一般、大学院生、若手研究者（ポスター）

一般、大学院生、若手研究者（ポスター）軸索輸送、細胞運動、細胞骨格、神経系の発生・再生、神経幹・前駆細胞と細胞分化

P-036（1） Analyses of mitochondrial changes in dysmyelinated axons グエンフィバング¹，ゼンホアイ¹，トゥックタイ¹，バオウー¹，齊藤成¹，齊藤百合花¹，池中一裕²，大野伸彦¹ ¹山梨大学大学院総合研究部　医学域　基礎医学系　解剖分子組織学教室，²生理学研究所　分子神経生理研究部門 In the nervous system, many axons are ensheathed by myelin, and dysmyelination disorders are congenital defects in myelin which often lead to loss of myelin called demyelination and axonal degeneration. Although mitochondria are important for axonal functions and survival, mechanisms and roles of mitochondrial alterations in dysmyelinated axons remained elusive. In this study, mitochondrial alterations in the dysmyelinated axons were studied in PLP transgenic mice with overexpression of proteolipid proteins（PLPtg）［1］. With serial block-face scanning electron microscopy（SBF-SEM）, the volume ratio of mitochondria（mitochondrial volume/axonal volume）at 1 month old（m.o.）is similar in myelinated axons of WT and PLPtg. The volume ratio is significantly increased in demyelinated axons but not in myelinated axons of 3 m.o. PLPtg, primarily by increasing the mitochondrial number. By contrast, at 5m.o. when most optic nerve axons are demyelinated in PLPtg, volume of each mitochondrion but not mitochondrial number is dramatically increased in the demyelinated axons, whereas cristae morphology of the large mitochondria was well maintained. Finally, we established organotypic cerebellar slice cultures, and observed robust myelination in WT slices and gradual myelin loss in PLPtg slices during maintenance. These results demonstrate acute mitochondrial accumulation and chronic mitochondrial enlargement upon myelin loss in dysmyelinated axons, and suggest that the enlargement is due to extensive mitochondrial fusion. The results also suggest that slice cultures can be useful to observe mitochondrial behaviour under dysmyelination in vitro. ［1］Kagawa et al. Neuron 13（1994）427-42.		P-037（1） TPT1 regulates the proliferation and/or survival of Neural stem/progenitor cells 大多茂樹¹，河上裕²，岡野栄之¹ ¹慶應大・医・生理学，²慶應大・医・先端研 MIF（Macrophage migration inhibitory factor）was identified as a functional molecule, which supports the proliferation and/or survival of murine neural stem/progenitor cells（NSPCs）using functional cloning strategy（S. Ohta et al., JSC. 2012）. In the functional cloning procedure, we also identified a new factor, TPT1（Tumor Protein, Translationally-Controlled 1）, which can increase the number of neurospheres. TPT1 is expressed in cultured NSPCs and the ventricular zone of mouse brain at embryonic day 14. Intriguingly, MIF-treated murine NSPCs increased the Tpt1 gene expression. Overexpression of murine Tpt1 in NSPCs increased the cell proliferation and the number of secondary neurospheres in vitro. In human ES-derived NSPCs, lentivirus-mediated gene silencing of MIF decreased the gene expression of TPT1, and over-expression of TPT1 increased the cell proliferation and in contrast, lentivirus-mediated gene silencing of TPT1 decreased the cell proliferation. In addition, the TPT1 gene silenced human ES-NSPCs showed the decrease of the S-phase fraction accompanied with the up-regulation of p21 and p27 gene expression, and increased the apoptotic activity. We also performed RNA sequencing and confirmed the gene expression changes of cell cycle related factors in the TPT1 gene silenced human ES-NSPCs. In addition, we tried to identify miRs regulated by TPT1 in human ES-derived NSPCs using Taqman array gene cards, and identified miR338-3p as a TPT1 down stream target. Finally, the TPT1-miR338-3p-SMO axis was newly identified as regulating the cell proliferation of human ES-NSPCs in vitro. Taken together, MIF-regulated TPT1 contributes to the proliferation and/or survival of NSPCs in both mouse and humans.

P-038（1） Role of Meis1 in the cerebellar granule cells 大輪智雄¹，田谷真一郎¹，宮下聡¹，西岡朋生²，中村卓郎³，五飯塚僚⁴，貝淵弘三²，星野幹雄¹ ¹国立精神神経・医療研究センター神経研究所病態生化学研究部，²名古屋大学大学院医学系研究科神経情報薬理学，³がん研究所　発がん研究部，⁴東京理科大学　生命医科学研究所　発生及び老化研究部門　In the nervous system, there are a wide variety of neuronal cell types that have morphologically, physiologically, and histochemically different characteristics and this diversity may enable us to elicit higher brain function. However, the molecular mechanisms underlying the developmental brain are largely unknown. The cerebellum is a good model system to study this issue because a variety of types of neurons are produced. In this study, we focused Myeloid ecotropic viral integration site 1（Meis1）that was expressed in the granule cells, Bergmann glias and ventricular zone of the developing cerebellum. To understand the function of Meis1, we generated Meis1 conditional knockout mice by crossing with Atoh1-Cre-Tg line, where Meis1 gene was specifically deleted in the granule cell lineage and by crossing with En1-Cre line, where Meis1 gene was specifically deleted in the whole cerebellum.In Meis1fl/fl；Atoh1-Cre-Tg mice, the size of the cerebellum was smaller than that of wild type littermates, yet the number of cerebellar lobules was increased in Meis1fl/fl；Atoh1-Cre-Tg mice. Interestingly, many Atoh1-positive cells were ectopically observed in deeper regions（parenchyma）of the postnatal cerebellum. These cells showed mitotic features and therefore we suspected they contributed to generating extra lobules. In Meis1fl/fl；En1-Cre mice, the size of the cerebellum was much smaller than that of wild type or Meis1fl/fl；Atoh1-Cre-Tg mice. Moreover, vermis and cerebellar lobules were disappeared. In contrast to wild type or Meis1fl/fl；Atoh1-Cre-Tg mice, defect of migration was observed in cerebellar granule cells. It has been reported that Bergmann glias regulate migration of granule cells. In Meis1fl/fl；En1-Cre mice, the abnormal position and morphology of Bergmann glias was observed.These results suggest that Meis1 plays important roles in development of granule cells and Bergmann glias, thereby Meis1 participates in correct development of cerebellum.		P-039（1）新生仔期に産生された海馬歯状回神経幹（前駆）細胞の発達過程における低分子量Gタンパク質Racの機能伊東秀記，森下理香，永田浩一愛知県心身障害者コロニー・研究所・神経制御海馬歯状回顆粒細胞は、約85％が出生後に産生されると言われているが、胎生期から生後発達期における神経幹（前駆）細胞の発達の分子機構については不明な点が多い。今回、私共は、独自に確立した新生仔マウスを用いたエレクトロポレーションによる遺伝子導入法を用いて、新生仔期に産生された歯状回顆粒細胞の神経幹（前駆）細胞の発達過程における低分子量Gタンパク質Racの機能解析を行った。Racには、Rac1、Rac2、Rac3の3種類の分子があり、脳組織ではRac1とRac3が発現している。そこで、Rac1の発現抑制（ノックダウン）実験を行った。新生仔マウス（生後0～1日）の脳室内にGFP発現ベクターとRac1のノックダウンベクターを注入し、エレクトロポレーションを行った。そして、21日後に固定脳組織切片を作製し、歯状回におけるGFP発現細胞の分布を蛍光顕微鏡あるいは共焦点レーザー顕微鏡により観察した。その結果、Rac1の発現を抑制すると、海馬歯状回顆粒細胞層に分布する細胞が減少し、顆粒細胞層と歯状回門の境界に分布する細胞が増加することがわかった。次に、これらの移動が障害された細胞における、歯状回顆粒細胞の分化マーカーの発現を組織化学的に解析した。その結果、calbindinおよびNeuNの発現が見られ、成熟した歯状回顆粒細胞の性質を有していることがわかった。また、Rac3の発現を抑制した場合にも、Rac1の発現を抑制した場合と同様に、神経幹（前駆）細胞の移動障害が見られた。一方、Cdc42の発現を抑制した場合には、神経幹（前駆）細胞の移動障害は見られなかった。これらの結果から、Rac1およびRac3は、海馬歯状回顆粒細胞の神経幹（前駆）細胞の発達過程において、重要な役割を果たしていると考えられた。

P-040（1） Functional role of connexin43-mediated gap junctional intercellular communication in proliferation of neural stem/progenitor cells generated after neuronal degeneration in the hippocampal dentate gyrus of adult mouse 米山雅紀，持田浩希，山口太郎，荻田喜代一摂南大学薬学部薬理学研究室 Various neurological injuries are widely recognized as promoting endogenous neurogenesis in the hippocampal dentate gyrus（DG）of adulthood. Gap junction is composed of connexin（Cx）proteins, thereby forming a functional syncytium which is mainly implicated in maintaining tissue homeostasis. Our previous studies demonstrated that the systemic treatment with trimethyltin chloride（TMT）causes the granule cell loss in the mouse hippocampal DG, with being regenerated in the dentate granule cell after the neuronal loss. To elucidate functional roles of Cx in neuroregeneration after neuronal degeneration, we determined the expression of Cx43 in the newly generated cells following neurodegeneration in the DG of adult mouse. In addition, we examined the effect of carbenoxolone（CBX, gap-junction blocker）on proliferation of the neural stem/progenitor cells（NPCs）derived from the adult mouse DG after neuronal degeneration. In vivo experiments, mice were given TMT（2.9 mg/kg, i.p.）to prepare slices for immunohistochemical analyses using antibody against nestin（NPCs marker）and Cx43. Cells positive for nestin and Cx43 markedly increased in the DG on day 3 to 7 after TMT treatment. In vitro experiments, immunostaining revealed that Cx43 was co-localized with most of nestin-positive cells. Exposure of the cells to CBX significantly attenuated the cell proliferation in a concentration-dependent manner. Moreover, CBX had no effect on lactate dehydrogenase release during the culture condition. Taken together, our results suggest that Cx43-mediated gap junctional intercellular communication has a critical role in proliferation of the NPCs generated following neuronal degeneration in the DG.