Poster Sessions

Poster Sessions グリア、ミエリン、神経免疫、その他

2P-37 Increased connection between mitochondria and endoplasmic reticulum in demyelinated axons Quynh Truc Thai^1,2，Bang Huy Nguyen^1,2，Yang Sui^1,2，Kazuhiro Ikenaka²，Nobuhiko Ohno^1,2,3 ¹Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan.，²Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences (NIPS), Aichi, Japan.，³Center for Multidisciplinary Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan. The connection between endoplasmic reticulum and mitochondria is known as the mitochondria associated membranes (MAM). This site has important roles for physiological functions, such as lipid metabolism, Ca2+ signaling and mitochondrial dynamics. Disruption of MAM has been implicated in mitochondrial dysfunction. Although mitochondrial dysfunction has been proposed as a major contributor of axonal degeneration in diseases of myelin, the pathophysiology of axonal degeneration is still unclear. In this study, we investigated changes of axonal MAM in demyelinated axons, using the serial block-face scanning electron microscopy (SBF-SEM) and a mouse model of chronic demyelination caused by extra-copies of proteolipid protein (PLP4e). Three-dimensional ultrastructural reconstruction of mitochondria and MAM in the optic nerves demonstrated that mitochondrial volume and surface areas and the total MAM areas on individual mitochondria were larger in the demyelinated axons of 5 month-old (mo) PLP4e compared with myelinated axons of 5mo wild-type mice. The increase of total MAM areas in demyelinated axons of PLP4e was attributable to enlargement of individual MAM, since MAM density (number of MAM / mitochondrial surface area) was similar. In the myelinated axons of 1mo PLP4e and wild-type mice, sizes of individual MAM and MAM density were similar. Immunostaining in optic nerves showed that immunoreactivity of Mfn2 is increased in demyelinated axons of 5mo PLP4e. These results suggest that enlargement of MAM is caused by chronic loss of myelin and mediated by increased expression of Mfn2 in the demyelinated axons.		2P-38 Exploring factors causing remyelination arrest by studying Cystatin F gene regulatory mechanism Jiayi Li¹，Wilaiwan Wisessmith¹，Takahiro Shimizu¹，Kenji F Tanaka^1,3，Kazuo Kunisawa¹，Takeshi Shimizu¹，Kazuhiro Ikenaka^1,2 ¹Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences，²Graduate University for Advanced Studies，³Department of Neuropsychiatry, Keio University School of Medicine Remyeliantion plays an important role in determining the fate of demyelinating disorders, however, it becomes arrested at the chronic phase. Cystatin F, a papain-like lysosomal cysteine proteinase inhibitor has been proved to be a crucial factor in regulating demyelination/remyelination. Using a chronic demyelination animal model, heterozygous proteolipid protein (PLP) transgenic 4e (PLP4e/-) mouse, we found cystatin F was induced and upregulated from the early phase of PLP4e/- mouse, when remyelination is active, and then decreased in the chronic phase, when remyelination is arrested. To explore the remyelination arrest mechanism in the chronic demyelinating disorders, we studied cystatin F gene regulatory mechanisms. We used a mouse line (CysF-STOP-tetO::Iba-tTA) in which the cystatin F gene expression is drived by the tetO promoter. We surprisingly found the cystatin F gene is forced to be expressed but its expression decreased later. Together with other data, we proved cystatin F expression was modulated at the post transcriptionally level. Then, we found two factors, embryonic lethal, abnormal vision, drosophila like RNA binding protein 1 (ELAVL-1) and miR29a, regulating cystatin F mRNA stability. ELAVL-1 as an AU rich elements binding protein, stabilized cystatin F mRNA. Its expression was downregulated together with cystatin F decreased level in both PLP4e/- mice and CysF-STOP-tetO::Iba-tTA mice. One the contrary, miR29a destabilized cystatin F mRNA. In vitro study showed ELAVL-1 siRNA and miR29a precursor downregulated cystatin F expression. All of these data revealed the important role of ELAVL-1 and miR29a in regulating cystatin F expression. It may provide a new sight in the therapy of demyelinating disorders.

2P-39 Alterations of synaptic terminals in the cerebellum under chronic demyelination Bang Huy Nguyen^1,2，Yang Sui^1,2，Quynh Truc Thai^1,2，Masahiko Watanabe³，Kazuhiro Ikenaka¹，Nobuhiko Ohno¹ ¹Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences.，²Department of Anatomy and Structure Biology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi.，³Department of Anatomy, Graduate School of Medicine, Hokkaido University, Sapporo, Japan Primary disease of myelin, including multiple sclerosis, leads to axonal degeneration and motor and sensory deficits but recent studies suggested that loss of synaptic connections in gray matter also contributes to the neurological disabilities. In this study, we investigated the synaptic changes in mouse cerebellum, using a progressive demyelination model caused by overexpression of myelin proteolipid protein (PLPTg) [1]. In PLPTg and wild-type (WT) mice at the different ages, changes of ultrastructures and molecular distribution in climbing fiber terminals were analyzed using serial block-face scanning electron microscopy (SBF-SEM) and immunohistochemistry. At 5 months of age, demyelinated axons and axons with abnormally thin myelin were prominent in the cerebellar white matter of PLPTg mice. In the molecular layers of cerebellar cortex, number and height of climbing fiber terminals were significantly reduced while quantitative SBF-SEM results showed mitochondrial volume in the terminals was increased in the PLPTg mice compared with age-matched WT mice. By contrast, the number and mitochondrial volume of the climbing fiber terminals were similar in PLPTg and WT mice at 1 month of age. In the PLPTg mice at 5 months of age, immunoreactivity of C1ql1, a member of C1q complement family regulating the formation and maintenance of climbing fiber – Purkinje cell synapses, was localized at the climbing fiber terminals, but significantly reduced compared with age-matched WT mice. These results demonstrated loss of synaptic terminals and enlargement of presynaptic mitochondria in climbing fibers of a progressive demyelination model, and suggest that regulation of synaptic organizer molecules is associated with the loss of synaptic terminals in the primary diseases of myelin.		2P-40 Unconventional myosin ID has an important role in oligodendrocyte during remyelination Reiji Yamazaki，Yoshihide Yamaguchi，Tomoko Ishibashi，Hiroko Baba Dept Mol Neurobiol. Sch Pharm Sci, Tokyo Univ of Pharm and Life Myelin is a multilamellar structure that ensheathes axon and is crucial for normal neuronal function. In the CNS, myelin is produced by oligodendrocytes (OLs) those wrap their plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we reported that myosin ID (Myo1d) is involved in formation and maintenance of myelin in cultured OLs. Myo1d is one of the unconventional myosin and our previous results suggest that it contribute to membrane dynamics either in wrapping or in transporting of myelin membrane proteins. However, the molecular mechanisms and role of Myo1d in vivo are still unclear. In this study, to clarify the function of Myo1d during remyelination in vivo, we injected siRNA in corpus callosum of cuprizone-treated demyelination mouse model using stereotaxic operation. While the intensity of NG2-positive signals and the number of CC1-positive cells were unchanged by Myo1d siRNA treatment, knockdown of Myo1d expression in vivo decreases the intensities of myelin basic protein (MBP) and proteolipid protein (PLP) immunofluorescence staining. Furthermore, Myo1d-knockdown increased the numbers of pro-inflammatory M1 type microglia and astrocytes during remyelination. In contrast, the number of anti-inflammatory M2 microglia was decreased after Myo1d-siRNA transfection. To examine the induction of apoptosis, we calculated the number of caspase3-positive cells. The percentage of caspase3-positive cells increased by Myo1d-knockdown. These results indicated that Myo1d-knockdown induces apoptosis of OLs, pro-inflammatory state, resulting in delay of remyelination. Therefore, Myo1d may be involved in survival of OL and formation of myelin during remyelination.

2P-41 Migration and differentiation of V-SVZ-derived oligodendrocyte progenitors in the injured cortex Mayaka Hashimoto¹，Naoko Kaneko¹，Seiya Yamamoto¹，Takuya Miyamoto¹，Hirohide Takebayashi²，Kazuhiro Ikenaka³，Kazunobu Sawamoto^1,4 ¹Dept Dev & Regen Biol. Grad Sch Med Sci, Nagoya City Univ，²Div Neurobiol & Anat. Grad Sch Med & Dent Sci, Niigata Univ，³Div Neurobiol & Bioinformat. National Inst Physiol Sci，⁴Div Neural Dev & Regen. National Inst Physiol Sci Oligodendrocyte progenitor cells (OPCs) distributed widely in the brain continuously generate oligodendrocytes that form myelin sheath. Small portion of the OPCs are produced in the ventricular-subventricular zone (V-SVZ), the most active neurogenic region located around the lateral ventricle. After brain injury, immature new neurons migrate from the V-SVZ toward the injured area using blood vessels and radial glial processes as scaffolds. V-SVZ-derived OPCs also migrate into the injured area, however, little is known about how they migrate and whether they contribute to oligodendrocyte regeneration after injury. Here, using a mouse model for neonatal cortical injury, we investigated migration and differentiation of the V-SVZ-derived OPCs in the injured area. V-SVZ-derived cells were labeled with GFP by electroporation at postnatal day 0 (P0)-P1, then cryogenic injury was applied to the cortex at P2. 24 hours after the injury, morphology and migration scaffolds of the GFP-labeled cells expressing the oligodendrocyte lineage marker Olig2 in the injured cortex were analyzed, and compared with those of immature new neurons by immunohistochemistry and time-lapse imaging. Most of the V-SVZ-derived OPCs showed simple monopolar or bipolar shape, and about 80% and 30% of these cells were observed in contact with radial glial processes and blood vessels, respectively, similar to those in the new neurons migrating toward the injured area. 28 days after the injury, about a half of the GFP-labeled V-SVZ-derived cells expressed a mature oligodendrocyte marker APC. These data suggest that V-SVZ-derived OPCs migrate efficiently using radial glial processes and blood vessels toward the injured cortex in neonatal mice, which contribute to oligodendrocyte regeneration.		2P-42 The effect of carba-derivative of cyclic phosphatidic acid on the repair of a stab-wounded cerebral cortex Mari Nakashima^1,3，Ayana Hamano^1,3，Kei Hashimoto^1,3,4，Hiroko Ikeshima-Kataoka^5,6，Mari Gotoh^2,3，Kimiko Murofushi²，Yasunori Miyamoto^1,3 ¹Division of Life Sciences, Graduate School of Humanities and Sciences, Ochanomizu University.，²Reserch Division of Human Welfare Science, Ochanomizu University.，³Institute for Human Life Innovation, Ochanomizu University.，⁴Research Fellow of Japan Society for the Promotion of Science.，⁵Faculty of Science and Engineering, Waseda University.，⁶Department of Pharmacology and Neuroscience, Keio University School of Medicine. Traumatic brain injury (TBI) induces blood brain barrier (BBB) break down and inflammation around the injury site. One of substances which has been known to suppress cuprizone-induced demyelination, a model of multiple sclerosis, is cyclic phosphatidic acid (cPA). cPA is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol, which inhibits chronic and acute inflammation. Therefore, it is expected that cPA would contribute to the repair of TBI. In this study, we aimed to characterize the effects of carba-derivative of cPA (2ccPA) on the repair of stab-wounded cerebral cortex.First, the effect of 2ccPA on the hemorrhage and recovery from BBB break down in the stab-wounded regions were assessed by serum immunoglobulin (IgG) extravasation. 2ccPA significantly suppressed the level of IgG extravasation in 1-7 days after the stab-wounded cerebral cortex. 2ccPA down-regulated not only inflammatory cytokine, Il-1β and Il-6 mRNA levels, but also a microglia marker, Iba1 mRNA level. Furthermore, 2ccPA down-regulated the Iba1 protein level, but not the cell number of microglia around the stab-wounded regions, suggesting that 2ccPA suppresses the activities of microglia. Activated microglia polarized to either M1 neurotoxic or M2 neuroprotective phenotype. 2ccPA remarkably down-regulated a M1 marker, Cd86 mRNA level around the lesions. In addition, the up-regulation of Il-1β, Il-6, and Cd68 mRNA by lipopolysaccaride in primary cultured microglia was suppressed by 2ccPA. Taken together, these results indicate that 2ccPA plays a critical role in the recovery of BBB breakdown and the suppression of M1 neurotoxic microglia phenotype, and contributes to the repair of stab-wounded cerebral cortex.

2P-43 Medium chain fatty acid attenuates lipopolysaccharide-induced NO production in cultured microglia Yasunori Nishimura，Mitsuaki Moriyama，Katsura Takano，Yoichi Nakamura Lab. Integrative Physiology, Vet. Sci., Osaka Pref. Univ. Microglia are brain macrophages and are major immunocompetent cells in central nervous system. In several neurodegenerative diseases such as Alzheimer’s disease (AD), microglia are activated to release nitric oxide (NO) and proinflammatory cytokines, resulting its neuropathology. Accumulating evidence suggests that dietary supplementation of coconut oil (CO) reduces the cognitive deficits associated with AD; however, the precise mechanism(s) underlying the beneficial effect of CO is unknown. CO contains large amount of medium chain fatty acids (MCFAs, C_8–12) reaching 60% of total fat, which is composed by 80% lauric acid (LA, C₁₂) and 10% octanoic acid (OA, C₈). In the present study, we examined the effects of MCFAs on microglia activated experimentally by lipopolysaccharide (LPS), using primary cultured rat microglia and mouse microglial cell line BV-2. LA attenuated the LPS (10 ng/ml)-induced NO production and the expression of inducible NO synthase protein in a dose-dependent manner, significantly more than 100 μM, although cell viability was not affected in both primary microglia and BV-2 cells. LA also decreased the LPS-induced production of intracellular reactive oxygen species at 6 h in BV-2 cells. Moreover, LA suppressed the LPS-induced phagocytosis of fluorescent-labeled 1μm microbeads. OA similarly attenuated these parameters induced by LPS at several-times higher concentrations than LA. These results suggest that attenuation of microglial activation by CO-derived MCFAs may alleviate glial and neuronal damage in AD.		2P-44 Effects of FGF2 on extracellular accumulation of adenosine by Ca²⁺ reduction in rat spinal astrocytes Ryota Eguchi，Soichiro Yamaguchi，Ken-ichi Otsuguro Lab Pharmacol. Facult Vet Med, Hokkaido Univ Extracellular adenosine (ADO) derived from astrocytes plays an important role as a neuromodulator in the central nervous system (CNS). Under ischemic conditions, it has been reported that extracellular ADO increases and mediates neuroprotective effects. It is also well known that astrocytes change their morphology and functions under pathological conditions. However, the effects of the astrocytic changes on ADO accumulation under ischemic condition remain unclear. In this study, we induced astrocytic changes by fibroblast growth factor 2 (FGF2) which increases after CNS ischemia, and investigated the effects of the changes on ADO accumulation induced by one of the ischemic stimuli, Ca²⁺ reduction. Cultured astrocytes isolated from rat spinal cord were treated with FGF2 and incubated with Ca²⁺-free artificial cerebrospinal fluid. Then purine (ATP, ADP, AMP, ADO and inosine) levels were measured by high-performance liquid chromatography analysis and luciferin/luciferase method. In the normal astrocytes, Ca²⁺ reduction evoked ATP release, resulting in ADO increase by ATP degradation. FGF2 treatment potentiated the ATP release but not the ADO increase. Potentiated ATP release was suppressed by the inhibitors of gap junction hemichannels (GJ HC). The expression and activity of GJ HC were increased by FGF2. FGF2 also potentiated the increase in ADO metabolite inosine. These results indicate that FGF2 increases expression and activity of GJ HC in astrocytes and potentiates ATP release via GJ HC. Furthermore, it is suggested that FGF2 also potentiates the degradation of extracellular purines. Under ischemic condition, astrocytes functionally changed by FGF2 may contribute to extracellular ADO accumulation.

2P-45 Analysis of neurotrophic factors in CNS-derived exosomes from plasma of patients with mood disorders. Shoshin Akamine¹，Akio Fukumori¹，Fumihiko Yasuno²，Takashi Kudo¹ ¹Department of Mental Health Promottion, Osaka University Graduate School of Medicine，²Department of Psychiatry, Nara Medical University Currently, biomarkers for mood disorders are obtained mainly by cerebrospinal fluid or positron emission tomography, which are highly invasive. Therefore, less invasive method for evaluating mood disorders is required. Peripheral blood analysis, which is less invasive, is influenced by various organs and is insufficient to reflect the state of CNS (Central Nervous System).In this study, we extracted CNS-derived exosomes from human plasma by combining exosome isolation techniques and immunoprecipitation method using anti-L1CAM (L1 cell adhesion molecule) antibodies, and compared a level of neurotrophic factors in CNS-derived exosome of mood disorders with that of normal controls. We found that IGF-1 in CNS-derived exosomes in patients with mood disorders are significantly lower compared to control.Density-gradient ultracentrifugation revealed that neuronal marker and exosomal marker proteins are present in the same density fraction, indicating that exosomes extracted in this method are CNS-derived.In previous reports, blood IGF-1 is significantly higher in mood disorder patients and it is explained that it may be compensatory regulation of the decreased bioavailability of IGF-1 in the CNS.We identified IGF-1 as a candidate for a biomarker of mood disorder, suggesting that reduction of IGF-1 signal is involved in mood disorder pathology.		2P-46 Inhibition of EGF receptor kinase activity by antipsychotic compounds Yutaro Kobayashi，Yuriko Iwakura，Hiroyuki Nawa Department of Molecular Neurobiology, Brain Research institute, Niigata University Epidermal growth factor receptor (EGFR) is expressed in dopaminergic neurons and GABAergic neurons in central nervous system. Many studies reported that the hyperactivity of EGFR is implicated in the pathology of schizophrenia. We reported that the quinazoline anticancer agents, which block EGFR kinase, can ameliorate abnormal behaviors of the animal models for schizophrenia. The main pharmacological action of antipsychotics is the competitive inhibition of monoamine neurotransmitter receptors, but many antipsychotics also have various side effects. The side effects aren’t ascribed to the inhibition of monoamine neurotransmitter receptors alone. Thus, we raised the hypothesis that commercial antipsychotic compounds also act on EGFR and contribute to their pharmacology. The inhibition of EGFR kinase was examined as follows ; Cultured cancer cells or rat cortical neurons pretreated with antipsychotic compounds, were stimulated with EGF, and subjected to immunoblotting with the anti-phospho EGFR antibody. Some of antipsychotic compounds decreased EGFR phosphorylation. In vitro kinase assay, their antipsychotic compounds directly acted on a synthetic tyrosine kinase domain of EGFR and attenuated its self-phosphorylation. The above results suggested that these antipsychotic compounds contribute to the improvement of psychiatric symptoms by not only antagonizing neurotransmitters but also inhibiting EGFR tyrosine kinase in the brain. Now, we plan to investigate inhibitory actions of those antipsychotic compounds on other tyrosine kinases as well.

2P-47 Amyloid β fibril formation on the reconstituted lipid bilayer and inhibitory effect of a GM1-cluster binding peptide Mako Nakai，Masaya Nishihara，Teruhiko Matsubara，Toshinori Sato Faculty of Sci. and Tech., Keio University The Alzheimer’s disease is the most common type of dementia associated with loss of memory and cognitive dysfunction. One of critical hallmarks of AD is deposition of the amyloid β protein (Aβ) in the brain. The Aβ has been reported to bind to the monosialoganglioside GM1 cluster in neuronal cell membranes and forms toxic oligomers and fibrils. The mechanism underlying Aβ deposition has been investigated, and the characterization of Aβ oligomers has been performed; however, a majority of therapeutic agent for AD showed only a moderate inhibition efficiency. We focused on the GM1 cluster to which Aβ binds because of its potential as a major therapeutic target against AD. In the present study, we used a GM1 cluster-binding peptide (VWRLLAPPFSNRLLP) identified by a phage display method as an inhibitor. We constructed a GM1-containing lipid bilayer (GM1/sphingomyelin/cholesterol, 10:45:45) on a Langmuir-Blodgett trough, and the interaction between Aβ and the peptide on the membrane. The surface topography was observed in the water phase by atomic force microscopy (AFM) and counted the number of Aβ fibrils to investigate the inhibitory effect of peptide. We found that the number of Aβ fibrils decreased in the presence of the GM1 cluster-binding peptide. We also showed that Aβ fibril formation was inhibited by the GM1 cluster-binding peptide before and after the administration of Aβ. Furthermore, a four-branched peptide dendrimer showed a high affinity for GM1 and inhibited Aβ fibril formation. These results suggest the potential of the GM1 cluster-binding peptide as a novel prophylactic and therapeutic agent for AD.		2P-48 A preliminary report of NAD precursors on social behaviors in mice Maria Nikolaevna Gerasimenko^1,2，Stanislav Cherepanov²，Anna Shabalova^1,2，Shigeru Yokoyama²，Haruhiro Higashida² ¹United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui，²Center for Child Mental Development. Kanazawa University NAD+ is one of the very important biomolecules, it participates in great number of vital processes like ATP synthesis, redox homeostasis and signal pathways. A lot of studies revealed positive effects of NAD+ precursors’ supplementation on physical condition in a case of neurodegenerative disorders, muscular dystrophy and senescence. In some studies anxiolytic effect of elevating NAD+ was observed. This effect can be explained by enhancing of energy metabolism and activation of sirtuins for which NAD+ is a co-factor and so influence on mTOR pathway regulation. NAD+ as a substrate in ADP-ribosyl cyclase reactions is one of the crucial components for oxytocin release. Through this pathway elevating of NAD+ contents possibly can lead to enhancing of oxytocin release and so can influence on social behavior. Recent findings suggest that hyper-activation of mTOR can play one of the crucial role in autism spectrum disorder (ASD). It makes mTOR the potential target for ASD therapy. On other hand, sirtuins use NAD+ as a co-factor and can lead to the mTOR pathway inhibition. There is another evidence that cADP-ribose works as an endogenous inhibitor of mTOR. Here we examined the role of NAD precursors in oxytocin release and the effect of them on social behaviors. For this purpose, we used two model mice lines with behavioral disturbances: First, CD38 knockout mice as a model of ASD via oxytocin release disruption; And CD 157 knockout mice as a model of anxiety. ICR and C57BL6N mice were used as control. In these mice treated with NAD precursors, first their social behaviors were observed.

2P-49 Amyloid Fibril Formation Mechanisms of αSyn in Caenorhabditis elegans Cesar Aguirre¹，Kensuke Ikenaka²，Masatomo So¹，Keiichi Yamaguchi¹，Hideki Mochizuki²，Yuji Goto¹ ¹Institute for Protein Research, Osaka University，²Graduate School of Medicine, Osaka University Ultrasonication is an experimental tool that has been widely applied to accelerate in vitro the fibril formation of amyloidogenic proteins, like α-Syn, β-2m, lysozyme, etc. The cavitation bubbles produced during ultrasonication facilitates the highly-ordered spatial arrangement that characterizes the amyloid fibrils. Then, ultrasonication is capable to break the metastable state known as supersaturation and leads to fibrillation. However, this technique cannot be applied directly to living organisms. In this work, we used a variation of ultrasonication, namely mild sonication, which enhances the formation of amyloid fibrils in alive C. elegans worms that overexpress αSyn-YFP fusion protein. Fibrils detection in vivo was performed by Confocal Microscopy and Western Blot analyses. The amyloid deposits generated inside the worms were extracted, fractioned using detergents (RIPA Buffer) and chemical denaturants (Urea and Gdn Buffers) throught ultracentrifugation, and amplified by seeding experiments, monitoring ThT fluorescence intensity. The morphology of the fibrils was studied using AFM and TEM. Our results also suggest that C-terminal truncation of α-synuclein is probably a normal cellular process. Ultrasonication seems to play the role of accelerating the aggregation of C-terminal-truncated  α-synuclein. Thus, truncation might take place naturally inside both sonicated and not sonicated worms. These processes would enrich the supersaturation metastable state in vivo leading to fibril formation.