e ポスター

e ポスター 6. グリア (オリゴデンドロサイト，ミエリン） e Poster 6. Glia (oligodendrocyte, myelin)

2020/9/11　14:00～15:00　オンデマンドB-1 P2-01 運動機能回復を基盤とする新規脱髄マウスモデルの開発 Development of a novel demyelination mouse model based on motor function recovery 山崎礼二^1,2、ジェフリーハング²、大野伸彦¹ 1. 自治医科大学医学部解剖学講座組織学部門、2. ジョージタウン大学生物学部 Reiji Yamazaki^1,2, Jeffrey Huang², Nobuhiko Ohno¹ 1. Department of Anatomy, Division of Histology and Cell Biology, School of Medicine, Jichi Medical University,, 2. Department of Biology, Georgetown University Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) characterized by progressive remyelination failure, axonal loss, and the accumulation of clinical disability However, whether remyelination promotes motor recovery following demyelinating injury remains unclear. Damage to the internal capsule (IC) is known to result in motor impairment in MS and stroke. Here, we induced focal IC demyelination in mice by lysophosphatidylcholine (LPC) injection using stereotaxic technique, and examined its effect on motor behavior. We also compared the effect of LPC induced IC damage to that produced by endothelin-1 (ET1), a potent vasoconstrictor used in experimental stroke lesions. We found that LPC or ET1 injections induced asymmetric motor deficit at 7 days post lesion (dpl), and that both lesion types displayed increased inflammation, myelin loss, and axonal dystrophy. The motor deficit and lesion pathology remained in ET1 injected mice at 28dpl. By contrast, LPC injected mice regained motor function by 28dpl, with corresponding reduction in activated microglia/macrophage density, and recovery of myelin staining and axonal integrity in lesions. These results suggest that LPC induced IC demyelination results in acute motor deficit and subsequent recovery through remyelination, and may be used to complement future drug screens and identify drugs for promoting remyelination.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-02 疾患特異的iPS細胞を用いたペリツェウス・メルツバッハ病（PMD）病態解析 Modeling and Phenotypic analysis of Pelizaeus-Merzbacher disease using patient-derived iPS cells 鈴木禎史¹、李コウ¹、後藤雄一¹、井上健¹ 1. 国立精神神経医療研究センター疾病研究第二部 Sadafumi Suzuki¹, Heng Li¹, Yu-ichi Goto¹, Ken Inoue¹ 1. National Center of Neurology & Psychiatry Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive leukodystrophy characterized by diffuse hypomyelination in the central nervous system (CNS). PMD is caused by mutations in the proteolipid protein gene PLP1, which encodes the major myelin protein of the CNS. Genomic duplications encompassing the entire PLP1 gene are the most frequent causative mutation. Although, the cellular consequences of how an extra-copy of wild-type PLP1 causes severe hypomyelination remains poorly understood, final differentiation and maturation of oligodendrocytes (OLs) have been thought to be primarily affected. Plp1-duplication model mice were generated, but they do not recapitulate the phenotype and pathophysiology of human PMD. To address cellular pathology of PMD more precisely, we have been generating human induced pluripotent stem cells (hiPSCs) from patients with PMD harboring PLP1 duplication and aim to differentiate them into oligodendrocytes to recapitulate the cellular pathology of PMD in culture. At this point we have generated hiPSCs from 8 PMD patients (5 duplication, 1 triplication, and 2 amino acid substitutions). We have established a protocol to differentiate hiPSCs into OLs by combining SOX10/Olig2 transduction and FACS. During the early stage of OL induction, we found abnormalities of induction efficiency, viability, mobilization, process number and length of oligodendrocyte precursor cells (OPCs) in a PMD-derived hiPSC line in comparison with a normal control. We also observed increase of reactive oxygen species (ROS) accumulation and a partially depolarized inner mitochondrial membrane in OLs in the early differentiation phase. These findings preliminarily suggest that the cellular phenotypes of PMD-derived hiPSCs may occur at early stage of OL differentiation, which is earlier than previously thought. We further seek to clarify if the pathophysiology underlying these early events is associated with PLP1 genotypes and gene dosage by analyzing other PMD-derived hiPSC lines.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-03* Teneurin-4による小径軸索の髄鞘形成を司るI/II型オリゴデンドロサイトの制御 Teneurin-4 regulates the generation of type I/II oligodendrocytes that myelinate small diameter axons 高橋りこ¹、林千香子¹、岩瀬未帆¹、鈴木喜晴¹ 1. 東京医科歯科大学大学院医歯学総合研究科遺伝子細胞検査学分野 Riko Takahashi¹, Chikako Hayashi¹, Miho Iwase¹, Nobuharu Suzuki¹ 1. Dept. of Mol. and Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University In the central nervous system (CNS), oligodendrocytes (OLs) form myelin around axons, which allows action potentials to conduct rapidly. Since the impaired myelin structure in the small diameter axons causes some neuronal disorders such as multiple sclerosis, the elucidation of the underlying mechanisms is required. OLs can be divided into 4 types with their morphology. As type I/II OLs myelinate small diameter axons preferentially, they may be critically involved in the myelin defects in the small diameter axons in these disorders. However, regulating molecules and localization of type I/II OLs have not been fully understood. In our previous study, we identified Teneurin-4 (Ten-4), a transmembrane protein on OLs, as a regulator of myelination in the small diameter axons. To elucidate the association of Ten-4 with type I/II OLs, we examined their distribution in the spinal cord in WT and Ten-4 deficient mice by electron microscopy analysis and immunohistochemistry using carbonic anhydrase II (CAII) as a specific marker for type I/II OLs. From our results of immunostaining at 7 weeks in WT mice, CAII-positive type I/II OLs were found in the entire regions in the white mater of the spinal cord, but dominantly localized in the corticospinal tract, fasciculus gracilis, and the inside part of ventral funiculus, where consist of small diameter axons. Furthermore, the number of CAII-positive OLs was remarkably decreased in Ten-4 deficient mice. CAII-negative OLs were also reduced in Ten-4 deficient mice, but the reduction in CAII-positive OLs was more prominent. The onset of the defect in type I/II OLs in Ten-4 deficient mice was from postnatal day (P) 7 to 11, when they emerged in WT mice. Consistent with these results, hypomyelination in the small diameter axons was already observed at P7. Our results suggested that Ten-4 was the regulator of myelination in the small diameter axons through the activation of type I/II OLs development. These findings may contribute to understanding the pathological mechanism of myelin-related disorders occurring in the axonal diameter-dependent manner.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-04* 髄鞘形成と軸索維持におけるteneurin-4とアクチン結合タンパク質CNPの相互作用 The crucial role of teneurin-4 associated with actin binding protein CNP in myelination and axonal maintenance 棟方勇貴¹、細田ゆき奈¹、車田博実¹、林千香子¹、鈴木喜晴¹ 1. 東京医科歯科大学大学院医歯学総合研究科遺伝子細胞検査学分野 Yuki Munakata¹, Yukina Hosoda¹, Hiromi Kurumada¹, Chikako Hayashi¹, Nobuharu Suzuki¹ 1. Dept. of Mol. and Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University Myelination by oligodendrocytes (OLs) in the central nervous system is essential for rapid conduction of action potential and axonal maintenance. In OL processes, actin polymerization and an increased level of calcium ions regulate the process extension on the axonal surface and myelination. Meanwhile, 2', 3'-cyclic-nucleotide 3'- phosphodiesterase (CNP) on OLs binds to either tubulin or actin, contributing to process elongation and establishment of the cytoplasmic channels as paths for nutrients from OLs to axons. CNP deficient mice display the axonal degeneration, although the mechanism with related molecules has not been fully understood. In our previous study, we identified that teneurin-4 (Ten-4), a transmembrane protein expressed in OLs, functioned as regulators of myelination in small diameter axons and actin cytoskeleton. From our recent screening experiment for identification of Ten-4 binding proteins, CNP was obtained as a candidate protein. In this study, we first co-transfected Ten-4 and CNP into COS-7 cells and carried out immunoprecipitation in the presence or absence of CaCl₂. As a result, CNP bound to Ten-4, depending on the calcium ion level. Then, to test the localization of Ten-4 and CNP in OLs, we performed immunocytochemistry and proximity ligation assay (PLA). The results showed that they were co-localized on OL processes, and also PLA signaling was detected on the cytoplasmic-rich regions with F-actin. We additionally hypothesized that Ten-4 is involved in axon maintenance, similar to CNP. The result of immunohistochemistry of neurofilament (NF), a marker for axons, in the Ten-4 deficient mouse spinal cord showed the abnormality of NF staining, and most of NF were phosphorylated. Also, the number of mitochondria per axons and activated microglia was increased, suggesting that the axonal degeneration occurred in Ten-4 deficient mice. The axonal degeneration was severer in small diameter axons than large axons. From these results, we conclude that Ten-4 forms a complex with CNP and its interaction may play an important role in axonal maintenance.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-05* オリゴデンドロサイト疾患変異をもつC11ORF73（Hikeshi）はリソソーム凝集を示し、細胞分化を阻害する Oligodendrocyte disease-associated C11orf73(Hikeshi) mutant proteins show lysosomal aggregations and inhibit oligodendroglial differentiation 服部耕平¹、山内淳司¹ 1. 東京薬科大学 Kohei Hattori¹, Junji Yamauchi¹ 1. Tokyo University of Pharmacy and Life Science In the development of brain and spinal cord, myelin sheath is derived from the differentiated plasma membrane of an oligodendroglial cell (oligodendrocyte). Through wrapping a neuronal axon with the myelin sheath, the myelinated axon can achieve rapid electrical conduction velocities. Also, its axon is more resistant to physical stress. Genetic hypomyelinating leukoencephalopathies are a heterogeneous group of the CNS disorders with white matter involvement and are often characterized by hypomyelinating phenotypes with periventricular cysts and vermian atrophy. The homozygous disease-related variant (Cys4-to-Ser [C4S]) of the c11orf73 (also called hikeshi) gene is known to be associated with infantile hypomyelinating leukoencephalopathy. Despite a rapidly increasing identification of the genes associated with leukoencephalopathies, the effects of their mutations on protein products and cellular behaviors still remain to be established. Herein we show that in mouse oligodendroglial FBD-102b cells, the C4S mutant proteins of C11orf73 are mislocalized in the lysosome where they can be aggregated. Expression of the C4S mutants leads to localization into the lysosome but not into the endoplasmic reticulum (ER) and the Golgi body whereas the wild types are localized in the nuclei and the cytoplasm. While parental FBD-102b cells exhibit morphological differentiation, being characteristic of myelin web-like structures, cells harboring the C4S mutants fail to exhibit it. Signaling through lysosome-related kinases is downregulated in cells harboring the C4S mutants. Together, leukoencephalopathy-associated C11orf73 mutant is mislocalized in the lysosome to inhibit morphological differentiation, presenting the part of potential cellular phathological mechanisms underlying hypomyelinating leukoencephalopathies.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-06* オリゴデンドロサイト疾患変異を有するAIMP2はゴルジ体に蓄積し、オリゴデンドロサイト前駆細胞の分化を阻害する Genetic leukodystrophy-associated AIMP2 accumulates in the Golgi body and inhibits differentiation of oligodendrocyte precursor cells 落合愛理沙¹、山内淳司¹ 1. 東京薬科大学 Arisa Ochiai¹, Junji Yamauchi¹ 1. Tokyo University of Pharmacy and Life Science The myelin sheath is differentiated from the plasma membran of an oligodendrocyte, wraping nerve axons to play an important role in achiving saltatory conduction and axonal protection. Inhypomyelinating leukodystrophy, the myelin sheath gradually disappears, possibly resulting in symptoms such as nystagmus and developmental delay of motor function. A nonsense mutation in the aimp2 gene (Tyr35-to-X [Y35X]) is known to be associated with hypomyelinating leukodystrophytype 17, but the pathological effect of mutation protein products and its cellular behavior remain to be known. Here, we show that the AIMP2 Y35X mutant protein is localized in an organelle other than the endoplasmic reticulum with aggregated forms in FBD-102b cells, a mouse oligodendrocyte precursor cell's cell line. Expression of the Y35X mutant leads to the Golgi body localization whereas the wild type is localized throughout the cytoplasm. Also, FBD-102b cells stably expressing the wild type AIMP2 undergo morphological differentiation; in contrst, cells expressing the Y35X mutant fail to do it. Together, mislocalization of the Y35X mutant in the Golgi body, as well as protein aggregation, could be associated with inhibition of morphological differentiation of oligodendrocytes, probably triggering the part of cell pathological mechanisms underlying hypomyelinating leukodystrophy type 17.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-07 ミエリン化オリゴデンドロサイトでの物理的な力を検出する牽引力センサーを用いた新しいアッセイシステムの構築 New assay system to detect mechanical force in myelinating oligodendrocytes using a tension sensor probe 清水健史¹、村越秀治^2,3、松本英俊⁴、石田章真¹、田尻直輝¹、飛田秀樹¹ 1. 名古屋市立大学医学研究科脳神経生理学、2. 生理研脳機能計測支援センター、3. 総研大生理科学、4. 東工大物質理工学院材料系 Takeshi Shimizu¹, Hideji Murakoshi^2,3, Hidetoshi Matsumoto⁴, Akimasa Ishida¹, Naoki Tajiri¹, Hideki Hida¹ 1. Dept of Neurophysiol and Brain Sci, Grad School of Med Sci, Nagoya City Univ, 2. Support Cent for Brain Res, Natl Inst for Physiol Sci, 3. Dept of Physiol Sci, SOKENDAI, The Grad Univ for Advanced Studies , 4. Dept of Mater Sci and Engineer, Tokyo Inst of Technol Oligodendrocytes (OLs) form myelin sheath around neuronal axons to increase conduction velocity of action potential. Although both thick and thin axons exist, the ratio of diameter of axon + myelin to axon diameter (g-ratio) is adjusted to optimum values for each axon, suggesting a factor that controls myelin formation in response to the axon diameter. The optimization of g-ratio is requisite for expressing higher brain functions. To clarify mechanisms underlying the optimum g-ratio, we try to investigate physical factors depending on its diameter. To visualize the OL generating force during myelination, a tension sensor based on fluorescence resonance energy transfer (FRET) was used: the efficiency of FRET decreases when the force is applied on it, setting two fluorescent molecules apart from each other. Polystyrene nanofibers with a diameter similar to neuronal axons were firstly prepared to investigate physical factors without neuronal activity. OLs could wrap myelin sheath around the nanofibers. The change of OL generating force during myelination depending on the nanofiber diameter was observed by FRET measurement. Cytochalasin D treatment significantly increased FRET index meaning lowered tension, indicating that intracellular force detected by the tension sensor was truly dependent on cellular cytoskeleton. These results lead to a novel and interesting conclusion that the physical factor controls myelin formation in response to the axon diameter. In addition, focal adhesion morphology could be investigated as the tension sensor is localized at focal adhesions. Thus, we can examine the correlation among the following factors using our FRET system: ‘axon diameter - OL generating force - focal adhesion morphology - myelin shape' in the same cultured OL cell.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-08 シャルコー・マリー・トゥース病モデルマウス:L-MPZマウスの病態進行の解析 Progressive pathology in Charcot-Marie-Tooth (CMT) disease model, L-MPZ mouse 大谷嘉典^1,2、崔晶晶²、山口宜秀²、藍郷加奈子²、原綾香²、坂剛太²、藤谷昌司¹、馬場広子² 1. 島根大学医学部、2. 東京薬科大学薬学部 Yoshinori Otani^1,2, Jingjing Cui², Yoshihide Yamaguchi², Kanako Aigo², Ayaka Hara², Gouta Ban², Masashi Fujitani¹, Hiroko Baba² 1. Faculty of Medicine, Shimane University, , 2. Tokyo university of Pharmacy and Lifesciences Large myelin protein zero (L-MPZ) is a translational readthrough isoform of myelin protein zero (P0, MPZ). Recently, translational stop codon readthrough has become a key mechanism to modulate canonical gene function in mammals. Previously, we reported that abnormal increase of L-MPZ caused Charcot-Marie-Tooth (CMT) disease-like neuropathy including demyelination and axonal damage, suggesting that aberration of translational readthrough may cause disease. However, the onset and progression of this neuropathy phenotype are still unclear. In order to clarify these disease processes during development and aging, histological analyses using immunostaining and electron microscopy were performed on L-MPZ heterozygous (Het) and homozygous (Hom) mice, in which canonical stop codon in P0 gene was mutated. Immunohistochemical analyses of L-MPZ Hom mice at postnatal day (P) 21 already demonstrated disruption of peripheral myelin structure, disorganization of the node and its surrounding area, increase of endoplasmic reticulum (ER) stress, and extensive macrophage infiltration. Electron microscopical analysis of P21 sciatic nerves indicated the increase of abnormal Schwann cells. Immunohistochemical analyses of 6-month- and 1-year-old L-MPZ Het and Hom mice exhibited more severe phenotypes compared to 10-week samples. Thus, excessive increase of L-MPZ during myelination causes CMT-like pathology and these changes gradually progress to become more severe phenotype in aging, which is similar to human CMT disease process.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-09* 翻訳リードスルー産物であるL-MPZは末梢神経系におけるミエリン形成・維持に重要である Translational readthrough molecule, L-MPZ, is essential for myelin formation and maintenance in the peripheral nervous system 後藤雅裕¹、崔晶晶¹、瀬戸口潔¹、林萌々花¹、平井大之¹、大谷嘉典²、山口宜秀¹、馬場広子¹ 1. 東京薬科大学、2. 島根大学 Masahiro Goto¹, Jingjing Cui¹, Yuki Setoguchi¹, Momoka Hayashi¹, Hiroyuki Hirai¹, Yoshinori Otani², Yoshihide Yamaguchi¹, Hiroko Baba¹ 1. Tokyo Univ. of Pharm. & Life Sci., 2. Shimane Univ. Translational readthrough is a key mechanism in translational regulation to expand the coding potential of a gene. Large myelin protein zero (L-MPZ), an isoform of peripheral myelin protein zero (MPZ, P0), is the first reported common mammalian protein synthesized by this mechanism in the physiological state. L-MPZ is present in compact myelin in the peripheral nervous system. Recently, we reported that abnormal increase of L-MPZ in genome-edited L-MPZ mice which synthesized L-MPZ but not P0, caused Charcot-Marie-Tooth (CMT) disease-like phenotype such as abnormal morphologies in myelin as well as in axons, and motor disturbances. At present, the physiological function of L-MPZ in the PNS myelin remain unknown. To investigate the functional significances of L-MPZ in vivo, we generated another genome–edited mouse line, P0 mouse that produced only P0 but not L-MPZ. Unlike L-MPZ mouse, P0 mouse exhibited no apparent motor symptoms but showed mild decrease of nerve conduction velocity compared to wild type. Immunohistochemical analysis of 10-week-old P0 mouse demonstrated disorganization of the node and its surrounding area, and macrophage infiltration in the sciatic nerve. P0 mouse also showed decrease of cytoplasmic channels in myelin, including E-cadherin-positive myelin paranode and Schmidt-Lanterman incisures, and Cajal bands. These abnormalities became more prominent in 6-month-old P0 mouse. Thus, loss of L-MPZ showed less but significant influence on myelin. Taken together with previous L-MPZ mouse study, these results suggest that appropriate level of L-MPZ is essential in the formation and maintenance of the PNS myelin.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-37 Teneurinファミリーを介したオリゴデンドロサイト-軸索間接着による髄鞘形成制御 Oligodendrocyte-axon Adhesion via Teneurin-4-Tenerin Binding Promotes Myelination 林千香子¹、高橋りこ¹、岩瀬未帆¹、鈴木喜晴¹ 1. 東京医科歯科大学大学院医歯学総合研究科遺伝子細胞検査学分野 Chikako Hayashi¹, Riko Takahashi¹, Miho Iwase¹, Nobuharu Suzuki¹ 1. Dept. of Mol. and Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University Myelin structure formed by oligodendrocytes (OLs) enables action potential to conduct rapidly. OLs extend their processes with sheet-like plasma membranes toward axons, which facilitates multi-lamellar myelin formation. During myelination, intimate interaction of OLs with axons is integral, but how this occurs at the molecular level is still not understood. To elucidate the molecular mechanisms of the neuronal recognition by OLs, we focused on teneurins (Ten-1~Ten-4), which are transmembrane proteins and have been predicted as cell adhesion receptors in vertebrates. In our previous study, we identified that Ten-4 was a key molecule for myelination due to the hypomyelination in Ten-4 deficient mice. As Ten-4 is expressed on oligodendrocyte precursor cells (OPCs) and OLs, it is conceivable that Ten-4 may work in the association of OLs with axons. When exploring the binding partners of Ten-4 using mass-spectrometry to identify co-immunoprecipitated proteins, intriguingly all teneurins were obtained as the candidates. Then, we assessed the cell-cell adhesion activity of Ten-4 with other teneurins by using each teneurin over-expressing cells and recombinant proteins of their extracellular domain (rTenECDs). Our results revealed homophilic and heterophilic interactions of Ten-4 with teneurins, and OPC attachment activity of rTenECDs, suggesting that Ten-4 on OPCs/OLs interacted with teneurins on axons. To examine the biological effects of the bindings, we cultured OLs on rTenECDs and found that OL process formation was promoted in the presence of rTenECDs. Further, using nanofibers coated with rTenECDs, especially rTen-4ECD, accelerated myelin formation. The expression of an essential structural protein in myelin, myelin basin protein (MBP), was increased through the activation of Src kinase when culturing OLs on rTen-4ECD. From these results, we conclude that Ten-4 positively regulates the oligodendrocyte-axon interaction and also functions as a platform of the signaling pathways for myelination.