e ポスター

e ポスター 1. グリア (オリゴデンドロサイト，ミエリン) e Poster 1. Glia (oligodendrocyte, myelin)

2020/9/10　14:00～15:00　オンデマンドB-1 P1-01 Olig2結合因子Obp2のオリゴデンドロサイト分化およびミエリン化における役割 Analysis of the effect of Obp2 on the oligodendrocyte differentiation and myelination in the central nervous system 竹林浩秀¹、シマンコワアンナ¹、備前典久¹、大野伸彦^2,3、阿部学¹、﨑村建司¹ 1. 新潟大学、2. 自治医科大学、3. 生理学研究所 Hirohide Takebayashi¹, Anna Simankova¹, Norihisa Bizen¹, Nobuhiko Ohno^2,3, Manabu Abe¹, Kenji Sakimura¹ 1. Niigata Univ, 2. Jichi Med Univ, 3. Natl Inst Physiol Sci Oligodendrocytes (OLs) wrap around neuronal axons to form myelin, which contributes to increased conduction velocity in the central nervous system (CNS). Myelination is the sophisticatedly orchestrated process requiring timely participation of many factors, and the disruption of precise control in the process leads to intractable neurological diseases with hypomyelination or demyelination. Olig2, a basic helix-loop-helix transcription factor, is indispensable for the production and differentiation of OLs. We have identified a novel Olig2 binding factor Obp2 (Olig2-binidng protein 2), which is known to play a role in the assembly of small nuclear ribonucleoprotein (snRNP) and transcriptional regulation, and demonstrated that Obp2 is indispensable for the survival of oligodendrocyte progenitor cells. In the current study, we generated Obp2 conditional knockout (cKO) mice in myelinating cells (Mbp-Cre; Obp2cKO mice) and then, demonstrated the involvement of Obp2 in the OL differentiation and myelination. Almost all these mice showed a lifespan of less than 7 weeks old. Immunohistochemistry and in situ hybridization demonstrated decreased number of oligodendrocytes in the spinal cords of Obp2 cKO mice. In addition, quantitative RT-PCR analysis supported the significant attenuation of the expression levels of mature OL-related genes. Moreover, electron microscopic analysis revealed thinner myelin sheaths in the Obp2 deficient spinal cords compared to those in the control spinal cords. Thus, these results suggest that Obp2 is essential for the OL differentiation and myelination in the CNS.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-02* オリゴデンドロサイト疾患変異を有するPOLR3Aはリソソームに蓄積し、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 POLR3A accumulates in the lysosome and inhibits differentiation of oligodendrocyte precursor cells 澤口粋¹、山内淳司¹ 1. 東京薬科大学 Sui Sawaguchi¹, Junji Yamauchi¹ 1. Lab. of Mol. Neurol., TUPLS Pelizaeus-Merzbacher disease (PMD) is an inherited hypomyelinating disorder in the central nervous system. PMD is caused by various types of mutations in the proteolipid protein 1 (plp1) gene whose gene products are involved in the interaction between myelin membranes. In some patients with PMD symptoms, an abnormality in the plp1 gene has not been identified. Therefore, there has been suggested to be the presence of the unidentified responsible genes for PMD or PMD-like diseases. Next-generation sequencing technology has identified that some mutations of polr3a is responsible for hypomyelinating-leukodystrophy 7 (HLD7), which is related to PMD. We introduced the wild-type or the disease-associated R140X mutant construct into oligodendrocyte progenitor cell line (FBD-102b) and investigated how the mutation is related to cell pathological phenotypes of HLD7. First, we have observed that the mutant proteins form aggregates whereas the wild type ones display a normal monomeric form in non-denaturing polyacrylamide gel electrophoresis. Second, the aggregated forms are mainly localized in the lysosome but not in the endoplasmic reticulum and the Golgi body. Concomitantly, lysosome-related signaling such as a translation initiation complex protein 4E-BP1 as well as ribosomal S6 proteins is downregulated. Finally, although cells stably harboring the wild type constructs trigger morphological changes following the induction of differentiation, cells stably harboring the R140X mutant constructs exhibit defective differentiated phenotypes. Together, mutant proteins are accumulated in the lysosome where signaling through mTOR is downregulated to decrease cellular differentiation. These results may provide one of the cell pathological processes of oligodendrocytes underlying HLD7.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-03* オリゴデンドロサイト疾患変異を有するEPRSはRab7に蓄積し、オリゴデンドロサイト前駆細胞の分化に影響する Rare oligodendrocyte disease-associated EPRS accumulates in Rab7-positive vesicles and affects its differentiation 田中満梨奈¹、山内淳司¹ 1. 東京薬科大学 Marina Tanaka¹, Junji Yamauchi¹ 1. Tokyo University of Pharmacy and Life Sciences Oligodendrocytes, one of the glial cells, provide structural support for neurons to function normally. At the same time, it forms a myelin sheath that acts as an insulator by wrapping around an axon and enables rapid information transmission in the brain. Pelizaeus-Merzbacher disease is a genetic disorder categorized neurodegenerative disease with white matter by insufficient myelin formation, currently known as hypomyelinating leukodystrophy 1 (HLD1). HLD15 is an autosomal recessive disorder bringing with motor and cognitive impairment in first or second decade of life. Its responsible gene encodes glutamyl-prolyl-tRNA synthetase (EPRS) for translation which is known to be a MSC component protein with seven other tRNA synthetases and three scaffold proteins, as well as the gamma-interferon-activated inhibitor of translation. Though five HLD15 mutations have been identified so far, including the homozygous Arg339-to-Ter (R339X) nonsense mutation, the effects of mutant proteins on the cellular behaviors remain largely unknown. Herein, we describe that in mouse oligodendrocyte cell line FBD-102b, the R339X mutant proteins are localized in Rab7-positive vesicleswhere they are aggregated. In contrast, wild type proteins have been indeed localized in the cytoplasm. While FBD-102b cells harboring stably wild type constructs had the ability to display differentiated phenotypes with web-like structures in cell periphery, cells harboring the R339X mutants affected differentiation. These results suggest that EPRS mutant proteins are accumulated in Rab7-positive vesicles and, having effects on cell morphological differentiation. These might be related to cell pathological mechanisms underlying HLD15.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-04* ラミニンE3ドメインによるオリゴデンドロサイト前駆細胞の増殖・生存活性 Promoted oligodendrocyte precursor cell proliferation and survival via Laminin E3 fragments 大石桃¹、恩知千菜美¹、山田桃奈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 Momo Ooishi¹, Chinami Onchi¹, Momona Yamada¹, Chikako Hayashi¹, 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, oligodendrocyte precursor cells (OPCs), which arise from the subventricular zone at the embryonic stage, proliferate and migrate along the blood vessels, and postnatally differentiate into oligodendrocytes. Meanwhile, a-dystroglycan (a-DG) on OPCs specifically adhere to laminins (LMs), ensuring proper differentiation into oligodendrocytes. LMs are extracellular matrix (ECM) proteins in basement membranes and composed of three subunits, a, b, and g chains. Nevertheless five a chains, whose E3 domains are the binding region to a-DG, have been identified, the biological activities of each LME3 on OPCs have not been well clarified yet. Since it was revealed that LMa1, LMa2, LMa4, and LMa5 were expressed in the CNS in our recent study, we prepared recombinant proteins of LMaE3 (LMa1E3, LMa2E3, LMa4E3, LMa5E3) to assess OPC attachment activity and biological effects on OPCs. As a result, high purity of LMa1E3, LMa2E3, and LMa4E3 were successfully obtained. As LMa5E3 was cleaved by endogenous proteases, it could not be prepared. Using the three LMaE3s, we found that the higher number of OPCs bound to LMa2E3. The attachment was inhibited by using anti-a-DG antibody, suggesting that OPCs adhered to LMa2E3 through a-DG on OPCs. Though OPCs attached to LMa1E3 more than a control, there was not a statistical difference. Finally, to evaluate the biological effects of the attachment on OPCs, we cultured OPCs for 3 days on LMaE3-coated wells and performed immunostaining with anti-Ki67 antibodies and TUNEL assay to examine the OPC proliferation and survival, respectively. After the culture, many Ki67-positive proliferating OPCs were observed on LMa2E3 and LMa4E3. In addition, less apoptotic OPCs were on LMa2E3, while the number of TUNEL-positive cells on LMa1E3 and LMa4E3 was comparable with poly-D-lysine as a control. These results revealed the LMaE3, particularly LMa2E3, promoted OPC proliferation and survival through the association with a-DG. Furthermore, LMaE3 may be applicable to developing of a new OPC culture substrate.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-05 脳微小血管内皮細胞由来の細胞外小胞の表面に存在するPDGF-Bはオリゴデンドロサイト前駆細胞の増殖を促進する PDGF-B on the surface of microvascular endothelial cell-derived extracellular vesicles stimulates proliferation of oligodendrocyte precursor cells. 倉知正¹、徐彬¹、大澤祥²、松崎利行³、好本裕平²、石崎泰樹¹ 1. 群馬大学大学院医学系研究科分子細胞生物学講座、2. 群馬大学大学院医学系研究科脳神経外科学講座、3. 群馬大学大学院医学系研究科生体構造学講座 Masashi Kurachi¹, Bin Xu¹, Sho Osawa², Toshiyuki Matsuzaki³, Yuhei Yoshimoto², Yasuki Ishizaki¹ 1. Dept Mol Cell Neurobiol, Gunma University Graduate School of Medicine, 2. Dept Neurosurg, Gunma University Graduate School of Medicine, 3. Dept Anat Cell Biol, Gunma University Graduate School of Medicine We previously reported that transplantation of brain microvascular endothelial cells (MVECs) promoted remyelination in the white matter infarct of a rat brain by increasing the number of oligodendrocyte precursor cells (OPCs) and improved the animal's gait disturbance. We further revealed MVEC-derived extracellular vesicles (MVEC-EVs) promoted survival and proliferation of OPCs in vitro. In this study, we investigated the molecular mechanism how MVEC-EVs contribute to OPC survival and proliferation in vitro. As EVs from Rat fibroblast-like cell line (Rat-1) cells (Rat-1-EVs) promoted neither survival nor proliferation of OPCs, we compared the contents of MVEC-EVs and those of Rat-1-EVs. Among over 800 proteins found only in MVEC-EVs by protein mass spectrometry, we focused on PDGF-B, which is well known to stimulate proliferation of OPCs. Enzyme-linked immunosorbent assay (ELISA) revealed the presence of approximately 10 pg PDGF-BB in 1 µg of MVEC-EVs, while it was undetectable in Rat-1-EVs by ELISA. Immunoelectron microscopy revealed that a part of PDGF-B was localized on the surface of MVEC-EVs. Treatment of MVEC-EVs with heparinase reduced the amount of PDGF-BB in EVs, suggesting that heparan sulfate proteoglycan on the surface of EVs plays a role for tethering PDGF-B to the surface of EVs. A neutralizing antibody against PDGF-B significantly reduced the promoting activity of MVEC-EVs on OPC proliferation. An antagonist (imatinib) against PDGF receptor showed the similar effect. Taken together, these results indicate that PDGF-B on the surface of MVEC-EVs plays a role for their promoting activity on OPC proliferation.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-06 オリゴデンドロサイト分化制御の新規分子機構解明へ向けて Towards understanding of novel molecular mechanisms regulating oligodendrocyte differentiation and myelination 石野雄吾¹、清水尚子¹、遠山正彌^1,2、宮田信吾¹ 1. 近畿大学、2. 大阪府立病院機構 Yugo Ishino¹, Shoko Shimizu¹, Masaya Tohyama^1,2, Shingo Miyata¹ 1. Kindai Univ., 2. Osaka Pref. Hosp. Org. Glial cells are recently recognized as essential components with numerous key roles within the nervous system. Among them, oligodendrocytes and Schwann cells have strong interactions with axons by wrapping them to form myelin sheath in the central nervous system and peripheral nervous system, respectively. Myelin provides support and insulation to the nervous system and is prerequisite for the smooth and high-speed nerve conduction. Therefore, defects and malfunctions of those glial cells have broad impacts within the nervous system. While structural alterations of myelin forming oligodendrocytes in several pathological situation have been illustrated, genetical aspects that account for those defective events remain to be fully understood. It is well known that post-translational modifications of transcription factors regulate oligodendrocyte differentiation and myelination. While large amount data have been accumulated about phosphorylations, the function of arginine methylation of transcription factors, which is mediated by protein arginine methyltransferases (PRMTs), is still poorly understood. To elucidate a novel molecular mechanism controlling oligodendrocyte differentiation and myelination, we screened protein-protein interactions among PRMTs and transcription factors and identified several partners. We'd like to propose arginine methylation as a novel and essential post-translational modification to understand more detail of normal development and pathological condition of oligodendrocytes.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-07 甲状腺機能低下症モデルマウスにおける性と週齢によるグリアの形態変化と行動変化 Sex- and age-differences in glial morphology and behaviors in a mouse model of hypothyroidism 野田百美¹、新山哲士¹、青井孝介¹ 1. 九州大学 Mami Noda¹, Tetsushi Niiyama¹, Kosuke Aoi¹ 1. Kyushu University Thyroid hormones (THs) are essential for the development and function of the central nervous system (CNS). It has been reported that the prevalence of hypothyroidism in adult is higher in women than in men and increases with age. Hypothyroidism may affect psychological condition, for example depression, and potentially increase the risk of cognitive impairment and neurodegeneration including Alzheimer's disease (AD). Microglial cells are involved in the removal of degenerating neurons and axons and maintains homeostasis in the CNS. It is known that various CNS diseases and neurodegenerative diseases occur due to dysfunction of microglial cells. We have reported sex- and age-dependent differences in microglia morphological changes in mouse model of hyperthyroidism. Here we analyzed the effects of hypothyroidism on glial cells in vivo and their behavior using young and old/male and female mice.