一般演題（ポスター）

一般演題（ポスター） Glia・Myelin II

2P-24 Analysis of PKC-dependent phosphorylation and cell adhesion property of myelin P0 readthrough isoform（L-MPZ） Yamaguchi Yoshihide，Yano Noriko，Sato Saki，Tabei Naruya，Nakanishi Hiroki，Baba Hiroko Dept. of Mol. Neurobio., Tokyo Univ. of Pharm. ＆ Life Sci. Homophilic interaction of PNS myelin protein zero（MPZ/P0）between the extracellular Ig domain leads to tight adhesion between each layers in myelin. This adhesion is affected by the PKC-dependent phosphorylation site in the cytoplasmic region of P0. A novel readthrough isoform of P0, large myelin protein zero（L-MPZ）, has the same PKC-dependent phosphorylation site as well as an additional putative PKC phosphorylation site in the extra L-MPZ specific domain. Since L-MPZ is localized in the PNS compact myelin and cell-cell adhesion sites in the L-MPZ transfected cells, L-MPZ may be potentially involved in cell adhesion and myelination. However, adhesion activity of L-MPZ and role of PKC-mediated phosphorylation are still unknown. To elucidate PKC phosphorylation of L-MPZ, we performed Western blot analysis of rat sciatic nerve homogenate using phospho-（Ser）PKC substrate antibody. PKC phosphorylation of L-MPZ was detected in the unique two-dimensional electrophoresis system using cationic detergent. The increase of phosphorylated L-MPZ was observed during early postnatal development of sciatic nerve. Additionally, two states of phosphorylation in L-MPZ were demonstrated by Western blotting using Phos-tag. Next, to clarify adhesion activity of L-MPZ, we performed the adhesion assay using HeLa cells which semipermanently expressed P0, L-MPZ, or phosphorylation site mutants. L-MPZ exhibited a cell adhesion activity which was clearly weaker than P0. This binding activity was affected by mutation of phosphorylation sites. Further, heterophilic binding of L-MPZ to P0 was demonstrated by fluorescence-labeled cells. These results suggest that content of L-MPZ in P0-rich myelin membrane may be related to flexibility of myelin structure which affects myelin function.		2P-25 Mechanical stress disrupts neuron-glia interactions at nodes of Ranvier Otani Yoshinori，Susuki Keiichiro Wright State University, Neuroscience, Cell Biology and Physiology Peripheral nerves are often exposed to mechanical stress leading to compression neuropathies such as carpal tunnel syndrome. The mechanisms of how mechanical forces cause peripheral nerve dysfunction still remain largely unknown. In myelinated nerve fibers, action potential conduction depends on high densities of voltage-gated sodium channels at the nodes of Ranvier. At paranodes flanking nodes, myelinating Schwann cells interact with axons and form junctions that restrict the mobility of sodium channel complex at nodes. We hypothesized that peripheral nerve compression alters the molecular composition of nodes and paranodes causing nerve conduction failure. To test this hypothesis, we utilized a chronic nerve compression model in which a silastic tube is placed around the mouse sciatic nerves. Two weeks after compression, motor nerve conduction velocity was significantly decreased across the compression site. Immunohistochemistry showed dispersed and reduced clusters of paranodal proteins that form axon-glial junctions. Clusters of nodal proteins were occasionally elongated in association with severely disrupted paranodal junctions. Quantitative PCR showed that cysteine protease calpain2 was up-regulated in the compressed nerves. Cytoskeletal proteins alpha II and beta II spectrins expressed in Schwann cells were down-regulated in the compressed nerves, whereas these spectrins were presumably proteolyzed by activated calpain2. These results suggest that the disruption of nodes and paranodes contributes to nerve conduction failure in compression neuropathies. Calpain-mediated proteolysis together with Schwann cell gene modulation may be involved in the disruption and/or rearrangement of paranodal structures during peripheral nerve compression.

2P-26 Phosphoglycerate mutase 1 is concentrated in the paranodal loops of myelinating Schwann cells Hayashi Akiko，Hata Ryosuke，Imabuchi Nobuto，Wakabayashi Ayumi，Baba Hiroko Dept. of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences Phosphoglycerate mutase 1（PGAM1）catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis.Recently, autoantibodies against PGAM1 have been found in sera from patients with multiple sclerosis by proteomics-based analysis. However, why the PGAM1 autoantibodies are produced in these patients is uncertain. In the present study, we examined distributions of PGAM1 and glycolysis related enzymes in rodent CNS and PNS to understand why the PGAM1 autoantibodies are produced and contribute to demyelinating disease.Western blot analysis showed that PGAM1 is abundantly detected in the PNS as well as the CNS. Immunohistological analysis showed that PGAM1 was present in GFAP-positive cell bodies and their processes including perivascular end feet and myelinated tracts in corpus callosum, white matter of spinal cord and cerebellum.In white matter, PGAM1 was co-localized with MBP-positive signals, indicating that this enzyme is enriched in myelin sheath. In contrast, PGAM1 was mainly observed in paranodal loops of the PNS myelin. No prominent staining was found in the cell bodies of non-myelinating Schwann cells, perinuclear cytoplasm of myelinating Schwann cells, nor Schmidt-Lanterman incisures where Schwann cell cytoplasm was present.Staining intensity was significantly reduced in the mice with disruption of paranodal axo-glial junction.Thus, present results suggest that PGAM1 is present in astrocytes and myelin and may contribute to glycolysis and energy metabolism in the CNS and PNS.Abundance of this enzyme in myelin membrane may be related to production of autoantibodies against PGAM1 in demyelination.		2P-27 DBZ, a CNS-specific DISC1 binding protein, positively regulates oligodendrocyte differentiation Shimizu Shoko，Miyata Shingo，Tanaka Takashi，Tohyama Masaya Div. of Mol. Brain Sci., Res. Inst. of Traditional Asian Med., Kinki Univ. Schizophrenia（SZ）is a serious and disabling mental disorder with a lifetime prevalence of about 1% of the population worldwide, and commonly has a chronic course. The underlying pathological mechanisms are still largely unknown, but a growing body of evidence suggests that it is a multifactorial disorder influenced by genetic, neurodevelopmental and social factors. Disrupted-in-schizophrenia 1（DISC1）is a gene disrupted by a（1；11）（q42.1；q14.3）translocation that segregates with major psychiatric disorders including schizophrenia, recurrent major depression and bipolar affective disorder in a Scottish family. Here we report that DBZ（DISC1 Binding Zinc-finger protein）, a brain-specific member of DISC1 interactome, positively regulates oligodendrocyte differentiation. In an in vitro oligodendrocyte primary culture, the expression of DBZ was increased after induction of differentiation of oligodendrocyte by deprivation of PDGF and siRNA knockdown of DBZ decreased the expression level of myelin related markers such as MBP, MAG and CNPase. In mouse corpus callosum, DBZ mRNA expression in oligodendrocyte was intense at P7, the period of myelination, and it was hardly detectable in adult by in situ hybridization. Furthermore, a delay of oligodendrocyte maturation in DBZ knockout mice was revealed by the electron microscope analysis. These results indicate that DBZ is involved in oligodendrocyte differentiation. As multiple lines of evidence obtained by brain imaging, studies in postmortem brains and genetic association studies have implicated oligodendrocytes and myelin dysfunction in SZ, these results may provide important clue about theunderlying etiology of SZ.

2P-28 Differential expression and distribution of myosin superfamily in oligodendrocyte Yamazaki Reiji，Yamaguchi Yoshihide，Ishibashi Tomoko，Baba Hiroko Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences Motor proteins are required for myelin formation and maintenance. One of motor proteins, unconventional myosin Va（Myo5a）has been reported to control morphology of oligodendrocytes（OLs）and CNS myelination. In the transcriptome database of OLs, another unconventional myosin VI（Myo6）mRNA has been found in O4-positive differentiated OLs. Recently, we revealed that the other unconventional myosin Id（Myo1d）is expressed in mature OLs（Yamazaki, R., et al., 2014）and is required for myelin-like-membrane formation in vitro. However, the expression and the localization of these three unconventional myosins in OLs are still unclear. To examine the expression of myosin superfamily in OLs in vivo, we performed immunofluorescence staining using cuprizone induced demyelination model mice. We revealed that all myosins are present in OLs in vivo. Their expressions in OLs are reduced in parallel with demyelination and then recovered in remyelination. To clarify the timing of expression during differentiation process and intracellular distribution, we performed double immunostaining with stage-specific OL makers and each myosin antibody using cultured OLs. Myo5a signals were detected in all stage of differentiation from A2B5-positive early progenitor to mature OL. Myo6 signals were present in most of O4-positive OLs but not in early progenitors. Myo1d were found only in mature OLs. In cultured mature OLs, Myo5a signals were distributed in main thicker processes, Myo6-positive signals were detected in MBP-positive myelin-like membrane sheets, whereas Myo1d was enriched in the leading edge of these membrane sheets. Therefore, these myosins may have different roles in developing OLs. Thus, these myosins may be involved in myelin formation and remyelination by the different ways.		2P-29 Kallikrein 6-mediated CNS myelin pathology in experimental autoimmune encephalomyelitis. Bando Yoshio¹，Bochimoto Hiroki²，Nomura Taichi¹，Tanaka Tatsuhide¹，Watanabe Tsuyoshi²，Yoshida Shigetaka¹ ¹Dept. of Functional Anat. ＆ Neurosci., Asahikawa Med. Univ.，²Dept. of Microscopic Anat. ＆ Cell Biol., Asahikawa Med. Univ. Multiple sclerosis（MS）is the most common chronic inflammatory demyelinating disease of the CNS. Demyelination and axonal damage are responsible for neurological deficits in MS. However, the mechanism of demyelination has not been fully understood. We have reported that Kallikrein 6（KLK6）, a serine protease secreted by mainly oligodendrocytes in the CNS, plays the crucial role in the EAE pathogenesis. Here, we examined KLK6-mediated morphological changes of myelin in MOG35-55-induced experimental autoimmune encephalomyelitis（MOG-EAE）. Osmium-maceration scanning electron microscopic（SEM）analysis displayed the ultrastructural abnormalities of myelin in the white matter of the EAE spinal cord. In the acute phase of EAE, myelin detachment from the axon was observed in wild-type mice at day 3 after MOG immunization. At this point, infiltrating immune cells into the CNS were not observed in the spinal cord of wild-type mice. On the other hand, KLK6 knock out mice inhibited myelin detachment from the axon even at day 7 after MOG immunization. These observations suggest that KLK6 plays a crucial role for demyelination at the acute phase of MOG-EAE.

2P-30 Brain microvascular endothelial cells promote survival of oligodendrocyte precursor cells Iijima Keiya¹，Kurachi Masashi²，Shibasaki Koji²，Naruse Masae²，Yoshimoto Yuhei¹，Mikuni Masahiko³，Ishizaki Yasuki² ¹Department of Neurosurgery, Gunma University Graduate school of medicine，²Department of Molecular and cellular Neurobiology, Gunma University Graduate school of medicine，³Department of Psychiatry and Neuroscience, Gunma University Graduate school of medicine Background and Purpose We previously showed that transplantation of brain microvascular endothelial cells（MVECs）stimulated remyelination in the white matter lesion induced by endothelin-1（ET-1）injection and improved the behavioral outcome. In the present study, we examined the effect of MVEC transplantation on the behavior of oligodendrocyte lineage cells（OLCs）in vivo. We also examined the effect of conditioned medium（CM）from MVEC cultures on survival of oligodendrocyte precursor cells（OPCs）in vitro. Additionally, we confirmed the therapeutic effect of MVEC transplantation on the white matter lesion using magnetic resonance（MR）imaging.Methods MVECs prepared from rat cerebral cortex were transplanted into ET-1-induced demyelinating lesion in the internal capsule（IC）of rat brains. Cell densities of OPCs, OLCs, immature oligodendrocytes（OLs）and mature OLs, apoptotic deaths of OPCs, and proliferative state of OLCs in and around the ET-1-induced lesions in IC of MVEC-transplanted animals were analyzed. The effect of CM from MVEC cultures on OPCs was analysed by counting pyknotic nuclei in OPC cultures. MR imaging was used to examine the changes in ischemic white matter lesions.Results MVEC transplantation reduced apoptotic death of OPCs and increased the number of OPCs and immature OLs in and around the ischemic lesion in the IC without increasing their proliferation. All these effects were independent of increased angiogenesis or blood flow.Conclusions Presence of MVECs per se has a beneficial effect on ischemic white matter damage in vivo. Further study of the molecular mechanisms by which MVECs inhibit apoptotic death of OPCs may lead to the establishment of a therapeutic strategy against ischemic demyelinating diseases.		2P-31 Effect of exosomes derived from vascular endothelial cells on OPC survival, proliferation and motility Kurachi Masashi¹，Mikuni Masahiko²，Ishizaki Yasuki¹ ¹Mol. Cell. Neurobiol., Gunma Univ. Grad. Sch. of Med.，²Psychiat. Neurosci., Gunma Univ. Grad. Sch. of Med. We examined the effect of brain microvascular endothelial cell（MVEC）transplantation on rat white matter infarction, and found that MVEC transplantation promoted remyelination of demyelinated axons in the infarct region. To help clarify the molecular mechanism of this phenomenon, we examined in vitro the effect of exosomes derived from MVECs and other cells on oligodendrocyte precursor cells（OPCs）. We isolated OPCs from postnatal day 0-2 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. Human umbilical vein（HUVECs）and aortic（HAECs）endothelial cells, immortalized mouse cerebral endothelial cell line（BEND3）and rat fibroblast-like cell line（Rat-1）cells were also used. We prepared exosomes from conditioned medium of each culture, using exosome precipitation solution. After 2 days in culture, there were significantly less number of pyknotic OPCs in the presence of exosomes derived from MVECs, HUVECs and HAECs when compared to control. A larger number of BrdU-positive OPCs were seen in the presence of exosomes derived from endothelial cells（MVECs, HUVECs, HAECs and BEND3）when compared to control. We also examined the effect of exosomes on motility of OPCs. OPCs migrated longer in the presence of exosomes derived from endothelial cells when compared to control. These results suggest that exosomes derived from endothelial cells promote survival, proliferation and migration of OPCs. Identification of molecules contained in the exosomes derived from endothelial cells may be useful for establishment of the therapeutic strategy against demyelinating diseases.

2P-32 Microglia-dependent neurodegeneration in demyelinating mouse model Shimizu Takeshi^1,2，Smits Ron³，Ikenaka Kazuhiro^1,2 ¹Natl. Inst. for Physiol. Sci. （NIPS）, Div. of Neurobiol. and Bioinfo.，²SOKENDAI（The Graduate University for Advanced Studies），³Dep. of Gastroenterology and Hepatology, Erasmus MC University Oligodendrocytes are glial cells that myelinate neuronal axons in the central nervous system. Neural network is disrupted when demyelinating diseases occur, such as multiple sclerosis. In the Experimental Autoimmune Encephalomyelitis（EAE）demyelinating mouse model, microglia respond to EAE-induced inflammation and produce various cytokines including IL-1β. Since expression of non-canonical Wnt signaling components in the EAE spinal neurons was observed beside cytotoxic M1 microglia, we postulate the interplay of the activated microglia and non-canonical Wnt pathway in the surrounding EAE spinal neurons. Application of recombinant IL-1β to cultured spinal neurons or co-culture of microglia up-regulated non-canonical Wnt signaling components. While neuronal degeneration was observed in the spinal cord of demyelinating EAE mice, IL-1β or activated microglia induced neuronal cell death via Ror2-c-Jun N-terminal kinase（JNK）pathway. The expression of non-canonical Wnt signaling components was significantly increased in the spinal cord of EAE mice. In vivo analysis of Wnt5a transgenic mice mating with a chronic demyelinating mouse model indicates that non-canonical Wnt signaling aggravates the demyelinating pathology through neurodegeneration. Activated microglia and Wnt-Ror2-JNK axis may provide a possible candidate target for therapeutic approaches to demyelinating disorders.		2P-33 Mitochondrial fission and elongation in microglia induced by activation with LPS Katoh Mitsuhiko¹，Bao Wu¹，Nguyen Bang Huy¹，Quynh Thai Truc¹，Sakoh Takashi¹，Saitoh Yurika¹，Saitoh Sei¹，Shinozaki Youichi²，Koizumi Shuichi²，Ohno Nobuhiko¹ ¹Departments of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi，²Departments of Neuropharmacology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi Macrophage/microglia activation is important for pathophysiology of central nervous system disorders including demyelinating diseases. Although mitochondrial biogenesis and metabolism modulate behavior of activated monocytes and peripheral macrophages, mitochondrial changes in activated microglia are largely unknown. In this study, we investigated mitochondrial morphology in lipopolysaccharide（LPS）-induced activation of primary microglia culture. Mixed glial cultures were prepared from neonatal C57BL/6 mice, and microglia were purified following 7-10 days of maintenance. The cultured microglia were activated by 1 μg/mL LPS with or without treatment with 1 mM N-acetyl-L-cysteine（NAC）, an antioxidant, and morphology of microglia and their mitochondria as well as phosphorylation state of mitochondrial fission protein, Drp1, were examined after fixation and immunostaining. Immunostaining for a microglial marker, Iba1, revealed that the culture contained ＞95% of microglia, and LPS stimulation induced typical changes of microglial morphology during the first few hours. Immunofluorescence of multiple mitochondria markers, including TOM20, COX I, COX Va and VDAC1, showed that microglial mitochondria after 1-3 hours of stimulation were significantly shorter compared with those under vehicle treatment and also those after 6-12 hours of stimulation. Consistently, the measurements of immunofluorescence intensity showed that phosphorylation of Drp1 at Ser 616, which induces Drp1 activation, was increased during the first few hours after stimulation. The decrease of mitochondrial length during the first few hours of stimulation was partially inhibited by treatment with NAC. These results indicate that mitochondrial fission and subsequent elongation are induced by stimulation with LPS, and at least partially mediated by phosphorylation of Drp1 at Ser616 and production of reactive oxygen species.

2P-34 Cell-cell interactions via CD47-SIRPα signal regulate microglial activation Hashimoto Miho¹，Nozu Tomomi¹，Urano Eriko¹，Saito Yasuyuki²，Kotani Takenori²，Murata yoji²，Matozaki Takashi²，Ohnishi Hiroshi¹ ¹Dept. Lab. Sci., Gunma Univ. Grad. Schl. Health Sci.，²Div. Mol. Cell. Signal, Dept. Biochem. Mol. Biol., Kobe Univ. Grad. Schl. Med. Signal regulatory protein α（SIRPα）, an immunoglobulin superfamily（IgSF）membrane protein, mediates cell-cell communication signal by interacting with CD47, another IgSF membrane protein. SIRPα is predominantly expressed in dendritic cells（DCs）or macrophages in the immune system, while both SIRPα and CD47 are predominantly expressed in neurons of the brain. In the central nervous system（CNS）, SIRPα is also expressed in microglia, while the functional significance of the CD47-SIRPα signal in regulation of microglial cell functions remains unclear. We found that whole-body knockout（KO）of SIRPα resulted in an increase in the number of cells that expressed CD11c, a cell marker of DCs, in the brain and spinal cord. These CD11c-positive cells were thought to be a subpopulation of brain-resident microglia, because they expressed microglial marker proteins, CD11b and Iba-1. These CD11c-positive microglia were predominantly found in white matter regions, such as corpus callosum, anterior commissure and fimbria. The number of the CD11c-positive microglia was also increased in the brain of CD47 KO mice, suggesting that the increase of CD11c-positive microglia was due to the lack of cell-cell interactions between CD47 and SIRPα. Subsequently, to evaluate inflammatory responses to lipopolysaccharide（LPS）in SIRPα KO mice, gene expression changes of proinflammatory cytokines were measured after 3hr and 24hr after LPS treatment in the brain and spinal cord. In SIRPα KO mice, LPS treatment induced elevated gene expression of proinflammatory cytokines, such as IL-1β and TNFα, compared with WT mice.These data demonstrate that deletion of CD47-SIRPα signal turns off the inhibitory control of microglial activity, leading to phenotypes such as an appearance of CD11c-positive microglia and hyper-responses to LPS in the CNS.		2P-35 Activation of mitochondrial transient receptor potential vanilloid 1 channel contributes to microglial migration. Shirakawa Hisashi¹，Miyake Takahito¹，Nakagawa Takayuki^1,2，Kaneko Shuji¹ ¹Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ.，²Dept. Clin. Pharmacol. Ther., Kyoto Univ. Hosp. Microglia, the resident immune cells in the brain, survey the environment of the healthy brain. Microglial migration is essential for many physiological and pathophysiological processes. Although microglia express some members of the transient receptor potential（TRP）channel family, there is little knowledge regarding the physiological roles of TRP channels in microglia. Here, we explored the role of TRP vanilloid 1（TRPV1）, a channel opened by capsaicin, heat, protons, and endovanilloids, in microglia. We found that application of capsaicin induced concentration-dependent migration in microglia derived from wild-type mice but not in those derived from TRPV1 knock-out（TRPV1-KO）mice. Capsaicin-induced microglial migration was significantly inhibited by co-application of the TRPV1 blocker SB366791 and the Ca²⁺ chelator BAPTA-AM. Using RT-PCR and immunocytochemistry, we validated that TRPV1 was expressed in microglia. Electrophysiological recording, intracellular Ca²⁺ imaging and immunocytochemistry indicated that TRPV1 was localized primarily in intracellular organelles. Treatment with capsaicin induced an increase in intramitochondrial Ca²⁺ concentrations and mitochondrial depolarization. Furthermore, microglia derived from TRPV1-KO mice showed delayed Ca²⁺ efflux compared with microglia derived from wild-type mice. Capsaicin-induced microglial migration was inhibited by membrane-permeable antioxidants and MAPK inhibitors, suggesting that mitochondrial TRPV1 activation induced Ca²⁺-dependent production of ROS followed by MAPK activation, which correlated with an augmented migration of microglia. Moreover, a mixture of three endovanilloids augmented microglial migration via TRPV1 activation. Together, these results indicate that mitochondrial TRPV1 plays an important role in inducing microglial migration.

2P-36 Neuroprotective function of microglia Fujita Yuki，Yamashita Toshihide Dept. of Mol. Neurosci., Grad. Sch. of Med., Osaka Univ. Microglia are the resident macrophages in the central nervous system（CNS）. It is known that microglia are involved in the surveillance of the CNS under the physiological conditions, and are required for the construction of neural circuitry during development. We previously reported the neuroprotective behavior of microglia in the developing brain. We found that microglia accumulated along subcerebral projecting axon, and their levels peaked at postnatal day 3 to 7. Inactivation or ablation of microglia increased apoptosis in layer V subcerebral and callosal projection neurons. Further, CX3CR1 is required for the survival effect of microglia. We tested candidate factors derived from microglia, and identified that microglia-derived insulin-like growth factor 1（IGF1）supports the neuronal survival. Thus, we demonstrated the mechanism of a trophic role of microglia in the developing brain. However the mechanism of neuron-microglia interaction during a specific period remains unclear. Although it is well established that Fractalkine（CX3CL1）-CX3CR1 signaling is involved in neuron-microglia interaction, the number of microglia did not decrease in the brain of Cx3cr1-deficient mice. This suggests that Fractalkine-CX3CR1 signaling is not required for the migration of microglia. We examined whether microglia distribute along postnatal axons depending on the axon-derived factor, and what factor attracts microglia to the axon.		2P-37 Functional analysis of protein arginine N-methyltransferase 8（PRMT8）in activated microglia that are induced by spinal cord injury. Mori Yasutake¹，Koyama Yoshihisa¹，Iguchi Tokuichi¹，Miyata Shingo²，Tohyama Masaya²，Sato Makoto¹ ¹Dept. of Anat. and Neurosci., Grad. Sch. of Med., Osaka Univ.,，²Div. of Mol. Brain Sci., Res. Inst. of Trad. Asian Med., Kinki Univ. Dendritic transport of αCaMKII mRNA is mediated by recognition of the cis-acting element located on its 3´-untranslated region（3´-UTR）by putative trans-acting factor（s）. To identify the factor（s）that directly bind to αCaMKII 3´-UTR and enable the dendritic translocation of αCaMKII mRNA, we affinity-purified proteins that bound to the 15 segments of αCaMKII 3´-UTR immobilized on streptomycin Sepharose through a Strepto-tag RNA aptamer. A list of proteins, including RNA-binding proteins（RBPs）, were identified with MALDI-TOF mass analyses. Among the detected proteins, hnRNP K, a multifunctional RBP, was rich in quantity in the purified fractions from the restricted fragments of the 3´-UTR, suggesting that it binds to the 3´-UTR in a sequence-specific manner. As expected, hnRNP K knockdown of rat primary culture neurons exhibited a significant reduction of localized αCaMKII mRNA signals in the dendrites, demonstrating an involvement of hnRNP K in the mRNA transport. In this study, we further analyzed the selective activities on the mRNA transport by two hnRNP K variants with different last exons, Ka and Kb, because merely anti-Kb immunoprecipitated αCaMKII mRNA, but not anti-Ka. Accordingly, impaired dendritic localization of αCaMKII mRNA induced by hnRNP K knockdown was rescued by Kb variant, but not by Ka. To address what makes the differences in binding competence between the variants, we focused on their methylated states on arginine residues. As a result, Kb turned out to be a major methylated target by the enzyme, protein arginine N-methyltransferase（PRMT1）, in the neurons. PRMT1 knockdown also impaired the dendritic distribution of αCaMKII mRNA, just as being observed in the hnRNP K knockdown. Since the mRNA delocalization induced by PRMT1 knockdown was recovered by protein transduction of in vitro methylated recombinant Kb protein fused with cell penetrating peptide, it is likely that the Kb-specific methylation is a vital event for transport of αCaMKII mRNA.

2P-38 Acidic pH inhibits interleukin-1β production by down-regulation of mitogen-activated protein kinase activity through the TDAG8/protein kinase A pathway in mouse microglia Sato Koichi，Jin Ye，Tobo Ayaka，Tobo Masayuki，Mogi Cihiro，Okajima Fumikazu Lab. of Signal Transduct., IMCR, Gunma Univ. Objective：OGR1 family G-protein coupled receptors（GPCRs）, including OGR1, GPR4, G2A, and TDAG8, sense extracellular protons, resulting in the stimulation of intracellular signaling pathways（Okajima F. （2013）Cell. Signal., 25, 2263-2271）. It is known that proinflammatory cytokines, such as interleukin-1β（IL-1β）, are released from activated microglia and involved in neurodegeneration of acute and chronic brain disorders, such as stroke and Alzheimer disease. Extracellular acidification in brain（pH=~6.5）has been observed in ischemia and neurodegenerative disorders, in which lactate and by-products of glycolysis are accumulated in association with impairment of mitochondrial function. Acidic pH is also shown to regulate microglia functions；however, the mechanism underlying acidic pH-induced actions remains unknown. Here, we examined whether extracellular acidic pH regulates IL-1β production, especially focusing on TDAG8 in mouse microglia. Results：（1）Extracellular acidification inhibited lipopolysaccaride（LPS）-induced IL-1β production, which was associated with the inhibition of IL-1β cytoplasmic precursor and mRNA expression. （2）The IL-1β mRNA and protein responses were significantly, though not completely, attenuated in microglia derived from TDAG8-deficient mice compared with those from wild-type mice. （3）The acidic pH also stimulated cellular cAMP accumulation, which was completely inhibited by TDAG8 deficiency. （4）Forskolin and a cAMP derivative, which specifically stimulates protein kinase A（PKA）, mimicked the proton actions, and PKA inhibitors reversed the acidic pH-induced IL-1β mRNA expression. （5）The acidic pH-induced inhibitory IL-1β responses were accompanied by the inhibition of extracellular signal-related kinase（ERK）and c-Jun N-terminal kinase（JNK）activities. （6）The inhibitory enzyme activities in response to acidic pH were reversed by the PKA inhibitor and TDAG8 deficiency.Conclusion：Extracellular acidic pH inhibits LPS-induced IL-1β production, at least partly, through the TDAG8/cAMP/PKA pathway, by inhibiting ERK and JNK activities, in mouse microglia. TDAG8 may be a potential target of neurodegenerative disorders.