Poster Sessions

Poster Sessions 神経系の発生・再生、神経細胞死

1P-22 TPT1 regulates the proliferation and/or survival of neural stem cells and glioma initiating cells Shigeki Ohta¹，Ayako Tokumitsu²，Takafumi Sone³，Ayuna Sakamoto¹，Yutaka Kawakami¹，Hideyuki Okano³ ¹Cell Info. Adv. Med. Res., Keio Univ. Sch. Med，²Div TR, Keio Univ. Sch. Med，³Dept Physiology, Keio Univ. Sch. Med In our previous study, MIF (Macrophage migration inhibitory factor) was identified as a functional molecule, which supports the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs) using functional cloning strategy. In the same functional cloning procedure, we also identified a new factor, TPT1 (Tumor Protein, Translationally-Controlled 1). Intriguingly, MIF-treated murine NSPCs increased the Tpt1 gene expression. Overexpression of Tpt1 in mouse NSPCs increased the cell proliferation in vitro. In human ES cell-derived NSPCs (hES-NSPCs), TPT1 regulated cell proliferation and neurogenesis. Importantly, the same phenomena were observed in human NSPCs derived from Cas9-mediated TPT1 targeted iPSCs compared with the wild-type cells. We also performed RNA sequencing and confirmed the gene expression changes of cell cycle related factors in the TPT1 gene silenced hES-NSPCs. Moreover, we tried to identify miRs regulated by TPT1 in hES-NSPCs using TaqMan Array Human MicroRNA Cards, and identified miR338 as a TPT1 downstream target. The TPT1-miR338-SMO axis was newly identified as regulating the cell proliferation of hES-NSPCs in vitro. Taken together, MIF-regulated TPT1 contributes to the proliferation and/or survival of NSPCs in both mouse and humans in vitro. In our other studies, we also reported the functions of MIF and CHD7 (Chromodomain-Helicase-DNA-binding protein 7) in glioma initiating cells, regulating the cell proliferation. Finally, we found that TPT1 gene expression was regulated by MIF and CHD7, respectively, and TPT1 gene silencing decreased the cell proliferation in the glioma initiating cells. Together, these results may contribute to the development of new therapeutic targeting for glioma.		1P-23 Protease-activated receptor 1 regulates neural progenitor proliferation through Rho kinase pathway Masanori Yoneyama，Taro Yamaguchi，Yusuke Onaka，Kiyokazu Ogita Lab Pharmacol. Facul Pharm Sci, Setsunan Univ It is now clear that there is a continual turnover of the mammalian hippocampal dentate gyrus neurons throughout life even in adult. Our previous studies demonstrated that thrombin-activated/protease-activated receptor-1 (PAR-1) attenuated proliferation of neural stem/progenitor cells (NPCs) derived from the hippocampal dentate gyrus of the adult mice. The molecular mechanism regulating their proliferation has not been well understood. In this study, we evaluated the involvement of Rho kinase pathway in proliferation of NPCs derived from the hippocampal dentate gyrus of adult mouse. NPCs prepared from the dentate gyrus were cultured in the neurobasal medium with B27 supplement, EGF, and bFGF for 14 days in vitro (DIV). After secondary replating nestin (NPCs marker)-positive cells were cultured for 5 DIV under the same conditions in the absence or presence of thrombin, Y27632, and/or fasudil for assessment of cell proliferation. Reverse transcription-PCR analysis showed the expression of mRNA encoding all subtypes of PAR in the NPCs. Immunostaining revealed that PAR-1 was co-localized with most of nestin-positive cells. Exposure of the cells to thrombin significantly attenuated the cell proliferation without cell damage. Next, thrombin-induced attenuation of proliferative activity was completely abolished by singly tested Y27632 and fasudil, which is Rho kinase inhibitor, respectively. However, neither inhibitor affected the proliferative activity of the cells. Taken together, our results support the possibility that thrombin negatively regulates cell proliferation through the Rho kinase pathway by activation of PAR-1 in the NPCs.

1P-24 The neurosphere culture period affects the yield of neurons after differentiation Kanako Takahashi¹，Hisashi Ohara²，Nozomi Kasahara²，Masahiro Takase²，Kaori Chujo¹，Yasunari Kanda¹，Yuko Sekino^1,3，Mitsuo Tanabe²，Kaoru Sato¹ ¹Div Pharmacol, Nat Inst Hlth Sci，²Lab Pharmacol, Sch Pharmacy, Kitasato Univ，³Lab Chemical Pharmacol, Grad Sch Pharm Sci, Tokyo Univ. Although protocols for mass production of human iPSC-derived neurons are necessary to apply these cells to the drug development process, little information is available about the culture protocols to produce neurons in high yield. In this study, we examined whether or not the length of the neurosphere (NS) culture period affects the number of neurons after differentiation by using rat NS prepared from E16 telencephalons. Two groups of NSs were used in this study with the culture periods of 11 and 25 days, respectively. Cells dissociated from each NS were differentiated in adherent condition. The numbers of neurons, neural stem cells, astrocytes, and radial glial cells on differentiation (diff.) day 1, 3 and 7 were quantified by immunostaining methods. At the start point of differentiation, the number of neurons in 25 day-group was smaller than that in 11 day-group. The increment rates of neurons from diff. day 3 to 7 were 1.1 times for 11 day-group and 15.1 times for 25 day-group, resulting in the increase in the neuronal number. On the diff. day 3, the percentage of radial glial cells was 52% in 11 day-group and 83% in 25 day-group. EdU incorporation assay (diff. day 3-7) showed that the percentages of EdU(+)Tuj1(+) cells in Tuj1(+) cells were 21.9% for the 11 day-group and 90.0% for the 25 day-group. These results suggest that longer NS culture period increases the percentage of radial glial cells, thereby resulting in the remarkable increase in the number of neurons differentiated from the radial glial cells.		1P-25 Region-dependent differences in the migratory capacity of hippocampal CA1 and neocortical neurons during brain development Ayako Kitazawa，Minkyung Shin，Kanehiro Hayashi，Ken-ichiro Kubo，Kazunori Nakajima Dept. Anatomy, Sch. Med, Keio Univ. Pyramidal neurons in the neocortex and hippocampus are born near the ventricle and migrate to their destinations during brain development. It has been known that neocortical neurons migrate in the mode called “locomotion”, in which they migrate along with a single radial glial fiber for long distances in the cortical plate. On the other hand, the hippocampal CA1 neurons migrate in the “climbing” mode, in which neurons migrate slowly using multiple radial glial fibers with multiple leading processes in the stratum pyramidale. However, the precise mechanisms of the “climbing” mode remain unknown. In this study, we further investigated differences and similarities of the migration between the hippocampal CA1 region and neocortex. We confirmed that the migrating neurons in the hippocampal CA1 region had adherens junction between the radial glial fiber and their leading processes and required N-cadherin as observed for the neocortical migrating neurons. However, when we transplanted mouse neocortical and CA1 neurons together into the developing mouse neocortex, the ectopically transplanted CA1 neurons did not migrate toward the cortical plate, while the transplanted neocortical neurons migrated normally into the cortical plate. Conversely, the ectopically transplanted neocortical neurons in the hippocampal CA1 region did not migrate toward the stratum pyramidale. These results indicate that the neocortical and hippocampal neurons might use distinct cues in their microenvironment for their migration.

1P-26 Enc1 controls neuronal migration and differentiation of excitatory neurons in the developing neocortex Yuki Hirota¹，Chikako Kudo-Tsurushige¹，Itsuki Ajioka²，Kazunori Nakajima¹ ¹Dept. Anatomy, Keio Univ. School of Med.，²Center for Brain Integration Research, Tokyo Medical and Dental Univ. The mammalian cerebral neocortex has a well-organized laminar structure, achieved by the highly coordinated control of neuronal migration. During neocortical development, excitatory neurons are generated from radial glial cells in the ventricular zone or from progenitor cells in the subventricular zone, and then radially migrate toward the pial surface of cortex. Finally neurons reach their final positions in the cortical plate, undergo terminal differentiation and maturation, and form functional circuits. In these processes, various molecular and cellular mechanisms are involved. Ectodermal-neural cortex-1 (Enc1), also known as a nuclear matrix protein termed “nuclear restricted protein in brain (NRP/B)”, was characterized as a Kelch-related protein expressed in the developing nervous system and colocalized with actin. Kelch family proteins are involved in various cellular processes including cell migration, cytoskeletal arrangement, protein degradation and gene expression. Enc1 is expressed in the telencephalon from early developmental stages and its possible roles in neuronal differentiation, neurite formation and neural protection from various insults were previously suggested by in vitro assays. However, its precise expression pattern and physiological functions during neocortical development have not been examined yet. Here, we studied the expression pattern and function of Enc1 during mouse neocortical development. Immunostaining analysis showed that Enc1 is strongly localized in the nucleus of layer Vb/VI neurons. Suppression of Enc1 disturbed radial migration and differentiation of early-born neurons. These results suggest that Enc1 controls neuronal migration and differentiation during cortical development.		1P-27 Development of human iPS cell lines with exceeding differentiation capacities and their differentiation into midbrain dopaminergic neurons Makoto Motono，Shizuka Funayama，Bagheri Mozhdeh，Hidemasa Kato Dept Functional Histology, Grad Sch Med, Ehime Univ Human induced pluripotent stem cell (hiPSC) capacity to differentiate into various somatic cell types varies substantially among the given cell lines. Recently, we and others have found that the DNA dioxygenase TET1 bears active role in epigenetically specifying the epiblast/primed pluripotency and during reprogramming of the mouse cells. In the current study, we have investigated the effect of TET1 when added to Yamanaka factors in hiPSC reprogramming and directly compared the differentiation capacities of the TET1-induced hiPSCs (T-iPSCs) with those of the conventionally-derived hiPSCs. Furthermore, we devised a novel differentiation evaluation system for both ectoderm (default-elite) and mesendoderm (primitive-streak-elite) which would facilitate the prospective elucidation of hiPSC lines with broader and higher differentiation capacities. In our multiple rounds of in-house derivation of T-iPSCs, we observed significant increase in the proportion of default-elites among the T-iPSCs. When these default-elite T-iPSCs were differentiated into midbrain dopaminergic neurons, we experienced high differentiation efficiencies for its progenitors which may outcompete the so-far obtained best results using human ES cells (over 50% NURR1-positive cells). Also, the induced neurons had longer TH-positive axons and more elaborate MAP2-positive dendrites than those of non-default-elite hiPSCs. Therefore, our novel evaluation system for prospectively depicting the differentiation capabilities of hiPSCs, when combined to the newly devised T-iPSC-protocol, represents promising measures in deriving and choosing iPSC clones with high differentiation ability which may ultimately offer unprecedented advantage for its application toward clinical and pharmaceutical usages.

1P-28 Microglial activation induces oligodendrogenesis from the subventricular zone after focal demyelination Masae Naruse¹，Koji Shibasaki¹，Hiroya Shimauchi^1,2，Yasuki Ishizaki¹ ¹Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine，²Department of Neurosurgery, Gunma University Graduate School of Medicine Neuroblasts derived from neural stem cells (NSCs) in the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) into the olfactory bulb to generate interneurons under normal physiological conditions. When demyelination occurs, neural stem or progenitor cells in the SVZ provide newly formed oligodendrocytes to demyelinated lesions. Plasticity of neural stem or progenitor cell lineage may tend to oligodendrogenesis under the influence of demyelinated lesions. The mechanisms, however, still remain unknown. The present study revealed that focal demyelination in the corpus callosum caused activation of microglia not only at the site of demyelination, but also in the SVZ, and dramatically increased generation of oligodendrocyte progenitor cells (OPCs) in the SVZ. Furthermore, inhibition of microglial activation by minocycline treatment decreased OPC generation in the SVZ, suggesting that activated microglia in the SVZ, induced by focal demyelination in the corpus callosum, regulate neural stem or progenitor cell lineage plasticity in situ. In contrast to finding with demyelination in the corpus callosum, inducing focal demyelination in the internal capsule did not induce either microglial activation or OPC generation in the SVZ. These results suggest that the mechanism of OPC generation in the SVZ following demyelinating lesions could be different among the demyelinated regions.		1P-29 The role of Fgf8 for development of the frontal cortex subdivisions Tatsuya Sato^1,2,3，Takako Kikkawa²，Tetsuichiro Saito⁴，Keiichi Itoi¹，Noriko Osumi² ¹Dept Info. Biol. Grad Sch Info Sci, Tohoku Univ，²Dept Dev. Neurosci. Grad Sch Med, Tohoku Univ，³FRIS, Tohoku Univ.，⁴Dept Dev. Biol. Grad Sch Med, Chiba Univ The frontal cortex (FC) consists of the prefrontal, premotor and motor cortices. The prefrontal cortex is subdivided into lateral, medial and orbital areas by their structure and function. While the premotor and motor cortices are responsible for animal movement, the prefrontal cortex in human contributes to higher brain functions, such as cognition, personality and decision making. Brain areas homologous to those of human exist in other mammals. Although the FC is important for animal behavior, developmental mechanisms of its subdivisions are not well understood. Here we show that an organizing molecule, fibroblast growth factor 8 (Fgf8), is a key regulator for development of the FC subdivisions. We misexpressed Fgf8 in the anterior pole of the telencephalon by exo utero electroporation at embryonic day 11.5 and analyzed morphological changes and expression of FC subdivision markers at postnatal day 0. We observed expansion of dorsomedial FC (anterior cingulate and prelimbic cortex) and lateral shifts of expression borders of dorsal FC subdivision markers. These results suggest that Fgf8 has an effect for medializing the dorsal FC. Remarkably, we observed lack of the almost entire ventral FC (orbital cortex). Taking into consideration the hypoplasia of the orbital cortex in Fgf8 hypomorph mutants (Cholfin & Rubenstein, 2008), we speculate that a moderate level of Fgf8 signal may be needed for proper development of the orbital cortex. Furthermore, we observed that the olfactory bulb conversely became larger than that of wild-type mice. Thus we conclude that Fgf8 may play a pivotal role in development of the FC subdivisions and the olfactory bulb.

1P-30 Phosphorylation of Id4 transcription suppressor by Cdk5-p35 at the time of neuronal differentiation Taro Saito，Toshinori Hisa，Kanae Ando Dept Biol. Sci, Tokyo Met Univ Differentiation from neural stem cells to neurons is achieved by alteration of gene expression specific to neurons. Basic helix-loop-helix (bHLH) transcription factors play a critical role in the regulation of cell differentiation including neurogenesis. Id (inhibitor of differentiation/DNA binding) is a family of bHLH transcription factors, lacking basic domain for binding to DNA, so it acts as an negative regulator of other bHLH transcription factors. Cyclin-dependent kinase 5 (Cdk5) is activated in differentiated neurons by the upregulation of its activator p35. While Cdk5 is necessary for neuronal differentiation, the role of Cdk5 in the regulation of neuronal gene expression have not been known yet. Recently, we found that Id2 was phosphorylated by Cdk5 among Id family, Id1-4. The phosphorylation site at Ser5 of Id2, is conserved in both Id3 and Id4. Then, Id4 has distinct structures, N-terminal Ala-rich and C-terminal Pro-rich regions, from other Id family. Furthermore, it is reported that Id4 KO mice exhibited reduction of the brain size. Here, we examined the phosphorylation of Id4 by Cdk5.Ser5 of Id4 was also phosphorylated by Cdk5 in transfected N2a cells. The expression of Id4 was increased during differentiation from neural stem cells to neurons. Id4 was phosphorylated by Cdk5 in neurons, but not in neural stem cells where Cdk5 activity is low. Phosphorylation of Ser5 did not affect the binding activity of Id4 to E2A and its cellular localization. Overexpression of neither wild type nor S5A mutant Id4 in primary neurons enhanced neurite extension, as was observed with wild type Id2. Now, we are investigating the function of Id4 and its Ser5 phosphorylation during neuronal differentiation.		1P-31 Functional analysis of Glial cell missing 1 in the mammalian brain Yoshitaka Hayashi，Satoshi Fuke，Takahiro Fuchigami，Naoko Morimura，Natsu Koyama，Seiji Hitoshi Dept Int Physiol. Grad Sch Med. Shiga Univ Med Sci Although glial cell missing (gcm) plays a critical role in the glial cell development in Drosophila, the function of Gcm1, one of orthologues of gcm in mammals, in the brain remains elusive. Overexpression of Gcm1 by retrovirus injected in the lateral ventricle was shown to promote the differentiation of neural precursor cells into astrocytes (Iwasaki et al., Development 130: 6027-6035, 2003) and we reported that Gcm1 and its homolog, Gcm2 , are required for the expression of Hes5 and the generation of neural stem cells in early embryos (Hitoshi et al., Nat Neurosci 14: 957-964, 2011). However, the function of Gcm1 in the adult mammalian brain remains to be investigated because Gcm1-deficient mouse embryos are lethal around E9.5 due to the placental dysfunction. In this study, we performed in utero electroporation (IUEP) studies to overexpress Gcm1 together with GFP in neural precursor cells at E14.5 and found that Gcm1 significantly promoted the emergence of GFAP(+)/GFP(+) cells . These GFAP(+) cells were also immune-positive for S100β, another marker of mature astrocyte, suggesting that they are astrocytes. Next, we investigated the differentiation into oligodendrocyte lineage cells by immunostaining the Gcm1 electroporated brains against Olig2, a marker of oligodendrocyte progenitor cells and mature oligodendrocytes. We observed a significant increase in number of Olig2(+) cells both in GFP(+) and in GFP(-) populations. Furthermore, we also noticed that Gcm1 overexpression resulted in more angiogenesis. Thus, our results suggest that Gcm1 plays an important role in the development of mammalian brain, and currently, we are exploring for the physiological function of Gcm1 in the adult brain.

1P-32 Involvements of acid-sensing ion channel-1a in hippocampal adult neurogenesis. Natsuko Kumamoto，Mariko Hoshikawa，Yasuhiro Shibata，Takashi Ueda，Shinya Ugawa Dept. of Anat. and Neurosci. Grad. Sch. of Med. Sci. Nagoya City Univ. Stroke-damaged adult brain attempts to repair itself by producing new neurons, regulatory mechanisms of which remain largely unknown. We speculate that local tissue acidosis in the lesion area affects adult neurogenesis through the activation of a neuronal acid-activated cation channel ASIC1a (acid-sensing ion channel-1a) involved in synaptic plasticity, learning and memory. To confirm the expression of ASIC1a transcripts in mouse hippocampal newborn neurons, we performed in situ hybridization combined with immunohistochemistry and identified substantial amounts of ASIC1a mRNA in the newborn neurons from early stages of the adult neurogenesis. These results raise the possibility that ASIC1a is involved in adult neural progenitor proliferation irrespective of local tissue acidosis. To investigate this hypothesis, we preliminarily measured neurogenesis by intraperitoneal injection of bromodeoxyuridine (BrdU), which labels newborn neurons, in wild-type and ASIC1a knockout (KO) mice. ASIC1a KO mice showed increased number of BrdU-labeled cells in the hippocampal dentate gyrus compared with age-matched wild-type mice. These results suggest that ASIC1a may inhibit proliferation of neural progenitor cells at the early stage of the neurogenesis in the absence of local tissue acidosis. Pathophysiological relationships between ASIC1a and adult neurogenesis in ischemic brain are currently under investigation.		1P-33 Effect of ER stress modulator on ER-Golgi SNARE expression and Aβ peptide secretion in NG108-15 cell Kei Suga^1,2，Masafumi Nishino²，Yasuo Terao²，Kimio Akagawa² ¹Dept. Chem. Kyorin Univ. Sch. of Med.，²Dept. Cell Physiol. Kyorin Univ. Sch. of Med. Endoplasmic reticulum (ER) stress and the activation of caspase3 have been implicated in neurodegenerative diseases such as Alzheimer’s disease (AD). We have been focusing on the neuronal function of ER-Golgi soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs). We previously showed that ER stress upregulates de novo synthesis of ER-Golgi SNAREs Syntaxin5 (Syx5) in Neuroblastoma-Glioma hybrid cell line NG108-15 (Suga K. et al., Exp. Cell Res., 2015). While ER stress caused the reduction of β-amyloid peptide (Aβ peptide), knockdown of Syx5 protein enhanced the secretion of Aβ. In addition, the reduction of Aβ secretion by ER stress was significantly suppressed by Syx5 knock down. Conversely, apoptosis induction by staurosporine resulted in down regulation of Syx5 proteins due to the degradation by activated caspase3. By using time lapse imaging analysis, we showed that sustained ER stress promotes caspase3-dependent apoptosis. Furthermore, we reported that a chemical chaperone 4-phenylbutyrate (PBA) showed alleviation of caspase3-dependent apoptosis induced by ER stress. In order to know the protective mechanism of the ER stress modulator, we present the effects of chemical compounds which modulate different sites in ER stress signaling on the expression of ER-Golgi SNAREs and the processing of βAPP in NG108-15 cells.