グリア性因子と脳機能制御 Glia-derived factors and brain function
O3-6-6-1 Dock7は座骨神経の軸索径の調節には関与せず、特異的にその髄鞘肥厚化を負に制御する Knockdown of Dock7 in vivo specifically increases myelin thickness in sciatic nerves without affecting axon thickness ○山内淳司1, 宮本幸1, 鳥居知宏1, 田上昭人1 ○Junji Yamauchi1, Yuki Miyamoto1, Tomohiro Torii1, Akito Tanoue1 国立成育医療センター研究所　薬剤治療研究部1, ヒューマンサイエンス振興財団2 Dept. Pharmacol., NICHD (JAPAN), Tokyo1, The Japan Human Health Sciences Foundation, Tokyo, Japan2 During development of the peripheral nervous system (PNS), Schwann cells (SCs) wrap individual axons to form myelin sheaths, which act as surrounding insulators and markedly enhance the propagation of the action potential. In peripheral neuropathies such as inherited demyelinating Charcot-Marie-Tooth (CMT) disease and diabetic neuropathies, chronic demyelination and defective remyelination are repeated, causing more severe neuropathies. It is thus thought that development of a drug that promotes proper myelination with minimal side effects could provide an effective therapy for these diseases. As yet, however, little is known about therapeutic target molecules and genetically-modified mice for testing such approaches. We previously cloned the dock7 gene and characterized Dock7 as the regulator controlling SC myelination; however, an important issue, whether knockdown of Dock7 specifically affects myelination by SCs but not leaves neurons unaffected, has remained unclear. Here, we generate newly-produced transgenic mice harboring short-hairpin RNA (shRNA) targeting Dock7. We also describe that Dock7 shRNA transgenic mice exhibit enhanced myelin thickness without affecting axon thickness in sciatic nerves of the PNS, as reduced thickness of the axon diameter is the primary indicator of denatured neurons. Similarly, purified in vitro SC-neuronal cocultures established from transgenic mice exhibit enhanced formation of myelin segments, suggesting that knockdown of Dock7 promotes myelination by SCs. Collectively, Dock7 knockdown specifically affects SC myelination in sciatic nerves, providing evidence that Dock7 may be a promising drug-target-specific molecules for developing a therapy for peripheral neuropathies that aims to enhance myelination.	O3-6-6-2 マウス脳内に発現する新規シアル酸化N結合型糖鎖の解析 Studies on a novel sialylated N-glycan in mouse brain ○鳴海麻衣1,2, 吉村武2, 鳥居知宏2, 池中一裕1,2 ○Mai Narumi1,2, Takeshi Yoshimura2, Tomohiro Torii2, Kazuhiro Ikenaka1,2 総研大・生理科学・分子神経生理1, 自然科学研究機構・生理学研究所・分子神経生理研究部門2 Div. of Neurobiol. and Bioinfo., Dep. of Physiol. Sci., Grad. Univ. for Adv. Studies1, Div. of Neurobiol. and Bioinfo., NIPS, NINS2 Glycosylation of proteins is one of the major posttranslational modifications.N-glycans harbored on glycoproteins profoundly affect the character of proteins by altering their structure or capacity to bind to other molecules. Sialic acid is an acidic monosaccharide present at the non-reducing terminal of sugar residues attached through α2,3-, α2,6- or α2,8-linkage. Sialic acid binding Ig-like lectin (Siglec) can distinguish these linkage types. Some Siglecs are crucial for brain function.We refined a method to purify and detect trace amounts of N-glycans and analyzed sialylated N-glycans from the mouse brain (Manuscript in preparation). In this study, 6-sialyl-LewisC ([Galβ1,3(NeuAcα2,6)GlcNAc-]) was detected, which is a novel sialylated N-glycan structure found in the mouse brain. Synthetic pathway to 6-sialyl-LewisC is unclear. 6-sialyl-LewisC-harboring glycoprotein and siglec that recognize 6-sialyl-LewisC are not identified. We found that ST6GalNAc4 mRNA was mainly expressed in cerebral cortex, hippocampus and cerebellar cortex. N-glycan containing 6-sialyl-LewisC was also highly expressed in these regions. These results suggest that ST6GalNAc4 is one of enzymes that are involved in the synthesis of 6-sialyl-LewisC.To identify 6-sialyl-LewisC-harboring glycoprotein, membrane proteins of mouse brain were separated by 2D-PAGE. After CBB staining, N-glycans were extracted from protein spots and analyzed by HPLC. We found that 6-sialyl-LewisC bound to specific acidic proteins with a molecular weight of 50~70kDa. These protein spots were analyzed by LC-MS. We detected Calreticulin and Nap22 (Basp1) as a candidate for 6-sialyl-LewisC-harboring glycoprotein.It is possible that there is a specific interaction between the 6-sialyl-LewisC-harboring glycoprotein and the siglec that recognizes 6-sialyl-LewisC.
O3-6-6-3 アストロサイトの脳型脂肪酸結合蛋白質(FABP7)は脂質ラフト形成を調節する Fatty acid-binding protein 7 (FABP7) regulates lipid raft formation in the astrocytes ○香川慶輝1, 宮崎啓史1, 安本有希1山本由似1, 原伯徳1, 澤田知夫1, 徳田信子1, 小林誠2, 向後寛3, 藤本豊士4, 吉川武男5, 大和田祐二1 ○Yoshiteru Kagawa1, Majid Ebrahimi1, Kazem Sharifi1, Hirohumi Miyazaki1, Yuki Yasumoto1, Ariful Islam1, Yui Yamamoto1, Tomonori Hara1, Tomoo Sawada1, Nobuko Tokuda1, Sei Kobayashi2, Hiroshi Kogo33, Toyoshi Fujimoto4, Takeo Yoshikawa5, Yuji Owada1 山口大院・医・器官解剖学1, 山口大院・医・生体機能分子制御学2, 群馬大院・医・生体構造学3, 名古屋大学・医・分子細胞学4, 理化研・分子精神科学5 Dept. Organ anatomy, Univ of Yamaguchi, Yamaguchi1, Dept. Molecular Physiology and Medical Bioregulation, Univ of Yamaguchi, Yamaguchi2, Dept. Anatomy and Cell Biology, Univ of Gunma, Maebashi3, Dept. Anatomy and Molecular Cell Biology, Univ of Nagoya, Aichi4, Lab. for Molecular Psychiatry, RIKEN BSI, Saitama5 Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acid (LCFA), controlling intracellular lipid dynamics. FABP7 has high affinity with omega-3 LCFA including docosahexaenoic acid, and is expressed by astrocytes and oligodendrocyte precursor cells in the brain. We previously showed that FABP7 is involved in the pathogenesis of schizophrenia, but the mechanism how FABP7 affects the astrocyte lipid homeostasis is still unknown. In this study, we sought to examine the role of FABP7 in the astrocytes focusing on the lipid raft formation which regulates various receptor-mediated signal transduction in response to extracellular stimuli.FABP7-KO astrocytes showed the decrease in the expression of TNF-α and the activation of MAPKs and NFκB was impaired after LPS stimulation compared with WT. In the membrane lipid raft fractions separated by sucrose gradient centrifugation, the accumulations of lipid raft-associated proteins including TLR4, EGFR, GFRα1, and caveolin-1 were decreased in FABP7-KO astrocytes. Moreover, the expression of caveolin-1 was found to be decreased at transcriptional level in FABP7-KO astrocytes. By using luciferase reporter assay, Cav-1 promoter region of approx. 200bp upstream from the translational start codon was identified to be associated with FABP7-dependent pathway.Our results suggest that FABP7 may control the lipid raft (caveolae) formation thorough regulating the expression of caveolin-1, thereby being involved in the response of astrocytes to various external stimuli.	O3-6-6-4 Neuronal plasticity is regulated by glial fatty acid binding protein (FABP7) ○Majid Ebrahimi1, Sharifi Kazem1, Yoshiteru Kagawa1, Yui Yamamoto1, Islam Ariful1, Yuki Yasumoto1, Hirofumi Miyazaki1, Tomonori Hara1, Tomoo Sawada1, Nobuko Tokuda1, Takeo Yoshikawa2, Yuji Owada11 Department of Organ Anatomy, Yamaguchi University Graduate School of Medicine, Ube, Japan1, Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Saitama, Japan2 Brain-type fatty acid binding protein (FABP7), an intracellular chaperon for polyunsaturated fatty acids (PUFAs), is strongly expressed by astrocytes of developing and mature brain and involved in the uptake, metabolism, signal transduction and gene regulating activities of PUFAs. We previously showed that FABP7 is implicated in the control of emotional behaviors and FABP7 knockout (FABP7-KO) mouse can be a model for human functional psychosis including schizophrenia. However, the mechanism by which astrocytic FABP7 modulates neuronal activity is still unknown. This study was undertaken to clarify the role of FABP7 expression on the neuronal plasticity control. In the layer II/III of medial prefrontal cortex (mPFC) of FABP7-KO mice, the number and length of dendritic branches and the spine density of apical and basal dendrites of pyramidal neurons were significantly decreased in comparison with their wild-type littermates. Consistent with these results, the primary cultured cortical neurons significantly decreased the length of the longest dendrite, the total dendritic length, and the extent of area covered by dendritic branches in the presence of FABP7-KO astrocytes. Furthermore, a decreased number of excitatory synapses as indicated by reduction in the co-localization of excitatory presynaptic (synaptophysin and VGlut-1) and postsynaptic (PSD95) protein aggregates was seen in the layer II/III mPFC of FABP7-KO mice as well as in the primary cortical neurons grown on FABP7 deficient astrocytes. Our results suggest that FABP7 may regulate the lipid homeostasis (metabolism/signal transduction) in the astrocytes, and subsequently affect the cortical neurons plasticity.

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