need to adequately proliferate and differentiate. RG cells show highly polarized morphology with long and thin processes. We have previously shown that mRNA of Cyclin D2, one of the cell cycle regulators, is transported by using its 3’ UTR element to the basal end-foot of RG cells (Tsunekawa et al., EMBO J, 2012). Cyclin D2 protein is asymmetrically inherited by one of the two daughter cells, which maintains its self-renewal capability. However, the machinery of Cyclin D2 mRNA transportation to the basal end-foot of RG cells remains unsolved. To address this question, we used the CRISPR/Cas9 genome editing system in mouse embryos and selectively removed the cis-regulatory sequence of Cyclin D2 mRNA transportation by introducing two single guide RNAs at around the both ends. We found that Cyclin D2 mRNA expression was detected in the ventricular zone but decreased in the basal end-foot of RG cells when the cis-regulatory sequence of Cyclin D2 mRNA was successfully deleted. These results suggest that the cis-regulatory sequence of Cyclin D2 mRNA is involved in the mRNA transportation to the basal end-foot of RG cells. We are currently producing various lines of the cis-regulatory sequence deleted mice, and analyzing whether the transportation system affects cell fate of RG cells and corticogenesis.		2P-31 Analysis of δ-catenin as a downstream gene of Pax6 in early development of the mammalian telencephalon SeungHee Chun，Takako Kikkawa，Noriko Osumi Dept Dev Neurosci. Grad Sch Med, Tohoku Univ Pax6 transcription factor plays a role in balancing cell proliferation and neuronal differentiation in the mammalian developing cortex via regulating expression of target genes. Various genes are reported to be up- or downregulated in the Pax6 mutant mouse cortex, and δ-catenin is one of such Pax6-downstream genes (Duparc et al., 2005; Osumi et al., 2008; Walcher et al., 2013). δ-catenin, also known as CTNND2, is a member of p120-catenin (p120^ctn) protein family containing ten Armadillo-repeats and constituting a cadherin-catenin cell adhesion complex (Kosik et al., 2005). Recently, a genetic study analyzing autism spectrum disorder (ASD) families enriched with female patients has reported CTNND2 gene as a novel ASD-related gene (Turner et al., 2015). However, the function of δ-catenin in cortical development is poorly understood. To elucidate the functions of δ-catenin in the developing brain, we observed δ-catenin expression in the telencephalon and found that it was strongly expressed in the ventricular zone (VZ), cortical plate (CP) and ganglionic eminence (GE) in wild type mice at embryonic day 12.5 and 14.5. Furthermore, δ-catenin established a honeycomb-like pattern, a typical adherens junction pattern, in the neuroepithelium of the VZ. In Pax6 homozygous mutants, δ-catenin  expressions was severely reduced in the apical surface of the VZ, but not in the CP and GE. These results suggest that δ-catenin expression in neuroepithelium of the VZ is regulated by Pax6, and it may also play a role in cortical development. To further examine the role of δ-catenin in the developing cortex, we are currently quantifying δ-catenin expression by Western blot analysis and knocking-down δ-catenin experiment by in utero electroporation.

2P-32 Mood stabilizing drugs activate adult neural cell-neurogenesis system Keita Nakaji，Natsu Koyama，Hiroaki Kitagawa，Takahiro Fuchigami，Seiji Hitoshi Dept Physiology. Shiga Univ of Medical Science Neural stem cells (NSCs) not only produce all neurons and glia in the developing brain but also reside in the adult brain and supply new neurons to the olfactory bulb and hippocampus, which play significant roles in the olfaction and some types of memory. NSCs attract much attention as a resource of cell replacement therapy for impaired central nervous system. However, efficient and clinically feasible strategy to activate endogenous NSCs is not currently available. We have previously demonstrated that mood stabilizing drugs, which are used to treat patients with bipolar disorder, enhance the self-renewal capability of mouse NSCs in vitro at therapeutically relevant concentrations in the cerebrospinal fluid. In this study, we examined the effect of a novel type of mood stabilizer, lamotrigine, on the self-renewal of NSCs in vitro and neurogenesis in the olfactory bulb, dentate gyrus and subependymal zone in vivo and also conducted behavioral battery after administration of lamotrigine and found some changes. Our data suggest that lamotrigine possesses similar pharmacological function to classical mood stabilizers, such as valproate, carbamazepine and lithium.		2P-33 The role of αvβ5 integrin in axon specification of cerebellar granule cell precursors. Ayaka Abe^1,2，Ayumi Akiyama^1,2，Kei Hashimoto^1,2,3，Yokichi Hayashi⁴，Yasunori Miyamoto^1,2 ¹Division of Life Sciences, Graduate School of Humanities and Sciences, Ochanomizu University.，²Institute for Human Life Innovation.，³Research Fellow of Japan Society for the Promotion of Science.，⁴Div. of Life Sci., Asahikawa Medical Univ. Vitronectin (VN), which is an extracellular matrix protein, is known to be involved in the regulation of proliferation and differentiation of cerebellar granule cell precursors (CGCPs) in mice. We previously revealed that VN promotes the progress of the initial differentiation stage of CGCPs. However, it has not been identified which receptor for VN is involved in the function of VN in CGCPs. A candidate of VN receptor is integrin (Itg). It is known that αvβ3 Itg and αvβ5 Itg are expressed in CGCPs. Then, we examined which Itg serves as a receptor for VN in CGCPs and what phenomenon the receptor contributes to in the initial differentiation stage of CGCPs.The effects of the knockdown (KD) of αv, β3, β5 Itg by the siRNAs were analyzed by immunohistochemistry of the CGCPs. The KD of αv Itg and β3 Itg up-regulated the number of proliferation marker-positive cells, but the KD of β5 Itg up-regulated the number of TAG1, a marker of initial differentiation, -positive cells. These results motivated us to check the role of β5 Itg as a receptor for VN. The KD of β5 Itg using shRNA-expression lentivirus suppressed the progress of initial differentiation stage of CGCPs by VN. Next, we speculated that αvβ5 Itg is involved in axon specification at the initial differentiation stage of CGCPs and examined the effect of β5 Itg on axon specification. The KD of αvβ5 Itg down-regulated the ratio of CGCPs with no axon, and the over-expression of αvβ5 Itg up-regulated the ratio of CGCPs with 2 or more axons. Similarly, loss and addition of VN affected the axon specification of CGCPs. Moreover, the effect of VN addition were inhibited by KD of β5 Itg. Taken together, αvβ5 Itg as a receptor for VN plays a critical role in axon specification in the initial differentiation stage of CGCPs.

2P-34 Fyn promotes detachment of chain-forming new neurons by controlling N-cadherin-mediated adherens junction in the postnatal brain Yayoi Seto¹，Kazuma Fujikake^1,2，Masato Sawada¹，Takao Hikita¹，Naoko Kaneko¹，Kazuya Sobue²，Kazunobu Sawamoto^1,3 ¹Dept Dev Regen Biol, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan，²Dept Anesth Intensive Care Med, Nagoya City Univ Grad Sch Med Sci, Nagoya, Japan，³Div Neural Dev Regen, NIPS, Okazaki, Japan In the postnatal brain, new neurons are generated from neural stem cells in the ventricular-subventricular zone throughout life. These new neurons form chain-like aggregates and migrate in a specialized route called rostral migratory stream toward the olfactory bulb (OB). After arriving at the OB, each new neuron detaches from chains and starts to migrate radially within the OB layers. However, the mechanism regulating detachment of chain-forming new neurons remains unknown. To identify the molecule that regulates detachment of new neurons from chains, we performed chemical screening and found that PP2, an inhibitor for Src family kinases, inhibits detachment of new neurons in vitro. To examine whether these kinases are expressed in the V-SVZ-OB pathway, we performed western blotting and immunohistochemistry, and found that Src and Fyn were expressed in new neurons in the V-SVZ and OB. Effects of in vivo Fyn knockdown (KD) suggest that Fyn promotes detachment of new neurons from chains. Since previous studies reported that chain-forming neurons attach each other via adherens junction (AJ)-like structures, we hypothesized that Fyn promotes detachment of new neurons by regulating N-cadherin-mediated AJ-like structures. To test this possibility, we performed Fyn and N-cadherin double KD experiments in vivo. The Fyn-KD-mediated suppression of detachment of new neurons in the OB was canceled by N-cadherin KD. Taken together, these results suggest that Fyn promotes detachment of new neurons from chains by modulating N-cadherin-mediated adhesion.		2P-35 Involvement of aerobic metabolism in direct reprogramming of mouse fibroblasts to neurons. Yuka Akagi，Yukihiro Harada，Tomoe Ueyama，Dai Ihara，Sae Nakagawa，Takahiro Sogo，Shu Nakao，Teruhisa Kawamura Dept Biomedical Sci. Grad Sch Life Sci, Ritsumeikan Univ It has been reported that direct reprogramming of human and mouse somatic cells into functional neurons by gene introduction of three factors, Pou3f2, Ascl1, and Myt1l (PAM). These reprogrammed neurons (iNs), which retain aging signatures from the donor, are presumed to be a good models to study age–associated disorders such as Alzheimerʼs disease. However, iNs almost loose proliferative capacity, and thus induction efficiency still remains insufficient. In addition, the mechanisms of direct conversion in iNs have yet been investigated. Recent studies including ours suggest the importance of metabolism shift on somatic cell reprogramming. Therefore, in the present study, we examine the role of cellular metabolism and its regulators in direct reprogramming to iNs.To induce direct reprogramming to iNs, we infected mouse embryonic fibroblasts (MEFs) with retrovirus vectors encoding PAM, and confirmed the expression of neuron specific marker genes including βIII–tubulin (Tuj1) and MAP2. The metabolism analysis exhibits that intracellular ATP concentration in the early phase of reprogramming was higher than mock–infected cells, whereas there was no difference in the copy number of mitochondria genomes. Furthermore, PAM-infected cells show higher expression levels of aerobic metabolism–related genes (e.g., ERRγ and PGC1α). On the other hand, expression levels of glycolysis–regulating factors (e.g., HIF1α and its downstream target gene, Glut1) were decreased. These results above suggest that neuron reprogramming factors, PAM would enhance aerobic metabolism rather than glycolysis. Although further studies should be needed, our findings may provide clues to improve the efficiency of direct reprogramming into iNs.

2P-36 Nesfatin 1-like-peptide and Neuronal cell death Noriko Marutani，Daisuke Kanayama，Akio Hukumori，Takashi Kudou Department of Mental Health Promotion, Osaka University Graduate School of Medicine Nesfatin 1-like-peptide (NLP) was first identified that processed from CALNUC (Nucleobindin 1) in 2015.The function of NLP is not known well, but Nesfatin1, homologue of NLP, has been reproted as an anorexigenic and insulinotropic peptide. NLP was also reported that was insulinotropic in mice and anorectic in fish and rat. CALNUC has been found within the nucleus, endoplasmic reticulum, cytoplasm of cells and extracellular space. It is presumed to be a functional protein for its Ca 2+ -binding domain and Leucine Zipper domain.It is suggested that CALNUC is secreted to extracellular space under ER stress condition and acts as an ER-stress-responsive negative regulator of ATF6 activation by inhibiting S1P mediated ATF6 cleavage at ER Golgi. In recent years, it is reported that CALNUC binds to multpule types of amyloid and inhibits its fibrillization.We have examined how CALNUC affects cell vulnerability to ER stress. The vulnerability to tunicamycin was elevated when CALNUC expression was knocked down by siRNA. On the contrary, cell cytotoxicity under ER stress was reduced in CALNUC overexpressing cell. We elucidate the effect of NLP on ER stress by cell-death assay using NLP overexpressing cells. We will report how NLP acts under ER stress and affect Aβ fibrillization.