一般演題（ポスター）

一般演題（ポスター） Neural Development・Neural Differentiation II

2P-18 Roles of the autism susceptibility candidate gene Auts2 for neuronal migration and neuritogenesis in the developing brain Hori Kei¹，Nagai Taku²，Shan Wei²，Sakamoto Asami¹，Taya Shinichiro¹，Hashimoto Ryoya¹，Hayashi Takashi¹，Abe Manabu³，Yamazaki Maya³，Nakano Keiko⁵，Nishioka Tomoki⁴，Sakimura Kenji³，Yamada Kiyofumi²，Kaibuchi Kozo⁴，Hoshino Mikio¹ ¹National Institute of Neuroscience, NCNP，²Dept. Neuropsychopharmacology ＆ Hospital Pharmacy，³Dept. Cell Neurobiol. Brain Res Inst., Niigata Univ.，⁴Dept. Cell Pharm., Nagoya Univ.，⁵Dept. Physiol., Saitama Sch of Med. Previous studies have demonstrated that mutations in Autism susceptibility candidate 2 gene were associated with multiple psychiatric illnesses including autism spectrum disorders（ASD）, intellectual disability（ID）. In developing brain, Auts2 mRNA is highly expressed at several brain regions responsible for the cognitive brain functions such as prefrontal cortex, hippocampus and cerebellum. The physiological roles of this gene in CNS, however, remain largely unknown. Here we reveal a novel function of the protein for AUTS2 in the cytoplasm, regulating cytoskeleton and neural development. Immunohistochemical analysis reveals that this protein is exclusively localized at nuclei of postmitotic neurons at cerebral cortex in an early embryonic stage. In later stage, however, it also appears at the neurites including axons. Immunocytochemistry shows, in neuronal cells, it localizes not only in nuclei but also in cytoplasm including growth cones. AUTS2 activates Rac1 via interaction with several Rac1-GEFs including P-Rex1 and Elmo2/Dock180 complex to induce lamellipodia in neuroblastoma cells and promote the neurite-outgrowth in primary hippocampal neurons. Our loss-of-function experiments revealed that AUTS2 participates in cortical neuronal migration and neuritogenesis by activating Rac1 signaling in the developing cerebral cortex. Moreover, the KO mice display behavioral abnormalities including anxiety-related emotion and memory formation. Thus, our findings indicate that AUTS2 contributes to cortical development and is critical for the acquisition of neurocognitive function.		2P-19 Acute inflammation induces the proliferation of radial glial cells in the optic tectum in response to traumatic brain injury Shimizu Yuki¹，Ueda Yuto¹，Ito Yoko¹，Tanaka Hideomi^1,2，Ohshima Toshio¹ ¹Dep. Life Sci. Med. Biosci., Grad. Sch. Adv. Sci. Eng., Waseda University，²Organization for European Studies, European Biomedical Science Institute, Waseda University Adult neurogenesis is a phenomenon that neural stem cells（NSCs）produce new neurons, astrocytes or oligodendrocytes in the adult brain. It is conserved among various vertebrates. In the adult mammalian brain, neurogenesis is restricted to the subventircular zone（SVZ）and the subgranular zone（SGZ）. In contrast, zebrafish have 16 NSCs niches and can continue to produce new neurons through life. In the optic tectum where optic nerves project, neuroepithelial-like cells in the dorsomedial margin of the perventricular gray zone（PGZ）have the property of self-renewal and multipotency and continue to supply new neurons, radial glial cells（RGCs）and oligodendrocytes. RGCs in the deeper layer expressing several stem cell markers as Sox2 and msi1 are quiescent, while RGCs in the telencephalon are proliferative and work as NSCs. In this study, we found that acute inflammation induced not only the proliferation of neuroepithelial-like NSCs but also that of RGCs in the PGZ. In addition, RGCs were also activated by stab injury, suggesting that RGCs in the deeper layer have a key role in the regeneration from the tissue damage in the PGZ.

2P-20 The role of natural killer cells in developmental brain Tanabe Shogo，Yamashita Toshihide Dept. of Mol Neurosci., Med., Osaka Univ During brain development, neural stem cells proliferate and generate neurons and glial cells in ventricular zone（VZ）and subventricular zone（SVZ）. As the proliferation and differentiation of neural stem cells determines the brain organization and function, it is important to understand the mechanism of neural stem cells activity for the treatment of psychiatric disorders. Recent study demonstrates that some immune cells such as microglia regulate the activity of neural stem cells. However, it has not been reported whether peripheral immune cells are involved in brain development. In current study, we examined the infiltration of peripheral immune cells to embryonic and postnatal mouse brain by flow cytometry analysis, and found that NK1.1+ natural killer（NK）cells infiltrate embryonic and postnatal brain. To identify the role of NK cells in developmental brain, we intraventricularly injected NK1.1 neutralizing antibody at E16 to deplete NK cells from embryonic brain, and found that the number of BrdU labeled mitotic cells around SVZ decreased by NK1.1 antibody injection. Moreover, BLBP+ astrocyte precursors and Tbr2+ basal progenitors were also reduced by NK1.1 antibody injection. These results suggest that NK cells promote neurogenesis and gliogenesis in postnatal brain by regulating the proliferation of neural stem cells.		2P-21 Role of the Meis1 in the development of cerebellum. Owa Tomoo¹，Taya Shinichiro¹，Nishioka Tomoki²，Nakamura Takuro³，Goitsuka Ryo⁴，Kaibuchi Kozo²，Hoshino Mikio¹ ¹Dept of Biochemistry andCellularBiology National Institute of Neurosucience NCNP，²Dept. of Cell Pharmacology, School of Medicine, Nagoya Univ，³Department of Carcinogenesis, Japanese Foundation for Cancer Research，⁴Division of Development ＆ Aging, Research Institute for Biological Sciences, Tokyo University of Science Meis1, which is a transcription factor, is involved in neural differentiation in neural stem cells, however the function of Meis1 in brain development remains unclear. We determine that Meis1 is expressed in the developing cerebellum, especially in granule cells. To investigate the role of Meis1 in granule cells, Meis1 granule cells-specific conditional knockout（cKO）mice are generated. Meis1 cKO mice exhibit the small cerebellum and abnormalities in cerebellar foliation. In addition, decreased proliferation of granule cells and increased number of immature granule cells are observed in Meis1 cKO mice. We reveal that these abnormal phenomena are regulated by Meis1 via the Pax6/Smad1/BMP signaling cascade. Our results suggest that Meis1 participates in granule cell development, including proliferation and differentiation, and is a key factor for correct cerebellar development.

2P-22 Knockdown of glycoprotein M6a in utero delayed the determination of neuronal polarity Ito Yasuyuki¹，Honda Atsuko¹，Takeuchi Kosei³，Matsushita Natsuki⁴，Igarashi Michihiro^1,2 ¹Department of Neurochemistry and Molecular Cell Biology, Graduate School of Medical and Dental Sciences, Niigata University，²Center for Transdisciplinary Research, Niigata University，³School of Medicine, Aichi Medical University，⁴Translational Research Center, Ehime University Hospital. Determination of the neuronal polarity is essential to the proper neural network organization of the developing brain. However, its molecular mechanisms have not been understood completely. We previously found by proteomics that glycoprotein M6a was one of the most abundant membrane proteins in the growth cone, and our many in vitro experiments indicated that M6a is involved in the determination of the neuronal polarization, thus, we suspected that M6a is responsible for the neuronal polarity in in vivo. To confirm this hypothesis, we investigated physiological roles of the M6a in the developing cerebral cortex using in utero M6a-knockdown（KD）. Since M6a is highly expressed in the developing cortical neurons（E12~）, in utero electroporation was performed at E14.5 and the fetus were fixed at E16.5 or P2.5. On two days after electroporation（E16.5）, the normal neurons migrated into the intermediate zone with changing their shapes, from the multipolar to the bi- or unipolar. In contrast, M6a-KD impaired the normal morphological transition, resulting in the increased proportion of the multipolar neurons. In addition, the number of neurites increased in M6a-KD neurons than the ones using the negative control shRNA. On seven days after electroporation（P2.5）, the axon elongation remained immature and the neurons with shorter axons increased in number. These results are consistent with our in vitro data, indicating that M6a participates in the physiological steps of neuronal polarization.		2P-23 Contactin associated protein（Caspr）4/LNX2 signaling pathway modulates neuronal differentiation of mouse neural progenitor cells Futagawa Toshitaka¹，Masuda Shogo¹，Takahama Kazuhiro¹，Ma Quanhong²，Xiao Zhi-Cheng³，Takeda Yasuo¹ ¹Dept. of Clin. Pharm. and Pharmacol., Grad. Sch. of Med. and Dent. Sci. of Kagoshima Univ.，²Jiangsu Key Lab. of Translational Research and Therapy for Neuro-Psycho-Diseases and Insti. of Neurosci., Soochow Univ.，³Shunxi-Monash Immune Regeneration and Neuroscience Laboratories, Dept. of Anat. and Dev. Bio., Monash Univ. [INTRODUCTION]The contactin-associated protein（Caspr）family includes 5 members：Caspr1, Caspr2, Caspr3, Caspr4 and Caspr5. Two of them, Caspr1 and Caspr2, have been well characterized as key molecules for central and peripheral myelination. However, the roles of Caspr4 have not been well characterized yet. Caspr4 is type I trans-membrane protein that have relatively large extracellular domain and short intracellular domain. It has been reported that Caspr4 is mainly expressed in specific neuronal subpopulations（Spiegel I et al, 2001）. Recently, it has also been reported that Caspr4 is a susceptibility gene of autism spectrum disorders（ASDs）（Karayannis T et al, 2014）. However, the molecular function of Caspr4 in the brain has yet to be identified. To understand the function of Caspr4, we have identified Ligand of Numb X2（LNX2）as a binding partner of Caspr4 intracellular domain using yeast-two hybrid analysis. LNX2 consists PDZ domains, the PDZ domains of LNX2 is a specific binding for PDZ binding motif of Caspr4. In this study, we focused on analyses of functional interaction of Caspr4 and LNX2, especially, the distribution in the developmental brain. Furthermore, we have investigated whether the interaction is related to neuronal differentiation. [RESULTS AND DISCUSSION]We have shown that both Caspr4 and LNX2 expressed in the ventricular zone（VZ）and neural progenitor cells（NPCs）isolated in embryonic 14 days from mice.In vitro differentiation assay, neuronal differentiation was significantly reduced when shRNAs were applied to decrease the expression of either Caspr4 or LNX2 and increased when either Caspr4 or LNX2 was overexpressed in NPCs. In utero electroporation, neuronal differentiation was also increased when either Caspr4 or LNX2 was overexpressed. These results describe positive modulation of neuronal differentiation by Caspr4/LNX2 signaling.