一般演題（ポスター）

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

1P-24 Maternal separated mice show the anxiety- and fear-related behavior and change neurogenesis in the limbic system Suzuki Harumitsu，Li Hongyu，Tanaka Aoi，Koyama Natsu，Hitoshi Seiji Dept. of Integrative Physiology, Shiga Univ. of Med. Sci. Early life stress is known to induce long-term alterations in emotional and anxiety-related behaviors. Rodent models of neonatal maternal separation（MS）stress have been used to explore the effects of early stress on changes in affective and cognitive behaviors. MS are associated with structural changes in brain regions linked to cognition and mood regulation. Here, we studied the effects of MS on the alteration of neurogenesis in the limbic system and anxiety-related behavior on C57Bl/6 mice. The MS was performed daily for 3 hr from P1 to P14 and behavioral test was started at 10 weeks of age. We used a battery of stress and anxiety-related behavioral tests in C57Bl/6 mice. （1）The open field test, which measures the basal anxiety level, showed that MS mice tended to spend shorter time in the center area, although total moving distance did not differ. （2）The acoustic startle response induced by the sudden loud tone stimulus was significantly elevated in MS mice. （3）The contextual and cued fear conditioning test provides a measure of memory by assessing a memory for the association between an aversive stimulus and a tone stimulus. MS mice showed decreased fear conditioning to the context and the tone compared to the control. While startle response was elevated in MS mice, freezing time during tone stimulus was significantly attenuated, suggesting that the fear memory formation or maintenance was impaired in MS mice. （4）Neurogenesis in the limbic system was increase in MS mice. These results suggest that neonatal MS treatment enhances the neurogenesis and alters the anxiety- and fear-related behavior. We are investigating whether or not MS treatment alters the differentiation of neural progenitor cells into excitatory and inhibitory neurons or even glial precursor cells, such as NG-2 positive oligodendrocyte precursor cells.		1P-25 Proteomic characterization during differentiation from human embryonic stem cells into early and late neural stem cells by neural stem sphere method Akama Kuniko^1,2，Shibasaki Gen²，Tomioka Tetsutaro²，Totsuka Akihiro³，Otsu Masahiro^4,5，Inoue Nobuo⁴，Nakayama Takashi⁶，Miura Yuri⁷，Iwamoto Machiko⁷，Tsumoto Hiroki⁷，Satoh Mamoru⁸，Kado Sayaka⁹，Suzuki Yutaka¹⁰，Kondo Yasushi¹⁰ ¹Cent. of General Educ., Chiba Univ.，²Dept. of Chem., Grad. Sch. of Sci., Chiba Univ.，³Dept. of Chem., Fac. of Sci. Chiba Univ.，⁴Lab. of Regen. Neurosci., Grad. Sch. of Human Health Sci., Tokyo Metrop. Univ.，⁵Dept. of Chem., Kyorin Univ., Sch. of Med.，⁶Dept. of Biochem., Yokohama City Univ. Sch. of Med.，⁷Res. Team Mech. Aging, Tokyo Metrop. Inst. of Geront.，⁸Clin. Proteomics Res. Cent., Chiba Univ. Hospital，⁹Center for Analytical Instrumentation, Chiba Univ.，¹⁰Regen. Med, Adv. Med. Res. Lab., Mitsubishi Tanabe Pharma Co. Understanding neurogenesis is valuable for the treatment of nervous system disorders. However, there is currently limited information about the molecular events associated with the transition from human ES cells to neural stem cells. We therefore investigated the differentially expressed genes during differentiation of highly homogeneous human embryonic stem cells to early and late neural stem cells by neural stem sphere method, using SDS-PAGE and liquid chromatography-tandem mass spectrometry. We identified 1145 differentially expressed proteins involved in these three differentiation stages. Together with the results of classification of protein functions and search of metabolic pathways related to differentially regulated proteins using DAVID bioinformatics tools and KEGG, respectively, suggested that ES cells differentiated to early neural stem cells via extracellular matrix-receptor interactions followed by their signal transduction, and that early neural stem cells differentiated to late neural stem cells via reorganization of cytoskeleton followed by extension of the cells with increase of differentiated neural stem cells.

1P-26 Role of Kruppel-like factor 5 in neural precursor cells during brain development Fuchigami Takahiro¹，Hayashi Yoshitaka¹，Kuroda Anri¹，Ishida Shouhei¹，Yamashita Yukako¹，Ema Masatsugu²，Hitoshi Seiji¹ ¹Dept. of Integ. Physiol., Shiga Univ. of Med. Sci.，²Res. Cent. for Animal. Life Sci., Shiga Univ. of Med. Sci. Kruppel-like factor（Klf）5 is a member of Klf family proteins, which are members of DNA-binding transcriptional factors with highly conserved sequences and redundant functions in the regulation of cell cycle, cell differentiation and tissue organization. Among Klf family, Klf4 is one of the defined factors which reprogram somatic cells to induced pluripotent stem cells（iPS cells）. Klf4 is a key regulator of pluripotency in embryonic stem cells（ESCs）, inner-cell mass（ICM）and neural precursor cells（NPCs）. Klf5 possesses overlapping function with Klf4 in the induction of iPS cells and ESCs self-renewal. Klf5 is shown to be essential for the blastocyst implantation, the three germ layers development, and the formation of cardiovascular system and optic vesicle. Although these preceding reports suggest that Klf5 has broad roles in the organogenesis, its function in the central nervous system has not been investigated despite of its expression in the developing brain. In this study, we have investigated roles of Klf5 in the proliferation and maintenance of NPCs in the developing cortex. When knockdown or overexpression of Klf5 was performed by in utero electroporation, aberrant differentiation and migration of NPCs were observed. We also found that BrdU incorporation of NPCs was altered after the knockdown/overexpression of Klf5. In addition, we performed a colony-forming neurosphere assay using NPC-specific Klf5 conditional knockout mice, which revealed the impairment of self-renewal of neural stem cells. To examine overlapping function of Klf4 and Klf5, we have also analyzed NPC-specific Klf4 and Klf5 double deficient embryos and observed accumulating effects of gene deletion in the brain development. Our data suggests that Klf5, as well as Klf4, plays an important role in the proliferation, differentiation and migration of NPCs.		1P-27 Role of Protease-activated receptor-1 in proliferation of neural stem/progenitor cells derived from the adult mouse hippocampal dentate gyrus Yoneyama Masanori，Tanaka Masayuki，Yamamura Shinjiro，Ogita Kiyokazu Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University It is now clear that there is a continual turnover of the mammalian hippocampal dentate gyrus neurons throughout life even in adult. Various neurological injuries are widely recognized as promoting endogenous neurogenesis in hippocampal dentate gyrus. Thrombin-activated/protease-activated receptor-1（PAR-1）is known to regulate proliferation of neural cells following brain injury including intracellular hemorrhage. To elucidate involvement of PAR-1 in neurogenesis occurred in the adult hippocampus, we evaluated the effect of thrombin and PAR-related peptides on proliferation of the neural stem/progenitor cells（NPCs）derived from the hippocampal dentate gyrus of adult mouse. Immunostaining revealed that PAR-1 was co-localized with nestin, which is a marker for NPCs. Reverse transcription-PCR analysis showed the expression of mRNA encoding all subtypes of PAR in the NPCs. Exposure of the cells to thrombin significantly attenuated the cell proliferation without morphological change and cell damage. However, the cell proliferation was not affected by the PAR-1 negative control peptide, RLLFT-NH2, which is an inactive peptide for PAR-1. Thrombin had no effect on lactate dehydrogenase release during the culture condition. In addition to thrombin, the PAR-1 agonist peptide, SFLLR-NH2, also attenuated the cell proliferation in a concentration-dependent manner. Moreover, the attenuation induced by thrombin was completely abolished by RWJ56110, which is a PAR-1 antagonist. These data suggest that PAR-1 negatively regulates proliferation of the NPCs in the adult hippocampus.

1P-28 The dorsoventral boundary of the germinal zone is a specialized niche for the generation of cortical oligodendrocytes Hitoshi Seiji^1,3，Asuke Jun¹，Naruse Masae^2,3，Ikenaka Kazuhiro³ ¹Dept Integrative Physiol, Shiga Univ Med Sci，²Dept Mol Cell Neurobiol, Gunma Univ，³Neurobiol Bioinformatics, NIPS Oligodendrocyte precursor cells（OPCs）appear in the late embryonic brain, mature to become oligodendrocytes（OLs）and form myelin in the postnatal brain. Recently, it has been proposed that early-born OPCs derived from the ventral forebrain are eradicated postnatally and that late-born OLs predominate in the cortex of the adult mouse brain. However, intrinsic and extrinsic factors that specify the ability of self-renewing multipotent neural stem cells in the embryonic brain to generate cortical OL-lineage cells remain largely unknown. Using an inducible Cre-loxP system to permanently label Nestin- and Olig2-lineage cells and using an in utero electroporation technique, we determined when and where cortical OL-lineage cells differentiate from neural stem cells in the developing mouse brain. We show that neural precursor cells in the dorsal VZ/SVZ are inhibited by Wnt signaling from contributing to cortical OLs in the adult brain. By contrast, neural precursor cells present in the dorsoventral boundary VZ/SVZ produce a significant amount of OLs in the adult cortex. Our results suggest that neural stem cells at this boundary are uniquely specialized to produce myelin-forming OLs in the cortex.		1P-29 Expression profiling of ubiquitin ligases with transmembrane domain in the brain Kaneko Masayuki¹，Wu Yan¹，Takai Tomoko¹，Nomura Yasuyuki²，Imaizumi Kazunori¹ ¹Dept. Biochem., Hiroshima Univ.，²Dept. Pharmacol., Kurume Univ. Sch. Med. Studies on endoplasmic reticulum（ER）-associated degradation（ERAD）, in which unfolded proteins accumulated in the ER are selectively transported to the cytosol for degradation by the ubiquitin-proteasome system, have been focused on molecular mechanisms in yeast. In human, disruption of the ER quality control system causes various diseases, such as neurodegenerative disease, lifestyle disease, and cancer. Furthermore, ER stress has become more important because it is also involved in cellular differentiation and tissue development. We have identified human 44 ubiquitin ligases（E3）with transmembrane domain, which are potentially involved in ERAD. As reason for so many genes in mammals compared with the yeast 3 ubiquitin ligases, they are assumed to have tissue-specific and/or developmental stage-specific roles. Here, we investigate the tissue distribution and cellular expression the ubiquitin ligases. First, we specialized high expression organization of each ubiquitin ligases in the human and the mouse tissues by quantitative PCR. Several kinds of tissue-specific and embryonic-specific ubiquitin ligases genes have been found, whereas most of ubiquitin ligases genes were expressed in nervous tissues. Furthermore some ubiquitin ligases were upregulated in the mouse brain at late embryonic stage. In addition, the expression levels of ubiquitin ligases were upregulated during retinoic acid-induced neural differentiation of mouse embryonal carcinoma P19 cells. Therefore, ubiquitin ligases with membrane may play roles in the regulation of neuronal differentiation and function in the brain development.

1P-30 Evidence for involvement of apoptosis in establishing proper cerebrospinal fluid hydrodynamics Yamaguchi Yoshifumi¹，Yoshida Ayako¹，Kawata Daisuke¹，Shinotsuka Naomi¹，Yoshida Mariko¹，Miura Masayuki^1,3 ¹Dept. Genet., Grad. Sch. Pharma. Sci., Univ. of Tokyo，²PRESTO, JST.，³CREST, JST. Apoptosis is a major form of cell death to remove unnecessary cells during development and adult tissue in many organisms. In vertebrate, a large number of cells undergo apoptosis during neural tube closure（NTC）in apoptosome-dependent manner. Mice lacking apoptosome activation often exhibit defects in NTC, which has hampered physiological roles of apoptosome-dependent caspase activation in brain development after NTC. We generated a transgenic mice in which broad spectrum of caspases can be suppressed in spatio-temporal pattern by the expression of p35, a pan-caspase inhibitor protein obtained from baculovirus. Mice expressing p35 by nervous-system specific drivers（NCre；p35V mice）were given birth at expected mendelian ratio, but most of them died by 1 month after birth. They showed severe postnatal growth retardation and hydrocephalus. Flow of cerebrospinal fluid（CSF）between 3rd and 4th ventricle was disturbed, while neither stenosis nor abnormality in ciliary morphology and motility was observed in the path of CSF flow including the aqueduct. The hydrocephalus and growth retardation of NCre；p35V mice was not rescued by simultaneous deletion of RIP3, an essential factor inducing necroptosis in the absence of capase-8 activation. The CSF of NCre；p35V mice contained a larger amount of secreted proteins than that of controls. These data suggest that establishment of proper CSF dynamics requires caspase activity during brain development after neural tube closure.