一般演題（口述）

一般演題（口述） Neural Development・Neural Differentiation

3O3-01 Cell-permeable p38 MAP kinase promotes migration of adult neural stem/progenitor cells Hamanoue Makoto¹，Morioka Kazuhito³，Ohsawa Ikuro⁴，Ogata Toru⁵，Takamatsu Ken^1,2 ¹Department of Physiology, Toho University School of Medicine，²Division of Chronic Inflammatory Diseases, Advanced Medical Research Center, Toho University Graduate School of Medicine，³Brain and Spinal Injury Center（BASIC）, Department of Neurological Surgery, University of California，⁴Research Team for Mechanism of Aging, Redox Research, Tokyo Metropolitan Institute of Gerontology，⁵Res. Inst., Natl. Rehab. Center, Dept. of Rehab. for the Move. Func. Endogenous neural stem/progenitor cells（NPCs）can migrate toward sites of injury, but the migration activity of NPCs is insufficient to regenerate damaged brain tissue. In this study, we showed that p38 MAP kinase（p38）is expressed in adult NPCs. Inhibitor experiments using the compound SB203580 revealed that endogenous p38 participates in NPC migration. To enhance NPC migration, we prepared a cell-permeable dominant-active version of p38（PTD-DA）consisting of the HIV protein transduction domain（PTD）fused to the N-terminus of p38. Treatment with PTD-DA protein significantly promoted the random migration of adult NPCs without disturbing cell survival or differentiation；this effect depended on the cell permeability and kinase activity of the fusion protein. These findings indicate that PTD-DA is a novel and useful tool for unraveling the roles of p38, and that PTD-DA provides a reasonable approach for regenerating injured brain by enhancing NPC migration.		3O3-02 Netrin-5 is highly expressed in neurogenic regions of the adult brain Yamagishi Satoru¹，Yamada Kohei²，Sawada Masato³，Mori Norio²，Sawamoto Kazunobu³，Sato Kohji¹ ¹Anatomy and Neuroscience, Hamamatsu University School of Medicine，²Center for Integrated Medical Research, School of Medicine, Keio University，³Nagoya City University Graduate School of Medical Sciences Mammalian netrin family proteins are involved in targeting of axons, neuronal migration, and angiogenesis and act as repulsive and attractive guidance molecules. Netrin-5 is a new member of the netrin family with homology to the C345C domain of netrin-1. Unlike other netrin proteins, murine netrin-5 consists of two EGF motifs of the laminin V domain（LE）and the C345C domain, but lacks the N-terminal laminin VI domain and one of the three LE motifs. We generated a specific antibody against netrin-5 to investigate its expression pattern in the rodent adult brain. Strong netrin-5 expression was observed in the olfactory bulb, rostral migrate stream（RMS）, the subventricular zone（SVZ）, and the subgranular zone（SGZ）of the dentate gyrus in the hippocampus, where neurogenesis occurs in the adult brain. In the SVZ and RMS, netrin-5 expression was observed in Mash1-positive transit-amplifying cells and in Doublecortin（DCX）-positive neuroblasts, but not in GFAP-positive astrocytes. In the olfactory bulb, netrin-5 expression was maintained in neuroblasts, but its level was decreased in NeuN-positive mature neurons. In the hippocampal SGZ, netrin-5 was observed in Mash1-positive cells and in DCX-positive neuroblasts, but not in GFAP-positive astrocytes, suggesting that netrin-5 expression occurs from type 2a to type 3 cells. These data suggest that netrin-5 is produced by both transit-amplifying cells and neuroblasts to control neurogenesis in the adult brain.

3O3-03 The role of Cdk5 in cell cycle arrest and neural differentiation Saito Taro，Hisa Toshinori，Ando Kanae Department of Biological Sciences, Tokyo Metropolitan University Cell cycle arrest occurs in advance of neural differentiation, however their coordination is not fully elucidated. Cyclin-dependent kinase（Cdk）is a Ser/Thr protein kinase family regulating cell cycle progression. Cdk5, an atypical Cdk, is solely activated in postmitotic neurons, while cell cycle machineries including other Cdks are inactivated in neural differentiation, suggesting that Cdk5 may have inhibitory activity for cell cycle progression. Here we examined the role of Cdk5 in cell cycle arrest and neural differentiation.Firstly, we selected SH-SY5Y human neuroblastoma cell, which is frequently used and differentiated by stimulation with retinoic acid（RA）, as model system. PI3K/Akt pathway is important for regulating cell growth and differentiation and also known to be activated in SH-SY5Y cells by RA stimulation. When SH-SY5Y cells were stimulated with RA, the up-regulation of p35, an essential activator for Cdk5, and Akt activation were observed. Next, we examined the expression of inhibitor of differentiation genes（IDs）, which are transcriptional suppressors for differentiation and down-regulated in the early step of neural differentiation. Stimulation by RA induced down-regulation of expression of Id1 and Id3 in transcriptional level in SH-SY5Y cells. Overexpression of p35 also induced both activation of Akt and down-regulation of Id1 without neurite outgrowth as was observed by RA stimulation. These results suggest that Cdk5 may induce early events in neural differentiation. Now, we are examining the pathway from Cdk5 activation to Akt activation or Ids down-regulation in both SH-SY5Y cells and neurons.		3O3-04 NRG1-ErbB4 signaling promotes generation of neurons from neural progenitor cells in the developing brain. Sato Tomomi^1,2，Sato Fuminori³，Kamezaki Aosa³，Sakaguchi Kazuya³，Tanigome Ryoma³，Kawakami Koichi⁴，Sehara Atsuko³ ¹Dept. of Anatomy, Sch. of Med., Saitama Med. Univ.，²Dept. of Ob. and Gyn., Sch. of Med., Saitama Med. University，³Dept. of Growth Reg., Inst. for Frontier Med. Sci., Kyoto Univ.，⁴Div. of Mol.and Dev. Biol., Natl. Inst. of Genet. Post-mitotic neurons are generated from neural progenitor cells（NPCs）at the expense of their proliferation. Molecular and cellular mechanisms that regulate neuron production temporally and spatially should impact on the size and shape of the brain. While transcription factors such as neurogenin1（neurog1）and neurod govern progression of neurogenesis as cell-intrinsic mechanisms, recent studies show regulatory roles of several cell-extrinsic or intercellular signaling molecules including Notch, FGF and Wnt in production of neurons/NPCs from neural stem cells/radial glial cells（NSCs/RGCs）in the ventricular zone（VZ）. However, it remains elusive how production of post-mitotic neurons from NPCs is regulated in the sub-ventricular zone（SVZ）. Here we show that newborn neurons accumulate in the basal-to-apical direction in the optic tectum（OT）of zebrafish embryos. While NPCs are amplified by mitoses in the apical VZ, neurons are exclusively produced through mitoses of NPCs in the sub-basal zone（SBZ）, later in the SVZ, and accumulate apically onto older neurons. This neurogenesis depends on Neuregulin 1 type II（NRG1-II）-ErbB signaling. Treatment with an ErbB inhibitor, AG1478 impairs mitoses in the SVZ of the OT. Removal of AG1478 resumes sub-ventricular mitoses without precedent mitoses in the apical VZ prior to basal-to-apical accumulation of neurons, suggesting critical roles of ErbB signaling in mitoses for post-mitotic neuron production. Knockdown of NRG1-II impairs both mitoses in the SBZ/SVZ and VZ. Injection of soluble human NRG1 into the developing brain ameliorates neurogenesis of NRG1-II-knockdown embryos, suggesting a conserved role of NRG1 as a cell-extrinsic signal. From these results, we propose that NRG1-ErbB signaling stimulates cell divisions generating neurons from NPCs in the developing vertebrate brain.

3O3-05 Involvement of novel mammalian transmembrane ubiquitin ligases in neuronal differentiation and function Takai Tomoko，Shiraishi Kanma，Imaizumi Kazunori，Kaneko Masayuki Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University The ubiquitin-proteasome system（UPS）is an essential process that regulates protein homeostasis, and is involved in the regulation of various cellular processes including cell proliferation, differentiation and survival. To date, it has been suggested that dysregulations in this system are implicated in the pathogenesis of neurodegenerative diseases, cancer and immune system disorders. In the UPS, ubiquitin ligases play an important role in the final step in the ubiquitination cascade for protein degradation. It has been shown that ubiquitin ligases are key molecules for neuronal differentiation and function. Recently, we identified 44 types of novel mammalian ubiquitin ligases which have the RING finger domain and the transmembrane domains. In this study, we performed gene expression profiling using human and mouse tissues and showed that several types of the ubiquitin ligases were predominantly expressed in the embryonic brain, compared with the adult brain. To identify novel ubiquitin ligases which are important for neuronal differentiation, maturation and function, we examined expression levels of ubiquitin ligases abundant in the brain during retinoic acid-induced neural differentiation of mouse embryonal carcinoma P19 cells. As a result, 3 types of ubiquitin ligases, RNF150, RNF152 and RNF182, were markedly up-regulated during neuronal differentiation. These genes have different expression patterns：RNF150 expression was highest at the neurosphere stage；RNF152 and RNF182 were predominantly expressed at the neuronal stages. These results suggested that these ubiquitin ligases may be involved in the regulation of neuronal differentiation and function, and provided further insights into mechanisms of brain development regulated by protein ubiquitination.		3O3-06 Neurogenesis from dying neurons by deregulated DNA repair pathway activation Ajioka Itsuki^1,2，Oshikawa Mio¹ ¹Center for Brain Integration Research（CBIR）, Tokyo Medical and Dental University（TMDU），²JST, PRESTO The cell cycle exit of progenitor cells must be temporally coordinated with the initiation of neuronal differentiation to control the number and ratio of different neuronal subtypes in the CNS. Once progenitor cells exit the cell cycle, their daughter neurons enter the post-mitotic G0 phase for terminal differentiation and lose their proliferative potential. This inability of differentiated neurons to undergo proliferation is one of the major reasons brain tissue cannot regenerate following injury. When mature neurons re-enter the S phase in pathological situations such as neurodegeneration, they undergo cell death. Thus, the regulatory networks that drive cell proliferation and maintain neuronal differentiation are tightly controlled. Tumor suppressor gene Rb and its family members（p107 and p130）are essential for regulating cell cycle in neuronal progenitors and neurons. Neuronal progenitor cells which lack all Rb family members initiate differentiation without exiting cell cycle and these proliferating neurons develop tumors in some cases. In contrast, when differentiating neurons lose all Rb family members after cell cycle exit, they undergo S phase but do not divide. We recently found the molecular mechanism by which differentiating neurons are tightly protected from cell division even in the absence of Rb family members. In this oral presentation, we show these data and discuss how neurons maintain post-mitotic and non-dividing feature.