Poster Sessions

Poster Sessions シナプス、細胞内・細胞間情報伝達

2P-12 Neuropsin dependent and independent behavioral tag setting. Yuka Suzuki，Yuya Yoda，Yasuyuki Ishikawa Dept Systems Life Engineering. Maebashi Institute of Technology Synaptic plasticity is widely accepted to provide a cellular basis for learning and memory. Synaptic associativity could be involved in activity-dependent synaptic plasticity, because it distinguishes between local mechanisms of synaptic tags and cell-wide mechanisms that are responsible for the synthesis of plasticity-related proteins. An attractive hypothesis for synapse specificity of long-term memory (LTM) is synaptic tagging: synaptic activity generates a tag, which captures the plasticity-related proteins derived outside of synapses. Previously we have been reported that neuropsin, a plasticity-related extracellular protease, was involved in synaptic tag setting. In the present study, we tested the hypothesis that neuropsin was engaged in behavioral tagging for LTM in vivo. Behaviorally, weak training inhibitory passive avoidance task (IA) or spatial object recognition task (SOR), which induces short-term memory (STM) but not LTM, can be consolidated into LTM by exposing animals to novel but not familiar environment 1 h before training. We found that neuropsin deficient mouse impaired such transformation short-term into long-term memory in IA but not SOR. These results suggest that both neuropsin dependent and independent mechanism of behavioral tag setting exist in vivo.		2P-13 Identifying Genetic Markers Associated with Treatment Response to Lurasidone via A Genome-Wide Association Study Akane Yoshikawa^1,2，Jiang Li¹，Herbert Y Meltzer¹ ¹Feinberg School of Medicine, Northwestern University, Psychiatry and Behavioral Sciences，²Bureau of SocialWelfare and Public Health, Tokyo Metropolitan Government Introduction:Schizophrenia (SCZ) is a heterogeneous disorder with a diverse genetic contribution to its etiology and treatment response to antipsychotic drugs (APDs). There are currently no biomarkers which can predict the response to APDs in SCZ. Therefore, to identify the genetic markers associated with treatment response is of great interest to triage the patients and disclose the fundamental mechanisms of drug efficacy. Methods: A genome-wide association study (GWAS) on two randomized clinical trials on Caucasian patients with SCZ was conducted by Affymetrix 6.0 SNP array to identify the genetic biomarkers associated with treatment response to lurasidone at last observation carried forward (LOCF). Treatment response was quantitatively evaluated by change in PANSS-Total from the baseline to LOCF. Results: We identified common variants in genes related to synaptic function as the top predictors of response to lurasidone in Caucasians patients. The identified genomic loci mainly resided in synaptogenic adhesion genes and scaffolding proteins, both essential for synaptic function. NRXN1 replicates previous findings for prediction of response to clozapine by us and others. Other synaptic genes predicting response included NRG1/3, KALRN, and neuron-specific splicing regulator, RBFOX1. Most of these genes and their associated pathways have been previously linked to SCZ. Some of the top SNPs are potential cis-eQTLs. Discussion: Our non-hypothesis driven GWAS study identified genetic biomarkers for prediction of treatment response. Replication study is currently going on. These findings suggest that synaptogenic adhesion and scaffolding proteins will be potential novel drug targets for SCZ.

2P-14 Alternative splicing of Neurexin plays a crucial role in Neuroligin-induced presynaptic differentiation on cerebellar GABAergic neurons in vitro Yuji Sato^1,2，Satoko Suzuki¹，Yoko Iijima ¹，Takatoshi Iijima¹ ¹Tokai University, Institute of Innovative Science and Technology, Medical Division，²Depatment of Chemistry and Bioresource, Faculty of Engineering, Tokai University Trans-synaptic interaction of synaptic cell adhesion proteins, Neurexins (NRXs) and Neuroligins (NLs), plays important roles in the specification and function of both inhibitory and excitatory synapses. Alternatively spliced segment 4 (AS4) of three NRX genes (Nrxn1/2/3) is a critical element for the selective trans-synaptic interaction. However, the splice code on NRXs and NLs on synapse specification and function is not fully understood. To clarify the exact role of splice code on NRX-NL interaction in specification on excitatory and GABAergic synapses, we evaluated synaptogenic receptor activity of NL1/2/3 isoforms in neuron-fibroblast co-culture system in which Nrxn AS4 are manipulated by ectopic expression of SLM2, which was recently identified as a selective and dominant regulator for AS4 splicing. We show that ectopically expressed SLM2 with viral infection induces the dramatic shift in splicing of all the three Nrxns into AS4 (-) in cultured cerebellar neurons. All the NLs preferentially induced excitatory presynaptic differentiation in both uninfected (control) cultures and SLM2-ectopically expressing (SLM2 E/E) ones, indicating that, closely correlated to the biochemical interaction reported previously, NLs can organize presynapses through both NRX AS4(-) and AS4(+). In contrast, NL-induced presynaptic differentiation of cerebellar GABAergic neurons is drastically abolished in SLM2 E/E culture independent of any NL isoforms, indicating that AS4 insertion of presynaptic NRXs is critical for NL-induced differentiation of GABAergic presynapses in cerebellar neurons. Therefore, this study may provide the new insights on splice-code of NRX-NL interaction for synapse specification and properties on particularly cerebellar GABAergic system.		2P-15 Alteration of neuronal circuit with autism-inducing drug administration in developing rat cerebellum Seita Sato¹，Yukiko Fueta²，Susumu Ueno²，Yuko Sekino³，Yasunari Kanda⁴，Sachiko Yoshida¹ ¹Dept Environ Life Sci, Toyohashi，²Univ Occupational Environmental Hlth,，³University of Tokyo，⁴Natl Inst Health Sci, Tokyo, Japan Valproate (VPA) is an antiepileptic drug known to correlate positively with the occurrence of autism in the offspring of mothers exposed to the drug during pregnancy. In autism, some structural abnormalities in the cerebellum have been reported. Especially, a reduction in size and number of Purkinje cells are revealed in both the postmortem human studies and VPA-administrated adult animals. We have observed the irregular elongation of Purkinje cell dendrites started earlier than control and excess folding in cerebellar lobules, especially between lobule V and VII (primary fissure) in VPA 600mg/kg at E16-administrated rat. In this report, we investigated the concentration dependency and timing dependency of VPA administration to rat embryo.VPA was once administrated to embryonic day (E) 14, 16 or 18 p.o. with 600mg/kg of mother weight, respectively. Some animals were administrated VPA at E 16 p.o. with 200, 300 or 400 mg/kg of mother weight, respectively. The cerebellum in pups was observed from postnatal day (P) 7 to P21.In VPA 200 mg/kg administrated rat, these abnormalities were not observed, while the animals administrated 300 or 400 mg/kg VPA showed irregular elongation of Purkinje cells and excess folding in the primary fissure. The pups administrated VPA 600 mg/kg at E14 showed similar abnormal development of the cerebellar cortex, however, the pups administrated VPA at E18 did not. We analyzed the cerebellar hyperplasia statistically using the ratio between the depth of primary fissure and the length of appropriate Purkinje layer. We suggest that the critical period of VPA neurotoxicity would be E14 to E16, and the critical concentration would be 300 mg/kg or more.

2P-16 Recovery effects from chemical-induced autistic degeneration with bumetanide or oxytocin in developing rat cerebellum Koichiro Ikai¹，Sarii Nakajima¹，Tatsuro Tomida¹，Susumu Ueno²，Yuko Sekino⁴，Yasunari Kanda³，Sachiko Yoshida¹ ¹Dept Environ Life Sci, Toyohashi Univ of Tech, Toyohashi, Japan，²Univ Occupational Environmental Hlth, Kitakyushu, japan，³Natl Inst Health Sci, Tokyo, Japan，⁴University of Tokyo,Tokyo, Japan Background: Autism Spectrum Disorder (ASD) is a disease characterized by repetitive behavior and interest, dysfunction of communication and social interaction. Sodium Valproate (VPA), one of the HDAC1 inhibitor, is known as an anti-epileptic drug, and a powerful inducer of human ASD. We have observed the irregular elongation of Purkinje cell dendrites started earlier than control and excess folding in cerebellar lobules, especially between lobule V and VII (primary fissure) in VPA 600mg/kg at E16-administrated rat. Recently, it was reported that oxytocin (OT), a neuropeptide hormone, or bumetanide (BU), a blocker of the cation-chloride co-transporter, could decrease ASDs repetitive behaviors. In this report, we investigated the recovery effects of Bu or OXT to rat autistic models. Methods: 600mg/kg P.O. VPA was administrated to pregnant rat at E16. Offspring was treated with 33µg/kg per day of OXT or Bu for some days from postnatal day 3 (P3) to P8. Some animals without VPA administration were treated with OXT during same period of VPA-administrated one. The cerebellum of pups were observed at P15, P21, P31 with immunohistochemistry and Hematoxylin/eosin staining.Result: The abnormal elongation of Purkinje cells and excess folding between lobule V and VII were relieved by one week OXT or Bu treatment. Single treatment showed little recovery effects. Conclusions: Both OXT and Bu would induce the function of γ-amino butyric acid (GABA) from excitation to inhibition in the developing cerebellum. Our results suggest the possibility of recovery treatment from ASD.		2P-17 Dendritic spine density and morphology of hippocampal neurons in arcadlin/protocadherin-8 deficient mice Kaede Yamamoto¹，Yuki Shin¹，Nanano Mizuta¹，Risako Harada¹，Kazuyoshi Kousaka¹，Keiko Tominaga²，Hiroko Sugiura³，Shin Yasuda³，Kanato Yamagata³，Hidekazu Tanaka¹ ¹Dept Biomed Sci. Grad Sch Life Sci, Ritsumeikan Univ，²Front. Biosci. Grad Sch, Osaka Univ.，³Synaptic Plasticity Prj. Tokyo Met. Inst. Med. Sci. Hippocampi of depressive patients and in animal models of depression become atrophic. In them, dendritic arbors shrink and spines decrease in number. On the contrary, successful treatments of depression with antidepressants or electroconvulsive seizure (ECS) recover hippocampal volume and spine density. Upon ECS, hippocampal neurons induce various immediate early genes, such as egr3/pilot, rheb, cpg15/neuritin/ieg68, narp, and arc/arg3.1. Among them, arcadlin/protocadherin-8 (acad) is a member of non-clustered-type δ2-protocadherin, and is induced transiently in dentate granule cells and CA1-CA3 pyramidal cells by ECS. Interestingly, we have found that long-term (18 days), but not short term (less than 18 days), treatment with an antidepressant, fluoxetine, induces acad in hippocampal neurons. In previous studies, we have also shown that acad negatively regulates the spine density of cultured hippocampal neurons by partially internalizing N-cadherin, which is known to be required for spine morphogenesis. To confirm these paradoxical responses in brain, we visualized the dendritic morphology of hippocampal neurons in acad^-/- mice by filling with Lucifer Yellow. Hippocampal neurons in acad^-/- mice showed higher density than in wild type (WT). The data suggest that acad suppresses spine density also in vivo. Although ECS and antidepressants elevate acad level in neurons, acad has rather a negative influence on the spine density. Acad may not mediate the anti-depression mechanism, but rather neutralize excess responses induced by anti-depression treatments. Detailed analyses of spine morphology and density of WT/acad^-/- mice treated with ECS will be also presented.

2P-18 Regulation of COPI vesicle transport via Scyl1 methylation in the pathogenesis of ER-stress induced neurodegenerative diseases Genki Amano¹，Shinsuke Matsuzaki²，Yasutake Mori³，Hironori Takamura¹，Sarina Han¹，Sho Shikada¹，Hiroki Sato¹，Mai Ito¹，Ko Miyoshi¹，Takeshi Yoshimura¹，Taiichi Katayama¹ ¹Molecular Brain Science, United Graduate School of Child Development, Osaka Univ，²Department of Pharmacology, Wakayama Medical University，³Department of Anatomy and Neuroscience, Graduate School of Medicine, International University of Health and Welfare Cumulative evidences have shown the importance of ER-stress in pathology of neurodegenerative diseases, such as Alzheimer’s disease, Amyotrophic lateral sclerosis, etc. Previous studies have indicated that accumulation of unfolded protein response (UPR) by ER-stress is related to the pathology of neurodegenerative diseases. To elucidate the pathogenesis of neurodegenerative diseases from the point of view of ER-stress, we investigated the altered gene expression in SK-N-SH cells under the condition of tunicamycin-induced ER-stress by the gene fishing method. As the result, we found that Protein arginine N-methyltransferase 1 (PRMT1) is up-regulated in SK-N-SH cells under ER-stress. Based on this result, we examined the role of PRMT1 in the ER-stress pathway. PRMT1-knockdown cells showed the abnormal Golgi formation and increased UPR. To elucidate the mechanism of these alterations, we screened the methylated proteins under ER-stress condition by immunoprecipitation-mass spectroscopy, and identified Scy1-like protein 1 (Scyl1). Scyl1, a member of the Scy1-like family of catalytically inactive protein kinases, was recently reported to function in retrograde COPI-mediated intracellular transport. Interestingly, Scyl1 has also been identified as a gene product that is lost in an animal model of motor neuron disease, the muscle-deficient mouse. In the motor neuron of the above model animal, the protein circulation system between ER and Golgi apparatus was abnormal due to dysfunction of COPI transport. In consequence, UPR may be accumulated. Thus, we present the effect of Scyl1 arginine methylation on the COPI vesicle transport. This study provides novel insights into the pathogenesis of neurodegenerative diseases by UPR accumulation.		2P-19 TULP3 regulates the formation of primary cilia in hTERT-RPE1 cells Sarina Han，Ko Miyoshi，Sho Shikada，Genki Amano，Hiroki Sato，Mai Ito，Hironori Takamura，Takeshi Yoshimura，Taiichi Katayama Dept Child Development & Mol Brain Sci, United Grad Sch Child Development, Osaka Univ The primary cilia are known as bio-sensors that receive extracellular signals and transduce them to the cell body in vertebrate cells. Impairment of cilia-mediated signaling causes ciliopathies characterized by cognitive deficits, obesity, retinal degeneration, etc. Some G protein-coupled receptors (GPCRs), such as appetite-regulating ciliary receptors, Somatostatin receptor 3 (Sstr3) and Melanin-concentrating hormone receptor 1 (Mchr1), are localized to primary cilia in rodent neurons. Similar to phenotype of ciliopathy, spontaneous “tubby” mice also show obesity, retinal dystrophy. Interestingly, in the tubby mouse brain, ciliary receptors Sstr3 and Mchr1 fail to localize to the primary cilia of neurons. Tub was identified as a responsible gene for tubby mouse. Tub and Tubby-like proteins (Tulp1~4) share the carboxy-terminal tubby domain and are collectively referred to as Tubby family proteins. Both Tub and Tulp3 are expressed in brain. In humans, a mutation in the TUB is associated with learning difficulty, obesity and retinal dystrophy. Recently, it is reported that TULP3 is a general adapter for trafficking of multiple ciliary GPCRs, as well as hedgehog signaling-regulating ciliary receptor, Gpr161. Disruption of mouse Tulp3 is embryonically lethal, including neural tube closure defect and so on. However, the underlying defects in the function of TULP3 at primary cilia remain unknown. In this study, we used the genome editing CRISPR/Cas9 system to disrupt the TULP3 gene in hTERT-RPE1 cells and measured cilium number, length and Gpr161 content by immunofluorescence in hTERT-RPE1cell lines with biallelic disruption of the TULP3 gene. TULP3 ablation affects cellular phenotypes such as cilium number, length and Gpr161 content.

2P-20 Exploring factors interacting with G-protein-coupled receptors at the primary cilium Sho Shikada，Ko Miyoshi，Takeshi Yoshimura，Sarina Han，Genki Amano，Hiroki Sato，Mai Ito，Hironori Takamura，Taiichi Katayama Dept Child Development & Mol Brain Sci, United Grad Sch Child Development, Osaka Univ On the surface of nearly every neurons in vertebrates there are protrusions of the cell membrane called primary cilia. In recent years, primary cilia have been studied as having a role as a non-synaptic sensory surveying various signals, and transmitting those signals into the cell. Defective formation or function of primary cilia is implicated in the pathogenesis of many human developmental disorders and diseases, termed ciliopathies. There are multiple G protein-coupled receptors (GPCRs) as signaling factors in primary cilia, and it has been clarified that many of them are important for various nerve functions. Certain GPCRs, including serotonin receptor 6 (HTR6) and somatostatin receptor 3 (SSTR3), selectively localize to neuronal primary cilia, and it has been suggested that these GPCRs are implicated in learning and cognition. Meanwhile, since serotonin receptor 7 (HTR7) and somatostatin receptor 5 (SSTR5) highly homologous to them do not localize to primary cilia, localization of GPCRs in primary cilia is specifically selected by other factors. Therefore, in this study, in order to identify regulatory factors of GPCR localization in primary cilia, we performed affinity column chromatography using GST-tagged fusion proteins of GPCRs, and attempted to search for factors that specifically bind to GPCRs localizing to primary cilia including HTR 6 and SSTR 3.		2P-21 Fibronectin on extracellular vesicles mediates their uptake into oligodendrocyte precursor cells Sho Osawa^1,2，Masashi Kurachi²，Hanako Yamamoto²，Yasuki Ishizaki² ¹Department of Neurosurgery, Gunma University Graduate School of Medicine，²Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine We previously demonstrated transplantation of brain microvascular endothelial cells (MVECs) into the white matter infarction reduced infarction area and improved animal’s behavioral outcome by increasing the number of oligodendrocyte precursor cells (OPCs). Our in vitro analysis revealed extracellular vesicles (EVs) from MVECs promoted OPC survival and proliferation. In this study, we analyzed the molecular mechanism how EVs from MVECs (MVEC-EVs) affect OPC behavior, focusing on proteins. We isolated OPCs from postnatal rat cerebral cortices by the immunopanning method, and cultured them in serum-free medium containing PDGF as a mitogen for several days. MVECs were prepared from adult rat cerebral cortices, and cultured in endothelial cell growth medium. We prepared EVs from conditioned medium of MVEC culture using the exosome precipitation solution. Protein mass spectrometry and ELISA revealed fibronectin is abundant on the surface of MVEC-EVs. Although fibronectin is reported to promote survival and proliferation of OPCs via integrin signaling pathway, we couldn’t attenuate the effects of EVs on OPCs by blocking the binding between fibronectin and integrins using RGD sequence mimics. Fibronectin is also reported to be involved in EV endocytosis. Flow cytometry and image analysis revealed EV uptake was decreased by interfering the interaction between fibronectin on EVs and heparin sulfate proteoglycan on OPCs. The decrease in the uptake of EVs attenuated their survival/proliferation promoting effect on OPCs. These results indicate fibronectin on the surface of EVs contributes to their internalization into OPCs, and thereby promotes OPC survival and proliferation, independent of integrin signaling pathway.

2P-22 Resveratrol suppresses brain tumor cell growth through the AMPK-mTOR signaling. Ayana Kawase，Hiroyuki Nawa，Nobuyuki Takei Dept Molecular Neurobiology, Brain Research Institute, Niigata Univ. Mammalian target of rapamycin (mTOR) is a kinase that is a master molecule of cell growth. Therefore, mTOR inhibitor, rapamycin and its analogs (Rapalogs) are used as an anticancer drug. However, because its crucial roles in cell growth, mTOR inhibitors often raise adverse side effects. Activity of mTOR is controlled by several upstream regulators. Among them, AMP-activated protein kinase (AMPK) suppresses mTOR. AMPK is known as a cellular energy sensor. Resveratrol is a polyphenol that is a plant cell origin. This substance is also famous as a polyphenol of red wine and also widely used as dietary supplements for a long time. So its safety for human has been established. Resveratrol is reported to activate AMPK, so that it is expected to suppress mTOR signaling pathways. In this study, we aimed to analyze the antitumor action of resveratrol, a safe dietary component. The strategy is targeted on upstream molecule, AMPK, not directly on mTOR itself, to reduce the severe side effects. Medullblastoma and glioblastoma cell lines (DAOY and U87MG, respectively) were used and analyzed. Resveratrol suppressed the cell growth of both DAOY and U87MG cells, as revealed by CCK-8 assay. Incorporation of BrdU was also inhibited by resveratrol. The results suggest resveratrol suppresses cell growth and inhibits cell proliferation. We also analyzed the changes of upstream and downstream molecules of mTOR by Western blot analysis. Resveratrol activated AMPK and its direct substrate, acetyl CoA carboxylase. Further investigation was performed on mTOR substrates, p70S6K and 4EBP. Protein synthesis rates were also examined. The results suggest that resveratrol suppresses tumor cell growth through AMPK-mTOR mediated translational control.		2P-23 Expression of Non-clustered δ-2 Protocadherins in the Central Nervous System Riku Itsugu¹，Natsumi Yamaguchi¹，Hironobu Takaya¹，Kazuyoshi Kousaka¹，Hiroko Sugiura²，Shin Yasuda²，Kanato Yamagata²，Hidekazu Tanaka¹ ¹Dept Bio Med Sci. Grad Sch Life Sci. Ritsumeikan Univ，²Synaptic Plasticity Prj. Tokyo Met. Inst. Med Sci During neural development, neurons are organized into sophisticated circuitry. Cadherin family is one of the candidate factors for the establishment of neural circuitry. Protocadherins (pcdhs) constitute large subfamilies within cadherin superfamiliy. They encode single-pass transmembrane proteins with 6 or 7 extracellular cadherin repeats, and grouped into two subfamilies, clustered and non-clustered. Clustered pcdhs are tandemly assembled on the chromosome 18 into α-, β-, and γ-Pcdh clusters in mouse. In contrast, non-clustered pcdhs include 15 genes scattered over different chromosomes. Non-clustered pcdhs are further classified into three subfamilies, δ1, δ2, and ε. Among the δ2-pcdhs, Arcadlin (Pcdh8/Acad) is unique in that it is expressed by neural activity such as epilepsy. Another δ2-pcdh member, Pcdh19, is also interesting; point mutation or deletion of Pcdh19 induces epilepsy. In addition, Acad, Pcdh17 and Pcdh19 have short and long spliced forms. In this study, we investigated the expression of δ2-pcdhs in the murine brain. We found that, δ2-pcdhs are broadly expressed in central nervous system, and that Acad and Pcdh17 is relatively enriched in the cerebral cortex. Among the δ2-pcdhs, Acad, but no other δ2-pcdhs, was induced by electroconvulsive seizure (ECS) specifically in the hippocampus. The induction was dominantly observed in the short form but little in the long form. Expressions of δ2-pcdhs except for acad did not differ between WT and Acad ^-/- mice, suggesting that acad is not compensated with other δ2 members.