若手道場口演（Wakate Dojo）

Wakate Dojo Development and Regeneration 3 若手道場口演発生・発達・再生3

7月26日（金）15:10～15:30　第10会場（万代島ビル　6F　会議室） 2WD10aa1-1 新規マイオカインPKM2の神経細胞における分子メカニズムの解明 Takahiro Kikuchi（菊池高弘），Atsushi Kodani（小谷篤），Chihiro Tohda（東田千尋）富山大学、和漢医薬学総合研究所、神経機能学分野、日本 Motor dysfunction of chronic spinal cord injury(SCI)is difficult to cure, even by several approaches effective at acute or subacute phase. We had focused on skeletal muscle atrophy, which is a characteristic detrimental factor in chronic phase of SCI, and explored drugs that improve both skeletal muscle atrophy and motor dysfunction. As a result, we found that intramuscular injection of acteoside improved muscle atrophy and motor dysfunction in chronic SCI model mice. By acteoside injection, the axonal density in the spinal cord was increased, and the presynaptic density terminating on motor nerves was increased (J. Neurotrauma, (2018) Kodani et al.,). Our previous investigation discovered for the first time that pyruvate kinase isoform M2 (PKM2) was secreted from skeletal muscle cells by acteoside stimulation. PKM2 has never been recognized as a myokine, therefore, the role of PKM2 as a myokine has not been known at all. Our previous study showed that extracellular PKM2 might cross the blood-brain barrier, suggesting the possibility of direct action of PKM2 to neurons. This study aimed to clarify effects and molecular mechanism of PKM2 on neurons. Treatment with recombinant PKM2 enhanced axonal growth in cultured cortical neurons at normal condition and also CSPG-coated inhibitory condition. Next, cell specificity of PKM2 secretion was investigated. Cultured primary cells were treated by acteoside, and PKM2 secretion in conditioned media was detected. PKM2 secretion was increased only in skeletal muscle cells, but not in cortical neurons, spinal cord neurons, astrocytes and microglia. The direct binding protein of PKM2 in neurons was explored using Drug Affinity Responsive Target Stability (DARTS) method. Valocin Containig Protein (VCP) was identified. VCP is expressed in various tissues and is involved in several functions such as apoptosis and endoplasmic reticulum-associated degradation (ERAD). However, association of VCP with PKM2 and its regulatory function for axonal growth are complentry unknown. This study is the first to show that a new myokine, PKM2, enhanced axonal growth and VCP may be involved in its molecular signaling.		7月26日（金）15:30～15:50　第10会場（万代島ビル　6F　会議室） 2WD10aa1-2 PRMT1によるScyl1アルギニンメチル化はゴルジ体形成を介した神経突起伸長に必須である Genki Amano（天野元揮）¹，Gneki Amano（天野元揮）¹，Takeshi Yoshimura（吉村武）¹，Yasutake Mori（森康丈）³，Ko Miyoshi（三好耕）¹，Sarina Han（韓薩日娜）¹，Sho Shikada（鹿田星）¹，Shinsuke Matsuzaki（松崎伸介）^1,2，Taiichi Katayama（片山泰一）¹ ¹大阪大院連合小児発達分子生物遺伝 ²和歌山県立医大医薬理 ³国際医療福祉大院医解剖神経 The Golgi apparatus plays a central role in the posttranslational modification, trafficking proteins and lipids. The Golgi apparatus regulates the development of apical and basal dendrite in embryonic pyramidal neurons. Protein arginine methylation is a common posttranslational modification to modulate protein function. Protein arginine methyltransferase (PRMT) 1 is required for the central nervous system development at embryonic and perinatal stages. SCY1 Like Pseudokinase 1 (Scyl1) interacts with γ₂-COP to form COPI vesicles and regulates Golgi morphology. However, the molecular mechanism by which Scyl1 is regulated remains unclear. Here, we report that Scyl1 is arginine methylated by PRMT1. The arginine methylation inhibitor decreased the Scyl1 arginine methylation, and the interaction between Scyl1 and γ₂-COP. The alanine replacement of arginine in the C-terminal region of Scyl1 decreased the Scyl1 arginine methylation, and the interaction between Scyl1 and γ₂-COP. Knockdown of PRMT1 inhibited Scyl1 arginine methylation and decreased the interaction between Scyl1 and γ₂-COP. In microsomal fraction, we found that PRMT1 was localized at Golgi fraction. In addition, abnormal Golgi morphology was observed in PRMT1 knockdown cells. Inhibition of arginine methylation resulted in decreased axon outgrowth and dendrite complexity. Thus, PRMT1 regulates Golgi morphogenesis via Scyl1 arginine methylation. We propose that Scyl1 arginine methylation by PRMT1 contribute axonal and dendritic morphogenesis in neurons.

7月26日（金）15:50～16:10　第10会場（万代島ビル　6F　会議室） 2WD10aa1-3 分泌タンパク質リーリンは脳でオリゴデンドロサイト前駆細胞の数と配置に影響を与える Himari Ogino（荻野ひまり）¹，Tsuzumi Nakajima（中島鼓美）¹，Kohki Toriuchi（鳥内皐暉）²，Mineyoshi Aoyama（青山峰芳）²，Mitsuharu Hattori（服部光治）¹ ¹名市大院薬病態生化 ²名市大院薬病態解析 Reelin is a secreted glycoprotein that is essential for normal brain development and function. Reelin binds to very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2, and induces phosphorylation of an intracellular protein Dab1. Previously, we showed that Reelin function is regulated by specific proteolysis and that ADAMTS-3 (A Disintegrin And Metalloproteinase with Thrombospodin motifs-3) cleaves and inactivates Reelin in the embryonic mouse brain. However, the physiological significance of the regulation of Reelin function by specific proteolysis for the brain development and function is still unclear. Oligodendrocytes (OLs) are the myelinating cells which arise from oligodendrocyte precursor cells (OPCs) in the central nervous system (CNS). Myelination occurs in a complex and dynamic manner throughout life and abundant OPCs remain in the adult CNS in order to generate new OLs, which repair the loss of myelin under the normal aging or pathological conditions. The molecular cues that regulate the development of OPCs and OLs in the brain are not fully understood. Previously, it was reported that the cultured OPCs and OLs express Reelin, Dab1, and VLDLR and that OLs and myelin are mislocalized in the brain of Reelin deficient (Reeler) mice or Dab1 mutant (Yotari) mice. However, because the layer formation and axon trajectory are severely disrupted in Reeler and Yotari mice, it remains unclear whether Reelin signaling is directly involved in the development of OPCs and OLs. In the present study, we show that the number and localization of OPCs are altered in the embryonic brain of two types of genetically modified mice; ADAMTS-3 knock-out mice and Reelin PADV knock-in mice in which the Reelin cleavage site was mutated. It suggests that Reelin functions are upregulated in these mice. We are also conducting the same line of experiments in the heterozygous Reeler and Yotari mice. The results obtained so far suggest that Reelin signaling affects the number and localization of OPCs by unknown mechanisms. We are currently investigating the effects of Reelin on the development of OPCs and OLs by using in vitro system in order to understand whether Reelin directly affects the development of OPCs and OLs, and to clarify the molecular mechanism.