髄鞘化、髄鞘-軸索相互作用 Myelination and Myelin-Axon Interaction
P1-2-15 Effect of peripheral neuropathy-inducible chemotherapeutic agents on cultured Schwann cells ○Ziauddin Azimi1, Meng Zhao1, Hisashi Shirakawa1, Takayuki Nakagawa1, Shuji Kaneko1 Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University1 Peripheral neuropathy is a major dose-limiting side effect of chemotherapeutic agents, including platinum-based drugs (cisplatin and oxaliplatin) and taxanes (paclitaxel). These agents exert direct and indirect toxic effects on peripheral sensory and motor nerves, resulting pain, sensory and motor loss. However, the mechanisms underlying chemotherapy-induced peripheral neuropathy remain unclear. Schwann cells (SCs) are the major glial cells in peripheral nervous system and play crucial roles in endogenous repair of peripheral nerve. Following nerve injury, differentiated myelinating SCs can dedifferentiate and recover the potential to proliferate. In this study, we examined the effects of cisplatin, oxaliplatin and paclitaxel, on cultured SCs. Cultured SCs were prepared from sciatic nerves of Wistar neonatal rats by MACS with p75 NTR antibody. The non-myelinating SCs was differentiated into myelinating SCs by forskolin. In MTT assay for assessment of SC viability, treatment with paclitaxel (0.01-0.3 μM), cisplatin (0.3-10 μM) and oxaliplatin (1-30 μM) for 1-2 days caused toxicity on SCs at concentration- and time-dependent manners. Immunostaining analysis revealed that paclitaxel at lower concentrations (0.001-0.1 μM) induced pronounced morphological changes of non-myelinating and meliating SCs, while cisplatin (0.3-10 μM) and oxaliplatin (1-30 μM) had little effect. Furthermore, the difference in the expression of myelin basic protein and protein zero between paclitaxel and cisplatin/oxaliplatin was observed. These results suggest that chemotherapeutic agents, such as paclitaxel, cisplatin and oxaliplatin, could affect on SC morphology, myelination and differentiation of SCs, which may contribute to the peripheral neuropathy, although the effects were different among agents. We are now investigating the effects on SC transcription factors, such as early growth response 2 (Egr2/Krox20) and POU domain class 3 transcription factor 1 (Oct6).	P1-2-16 中枢および末梢神経系におけるホスホグリセリン酸ムターゼ1の分布 Distribution of phosphoglycerate mutase 1 in the central and peripheral nervous systems ○林明子1, 今渕信登1, 若林あゆみ1, 馬場広子1 ○Akiko Hayashi1, Nobuto Imabuchi1, Ayumi Wakabayashi1, Hiroko Baba1 東京薬科大学　薬学部　機能形態学教室1 Dept Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences, Tokyo1 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. PGAM1 is enriched in brain, however, distribution of this enzyme as well as how the autoantibodies are produced in these patients is not uncertain. In the present study, we examined distribution of PGAM1 in the CNS and PNS from normal rats and myelin mutant mice to understand the mechanism of antibody production and contribution to demyelinating disease. Western blot analysis showed that PGAM1 is abundantly detected in the PNS in addition to 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, anti-PGAM1 antibody strongly stained paranodal regions 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 be contributed 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.
P1-2-17 DEAD-box RNA helicase Ddx54は中枢でのミエリン形成に必須である A DEAD-Box RNA Helicase Ddx54 protein in oligodendrocytes is indispensable for myelination in the central nervous system ○山本雅浩1,2, 植木俊之1, 吉岡望3, 森崎博実4, 清和千佳2, 鶴尾吉宏5, 阿相皓晃2 ○Masahiro Yamamoto1,2, Toshiyuki Ueki1, Nozomu Yoshioka3, Hiromi Morisaki4, Chika Seiwa2, Yoshihiro Tsuruo5, Hiroaki Asou2 （株）ツムラ　ツムラ研1, 慶應大医・漢方医学センター2, 都神経研・発生形態3, シスメックス・中央研4, 和歌山医大・解剖5 Tsumura Res Labs, Tsumura & CO., Ami1, Center for Kampo Medicine, Keio Univ School of Medicine, Tokyo2, Dept Brain Development, Tokyo Metropolitan Institute of Neuroscience, Tokyo3, Central Res Lab, Sysmex, Kobe4, Dept Anatomy Cell Sci, Wakayama Med Univ, Wakayama5 We developed a new monoclonal antibody, 4F2, using cultured oligodendrocytes as an antigen. 4F2 specifically labels oligodendroglial lineages in rat brains since embryonic day 9 to old age as well as in vitro neural cell culture. The 4F2 antigen was associated with all four isoforms of myelin basic protein (MBP) in coimmunoprecipitation experiments using brain homogenates or cell lysates of cultured oligodendrocytes. Immunoscreening of a brain cDNA library identified the antigen as DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases involved in RNA metabolism, transcription, and translation. A recombinant adenovirus expressing a short hairpin RNA to silence endogenous Ddx54 protein was intraventricularly injected into the brains of mice on postnatal day 2. After 1 week, a substantial amount of myelin- associated glycoprotein (MAG) and MBP immunosignals were present but MBP+ cells accumulated in the subplate layer and did not intrude into the emerging white matter, resulting in extensive demyelination. Immunoblot analysis revealed that Ddx54 knockdown caused a significant decrease in the level of 21.5 kDa MBP isoform and Ddx54, but the amount of other oligodendrocyte-specific proteins including MAG, QKI proteins and three MBP isoforms (14, 17.5 and 18 kDa) remained unchanged. Transfection of the Ddx54 expression vector into luciferase reporter-introduced neuroepithelial cells resulted in upregulated MBP promoter activity. Cotransfection of the Ddx54 gene with MBP isoform genes increased the nuclear localization of the 21.5-kDa MBP isoform, which has been reported to function as a nuclear signal transduction molecule. Furthermore, Ddx54 protein was indicated to directly interact with MBP mRNA. These results suggest that Ddx54 protein play an important role in central nervous system myelination, presumably in myelin sheath formation after the differentiation of oligodendrocytes.	P1-2-18 Minocyclineによって脱髄後のCNTFの発現が抑えられることで再ミエリン化が抑制される Minocycline Reduces Remyelination by Suppressing Ciliary Neurotrophic Factor Expression after Cuprizone-induced Demyelination ○田中達英1, 村上公一1, 板東良雄1, 吉田成孝1 ○Tatsuhide Tanaka1, Koichi Murakami1, Yoshio Bando1, Shigetaka Yoshida1 旭川医科大学 医学部 解剖学講座 機能形態学分野1 Dept. of Functional of Anatomy and Neuroscience, Asahikawa Medical University1 Remyelination is a regenerative process of demyelinated axons and sometimes limited in demyelinating diseases such as multiple sclerosis. However underlying molecular mechanisms of such limitation are unclear. In this study, we employed the murine cuprizone model of demyelination, in which considerable remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. We gave cuprizone in the diet for 4 weeks to mice and then allowed animals to remyelinate with normal diet. Microglia accumulated during 2-4 weeks of cuprizone diet in the corpus callosum of mice, and these cells remained activated 2 weeks after the change to normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone-induced demyelination. Minocycline treatment reduced the number of CC1-positive oligodendrocytes, as well as levels of MBP, CNPase in the remyelination phase. The expression of ciliary neurotrophic factor (CNTF) mRNA increased by 4 weeks of cuprizone diet and remained at high 2 weeks after the change to normal diet in the corpus callosum. Immunohistochemistry revealed that microglia expressed CNTF in the corpus callosum during and after cuprizone is given. Minocycline treatment suppressed CNTF expression during remyelination phase with normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro experiments showed CNTF directly affected the differentiation of oligodendrocytic cells. Together, these findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone-induced demyelination.
P1-2-19 有随神経線維パラノードタンパクに遺伝子変異を持つshamblingマウスにみられる神経軸索変性の分子基盤 Molecular basis for the axonal degeneration of myelinated nerves in shambling mice with mutation of a Caspr gene encoding an axonal membrane protein at the paranode ○高岸芳子1, 佐々木郁磨1, 村田善晴1 ○Yoshiko Takagishi1, Ikuma Sasaki1, Yoshiharu Murata1 名古屋大学　環境医学研究所　発生・遺伝分野1 Department of Genetics, Research Institute of Environmental Medicine, Nagoya University, Nagoya1 Neurological mouse shambling (shm) has mutation of a gene encoding Caspr, an axonal membrane protein at the paranode of myelinated nerves. The paranode is a site of axon-glia interaction, which is crucial for organization and maintenance of myelinated nerves. The shm mice develop ataxic gait at birth 2~3 weeks of age after birth and thereafter exhibit hind limb paresis and trembling body. Our study showed that paranodal junctions between the axolemma and myelin loops were not formed and possible thereby the excitation-conduction velocity was reduced in myelinated nerves form both peripheral (PNS) and central (CNS) nervous systems. The neurological defects of shm mice progress as mice age. Our EM analysis in aged shm mice at 10 months and older demonstrated that neurons and myelinated nerves in both CNS and PNS were degenerated and contained various altered organelles; autolysosome-like structure and autophagosome-like structures. In this study we aimed to identify the molecular constituent of degenerated organelles by immunohistochemistry and evaluate the molecular mechanism of neuronal degeneration undergoing in aging of shm mice. The positive staining of microtubule-associated protein light chain 3 (LC3), an essential component of autophagy, was detected in myelinated nerves of the cerebellum, which was affected with a loss of Purkinje cells and extensive axonal swellings in aged shm mice. The LC3 positive nerves were also present in aged sciatic nerves where a number of axons were degenerated. Myelinated nerves in these areas were also immunopositive for beta-amyloid precursor protein (APP) and nonphosphrylated neurofilament (NF-H). The LC3 and APP staining was not found in the normal mice as well as in young normal and shm mice. The present findings suggest that LC3, APP and nonphosphrylated NF-H is involved in axonal degeneration in shm mice.	P1-2-20 成体マウス脳弓におけるオリゴデンドロジェネシス; 海馬神経交連・投射線維のミエリン化制御 Oligodendrogenesis in the fornix of adult mice; dynamic myelination of axonal fibers of hippocampal neurons ○福島翔平1, 西川和範1,2, 小野勝彦2,3, 竹林浩秀3, 宮田清司1 ○Shohei Fukushima1, Kazunori Nishikawa1,2, Katsuhiko Ono2,3, Hirohide Takebayashi3, Seiji Miyata1 京都工芸繊維大院・応用生物1, 京都府立医科大院・生物2, 新潟大院・医歯学総合・神経生物3 Dep of Appl Biol, Kyoto Inst of Technol, Kyoto, Japan1, Dep of Biol, Kyoto Pref Univ of Med, Kyoto, Japan2, Div of Neurobiol & Anat Grad Sch of Med & Den Sci Niigata Univ, Niigata, Japan3 Oligodendrocytes, the myelin-forming cells of the CNS, are generated from proliferating oligodedrocyte progenitor cells (OPCs) during the first few postnatal weeks in the rodents and peak at the second week, but recently oligodendrogenesis occurs at several brain regions in adult rodents. In the present study, we aimed to elucidate the possibility whether or not oligodendrogenesis occurs in the fornix where passing fiber of hippocampal neurons locates and include the ventral hippocampal commissure (vhc), fimbria of the hippocampus(fi), triangular septal nucleus (TS), and dorsal part of lateral septal nucleus (LSD). First, we observed many proliferation cells in the fornix of adult mice by using a mitotic marker bromodeoxyuridine (BrdU). The number of BrdU-positive cells was higher in the vhc and fi than that of the TS and LSD. There were many plated-derived growth factor receptor A (PDGFRA)-positive and NG2-positive OPCs in the fornix. Double labeling immunohistochemistry further revealed that BrdU-labeled nuclei were often seen at PDGFRA- and NG2-positive OPCs For determining fate of proliferating cells into oligodendrocytes, astrocytes, or neurons, we performed double labeling immunohistochemistry of BrdU with an oligodenrocyte marker Olig2, an astrocyte marker S100B, and a neuronal marker HuC/D 4 weeks after 7-day BrdU administration. The percentage of BrdU-labeled nuclei were more than 90 in Olig2-positive oligodendrocyte/OPCs, but rare in S100B-positive astrocytes and scarcely seen in HuC/D-positive neurons. In conclusion, the present study demonstrates the occurrence of oligodendrogenesis in the fornix of adult mouse brains under normal condition, indicating myelination of hippocampal neuronal fibers is dynamically regulated by oligodendrogenesis.
P1-2-21 慢性ストレスによるランビエ構造変化はうつ病発症に関連する Pathogenesis of major depressive disorder is associated with structural abnormality of node and parenode of Ranvier by chronic stress exposure ○宮田信吾1, 小山佳久2, 谷口学2, 遠山正彌1,3 ○Shingo Miyata1, Yoshihisa Koyama2, Manabu Taniguchi2, Masaya Tohyama1,3 近畿大・東洋医学・分子脳科学1, 阪大院・医・神経機能形態学2, 阪大院・連合小児発達学・分子生物遺伝学3 Div Mol Brain Sci, Res Ins Tra Asian Med, Kinki Univ, Osaka1, Dept Anat&Neurosci, Med, Osaka Univ, Osaka2, Dept Child Develop and Mol Brain Sci, United Child Develop, Osaka Univ, Osaka3 Major depressive disorder is probably the oldest and still one of the most frequently diagnosed psychiatric illnesses. Major depressive disorder is one of the leading causes of disturbances in emotional, cognitive, autonomic, and endocrine functions, affecting nearly 7% of the population in Japan. According to the large amount of information on depressive diseases that has been accumulated during recent years, patients with major depressive disorder show an enhanced biologic stress-response mechanism, especially a hyperactive hypothalamic-pituitary-adrenal (HPA) axis and high levels of circulating cortisol. Although dysregulation of the HPA axis by chronic stress is indicative of major depressive disorder, the molecular mechanisms and functional changes in the brain underlying depression are largely unknown. In this study, by using repeated water-immersion and restraint stress (WIRS) as a stressor for mice, we attempted to elucidate the molecular pathway induced by elevated plasma corticosterone levels. We observed the following effects in vivo: the activation of SGK1 signaling pathway induces morphological changes in oligodendrocytes; and after recovery from chronic stress, morphological changes in oligodendrocytes and after recovery from chronic stress, the abnormal arborization of oligodendrocytes return to the control levels. Furthermore, we found the structural abnormality of node and parenode of Ranvier by chronic stress exposure.	P1-2-22 発達期小脳プルキンエ細胞軸索におけるパラノーダルジャンクションの重要性 The importance of paranodal axo-glial junctions in Purkinje neurons during early development ○石橋智子1, 小泉綾1, 馬場広子1 ○Tomoko Ishibashi1, Aya Koizumi1, Hiroko Baba1 東京薬科大学　薬学部　機能形態教室1 Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences1 Myelin sheaths are attached to axons on both sides of each node of Ranvier by means of highly specialized paranodal axo-glial junctions (PNJs). To understand the importance of the PNJs, we investigated cerebroside sulfotransferase (CST; a sulfatide synthetic enzyme) deficient mice that partially lack PNJs in both the CNS and PNS. These CST-deficient mice were born healthy, but began to display hind limb weakness by 4 weeks of age and subsequently showed a pronounced tremor and progressive ataxia (Honke et al., 2002). Aside from the lack of PNJs, compact myelin was intact in these mutant mice. However, in addition to the abnormality of the PNJs, myelinated internodal axons showed many focal swellings in Purkinje cell axons in the cerebellum. Immunohistochemical analysis by means of the antibody against type I IP3 receptor (IP3RI) revealed high immunoreactivity within the swellings, and this IP3R1 accumulation was the first change we observed at the swellings. Ultrastructural studies of the cerebellum at 18 days of age revealed that focal axonal degenerations such as vacuoles were already present. Non-uniformly-distributed microtubeles and neurofilaments were sometimes observed at the same age. These findings suggest that the formation of the PNJs is one of the critical events for the fine structure of axons during the developmental stages.

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