神経化学教育口演セッション3　Neurochemistry Educational Oral Session3

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神経化学教育口演セッション3 Neurochemistry Educational Oral Session3

EO3-1 Myosin IDはオリゴデンドロサイトのミエリン形成に重要な役割をしている山崎礼二，山口宣秀，石橋智子，馬場広子東京薬科大学薬学研究科機能形態学教室 Myelin is a unique multilamellar structure and crucial for normal neuronal function. In the CNS, oligodendrocytes generate myelin, which extend several processes and wrap many layers of plasma membrane around axons. At present, the mechanisms of this myelin formation including membrane trafficking and wrapping remains unknown. Myosin ID（Myo1d）which is an unconventional class I myosin is a candidate motor protein involved in myelin formation. Previously, we reported that Myo1d was expressed in mature rat oligodendrocytes expressing myelin proteolipid protein（PLP）. In this study, to clarify the function of Myo1d in oligodendrocyte, we performed siRNA-mediated knockdown of Myo1d in oligodendrocytes. At three days after differentiation, cultured rat oligodendrocytes were treated with Myo1d-specific siRNA or control siRNA for 48 hours. The expression of Myo1d mRNA in oligodendrocyte was effectively reduced by the Myo1d-siRNA-treatment. Morphological changes were examined by immunostaining using O4 antibody. Retraction of processes and/or degeneration of myelin-like membrane were seen in Myo1d-siRNA-treated oligodendrocytes. The morphologies of non-treated and control-siRNA-treated oligodendrocytes looked normal. Morphological changes in Myo1d knockdown oligodendrocytes were confirmed using the fluorescence-labeled Myo1d-siRNA. These results suggest that Myo1d may play a role in the formation and/or maintenance of myelin membrane in oligodendrocytes．		EO3-2 神経サブタイプ及び活動電位に依存したオリゴデンドロサイトによる髄鞘形成の解析長内康幸¹，清水健史^1,2，森琢磨^1,3，吉村由美子^1,3，畑中伸彦^1,4，南部篤^1,4，小林憲太^1,5，池中一裕^1,2 総合研究大学院大学生命科学研究科生理科学専攻¹，生理学研究所　分子神経生理研究部門²，生理学研究所　視覚情報処理研究部門³，生理学研究所　生体システム研究部門⁴，生理学研究所　ウイルスベクター開発室⁵ Oligodendrocyte myelinates multiple axons in the central nervous system. Recent reports have revealed that depolarization of oligodendrocytes increases axonal conduction velocity, suggesting that multiple axons ensheathed by a depolarized oligodendrocyte is modulated synchronously. We examined whether oligodendrocytes preferentially myelinate particular axons in the white matter. Neuronal axons in the corpus callosum are derived from distinct brain regions. We focused on callosal oligodendrocytes and established a novel method to observe interaction between oligodendrocytes and neurons by the use of modified rabies virus（RV）and adeno-associated virus type2（AAV2）encoding different fluorescent markers. While we labeled axons derived from the motor cortex and the sensory cortex by AAV-DsRed2 and AAV-BFP respectively, oligodendrocytes were labeled by RV-GFP. We found that percentage of myelinated axons derived from the sensory cortex is higher than that from the motor cortex. We are now applying this technique to the optic chiasm to analyze changes of myelination after deprivation of light stimulation. We plan to block light stimulation by suturing eyelid of either eye. After the operation, we will label axons from right eyeball and left eyeball by the means of injection of AAV-DsRed2 and AAV-BFP, while oligodendrocytes are labeled by injection of RV-GFP to the optic chiasm. This newly established technique enables us to observe direct interaction between individual oligodendrocyte and neuron．

EO3-3 Protease activity of DINE is vital for motor nerve arborization and neuromuscular junction formation Matsumoto Sakiko，Kiryu-Seo Sumiko，Kiyama Hiroshi Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine Damage-induced neuronal endopeptidase/Endothelin-converting enzyme-like 1（DINE/ECEL1）is a membrane-bound metalloprotease that we originally identified as a nerve injury-inducible gene. The abundant expression of DINE is also observed in developing motor neuron. We previously demonstrated that DINE-deficient mice（KO）died at birth because of respiratory failure, due to the insufficient arborization of phrenic motor nerve terminal and the remarkable loss of neuromuscular junction（NMJ）in embryonic diaphragm. Recently, some reports have showed that the mutation of DINE/ECEL1 causes a congenital distal arthrogryposis in human. These findings suggest that DINE plays an important role in development of NMJ in both mice and human. Since some of metalloprotease family members function independent of protease activity, we have attempted to clarify whether DINE functions as protease or not in vivo. For this purpose, we performed the rescue experiment of DINE KO. We first generated two types of DINE transgenic mice（Tg）under the control of motor neuron-specific Hb9 promoter, which drove either the wild type（WT）DINE or protease active site-mutated（mut）DINE. We confirmed that both Tg expressed the exogenous DINE specifically in developing motor neurons. We next crossed both Tg with DINE KO to examine which type of DINE could rescue the phenotype of DINE KO. WT DINE expression in DINE KO contributed to succeeding in the nerve terminal arborization of phrenic motor nerve and the appropriate NMJ formation, which led them to survive after birth. In contrast, the mut DINE failed to rescue the phenotype of DINE KO, leading to death at birth. We conclude that the protease activity of DINE is essential for proper development of nerve terminal and NMJ.		EO3-4 小脳の発生に伴うMLC1の発現解析菊地原沙織^1,2，稲村直子^1,2，杉尾翔太¹，田中謙二³，渡辺雅彦⁴，池中一裕^1,2 自然科学研究機構生理学研究所分子神経生理研究部門¹，総合研究大学院大学生命科学研究科生理科学専攻²，慶應義塾大学医学部精神・神経科学教室³，北海道大学大学院医学研究科/医学部医学科解剖学講座解剖発生学分野⁴ MLC1 has been reported to be a causative gene for megalencephalic leukoencephalopathy with subcortical cysts（MLC）. Previous researches on the cerebral cortex showed that MLC1 is localized in the astrocyte cell membrane. However, MLC1 function is not yet totally clear. In the cerebellum, MLC1 is also expressed in the Bergmann glia（BG）, which are a type of astrocytes that develop from radial glia. The function of BG has attracted interests of many people because they play a key role in the development of cerebellum. For example, they are responsible for the precise localization of various cerebellar cells, formation of lobes, and formation and maintenance of synapses. In MLC1 overexpressing mice, our group previously observed that BG soma was ectopically localized in the molecular layer and its morphology also changed. This result suggests MLC1 might have a relationship with development and maturation of BG. In this research, MLC1 expression during cerebellar development of wild type mice was examined by immunohistochemistry. As a result, MLC1 expression and its localization in the cell changed during development. At embryonic day（E）14.5, MLC1 was expressed in the radial glia soma in the ventricular zone. At E16.5 days, MLC1 expression differed between the areas in the ventricular zone. From E16.5 to postnatal day（P）0, MLC1 expression was observed in the radial glia soma, some part of the fibers and the endfeet attached with vessels. At P5, MLC1 was specifically expressed in the BG fiber in the fissure and the endfeet attached with basement membrane. After P10, the lamella structure also expressed MLC1. These results suggest that MLC1 expression and localization would be responsible for regulation of the BG development.

EO3-5 数理モデルと再凝集培養系を用いた大脳皮質神経細胞の接着様式の解析松永友貴¹，野田万理子¹，村川秀樹²，三浦岳³，久保健一郎¹，仲嶋一範¹ 慶應義塾大学医学部解剖学教室¹，九州大学大学院数理学研究院²，九州大学大学院医学研究院系統解剖学分野³ The mammalian neocortex has a highly organized 6-layered structure of neurons. Cortical neurons are generated within the ventricular zone（VZ）or subventricular zone（SVZ）, and migrate along radial fibers toward the pial surface. Newly born excitatory neurons migrate radially into the cortical plate（CP）past the earlier-born neurons, resulting in the birth-date-dependent"inside-out"alignment of neurons in the CP. Although the Reelin-deficient mouse, reeler, has been studied for more than 50 years and Reelin is indispensable for the establishment of the"inside-out"neuronal layers, cellular and molecular functions of Reelin for layer formation are still largely unknown. Reaggregation culture is a tool for studying intercellular adhesion. In the previous study, several clusters of MAP2-positive neurons were abnormally observed in the reaggregates of the reeler cerebral cortical cells. This result suggests the possibility that intercellular adhesion is altered in the reeler cerebral cortex. In the present study, to uncover how Reelin controls the intercellular adhesion among cortical cells, we performed reaggregation culture of the cells from reeler cerebral cortex. Dissociated cells from embryonic day（E）12 cerebral cortex were incubated in tubes on a rotating holder for 5 days. Reelin was supplied in two ways：i）we added Reelin that was produced and secreted from the reelin-transfected HEK293T cells, or. ii）we transfected an expression vector for Reelin into the cortical cells in the reaggregates. In addition, we made mathematical models of cell sorting, and examined the factors necessary and sufficient for the formation of cell reaggregation patterns in the presence or absence of Reelin.		EO3-6 大脳新皮質においてリーリン分子により誘導される神経細胞凝集を担うシグナル伝達経路の探索 Inoue Seika，Hayashi Kanehiro，Kubo Ken-ichiro，Nakajima Kazunori 慶應義塾大学医学部解剖学教室仲嶋研究室　Reelin is secreted by Cajal-Retzius cells in the marginal zone of the developing neocortex. This molecule is essential for the formation of the neocortex, because the layered structure is basically inverted in the Reelin-deficient mouse, reeler. We previously reported neuronal aggregation induced by the ectopic expression of Reelin, and this neuronal aggregation showed a birthdate-dependent inside-out alignment（Kubo, et al. J. Neurosci., 2010）. These characteristics of the neuronal aggregation are similar to those of the outermost structure of a cortical plate in the developing neocortex. Therefore, molecular machinery in this directed arrangement of neuronal aggregation caused by Reelin may also work in the layer formation of the neocortex. Although various molecules are reported as the downstream of Reelin, signaling pathways of the neuronal accumulation induced by Reelin are still unknown. Thus, we investigate the molecular mechanisms to regulate this neuronal accumulation. 　In the present study, we expressed Reelin and knockdown（or dominant-negative）vectors for molecules that are already known as downstream mediators of Reelin signaling in the mouse embryonic neocortex using in utero electroporation. Then, we examined how these molecules affect the neuronal accumulation caused by Reelin. 　As a result, we observed abnormalities in the formation of the neuronal aggregation by knockdown of Crk, C3G, Nckβ, and that these phenotypes were different among molecules. On the other hand, Akt knockdown didn't affect the neuronal aggregation. These results suggest that several signal pathways are associated with the neuronal aggregation evoked by Reelin. We will further analyze how each signaling contributes to the formation of this neuronal aggregation.