Akiko Miwa

Publications (15)75.11 Total impact

• Article: RP58 Regulates the Multipolar-Bipolar Transition of Newborn Neurons in the Developing Cerebral Cortex.

  Chiaki Ohtaka-Maruyama, Shinobu Hirai, Akiko Miwa, Julian Lk-Tsen Heng, Hiroshi Shitara, Rie Ishii, Choji Taya, Hitoshi Kawano, Masataka Kasai, Kazunori Nakajima, Haruo Okado
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  ABSTRACT: Accumulating evidence suggests that many brain diseases are associated with defects in neuronal migration, suggesting that this step of neurogenesis is critical for brain organization. However, the molecular mechanisms underlying neuronal migration remain largely unknown. Here, we identified the zinc-finger transcriptional repressor RP58 as a key regulator of neuronal migration via multipolar-to-bipolar transition. RP58(-/-) neurons exhibited severe defects in the formation of leading processes and never shifted to the locomotion mode. Cre-mediated deletion of RP58 using in utero electroporation in RP58(flox/flox) mice revealed that RP58 functions in cell-autonomous multipolar-to-bipolar transition, independent of cell-cycle exit. Finally, we found that RP58 represses Ngn2 transcription to regulate the Ngn2-Rnd2 pathway; Ngn2 knockdown rescued migration defects of the RP58(-/-) neurons. Our findings highlight the critical role of RP58 in multipolar-to-bipolar transition via suppression of the Ngn2-Rnd2 pathway in the developing cerebral cortex.
  Cell reports. 02/2013;
• Article: RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1-4 genes in the developing cortex.

  Shinobu Hirai, Akiko Miwa, Chiaki Ohtaka-Maruyama, Masataka Kasai, Shigeo Okabe, Yutaka Hata, Haruo Okado
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  ABSTRACT: Appropriate number of neurons and glial cells is generated from neural stem cells (NSCs) by the regulation of cell cycle exit and subsequent differentiation. Although the regulatory mechanism remains obscure, Id (inhibitor of differentiation) proteins are known to contribute critically to NSC proliferation by controlling cell cycle. Here, we report that a transcriptional factor, RP58, negatively regulates all four Id genes (Id1-Id4) in developing cerebral cortex. Consistently, Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of NSCs. These phenotypes were mimicked by the overexpression of all Id genes in wild-type cortical progenitors. Furthermore, Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. Finally, we determined p57 as an effector gene of RP58-Id-mediated cell fate control. These findings establish RP58 as a novel key regulator that controls the self-renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis.
  The EMBO Journal 01/2012; 31(5):1190-202. · 9.20 Impact Factor
• Article: The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex.

  Haruo Okado, Chiaki Ohtaka-Maruyama, Yoshinobu Sugitani, Yuko Fukuda, Reiko Ishida, Shinobu Hirai, Akiko Miwa, Akiyo Takahashi, Katsunori Aoki, Keiji Mochida, Osamu Suzuki, Takao Honda, Kazunori Nakajima, Masaharu Ogawa, Toshio Terashima, Junichiro Matsuda, Hitoshi Kawano, Masataka Kasai
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  ABSTRACT: The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.
  Developmental Biology 06/2009; 331(2):140-51. · 4.07 Impact Factor
• Article: Co-localization of a novel transcriptional repressor simiRP58 with RP58.

  Akiyo Takahashi, Shinobu Hirai, Chiaki Ohtaka-Maruyama, Akiko Miwa, Yutaka Hata, Shigeo Okabe, Haruo Okado
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  ABSTRACT: We have cloned a novel transcriptional repressor protein, termed simiRP58, which has high homology to RP58. Both simiRP58 and RP58 belong to the POZ domain and Kruppel Zn finger (POK) family of proteins. Using the luciferase assay system, we found that simiRP58 also has transcriptional repressor activity like RP58. Northern blotting and quantitative RT-PCR showed that simiRP58 was expressed in testes at the highest level. In situ hybridization of testes showed that simiRP58 is expressed by spermatocytes in only a portion of the seminiferous tubules. In contrast, expression of RP58 by spermatocytes was ubiquitous in all seminiferous tubules. Using COS-7 cells, we observed that simiRP58 was localized in the cytoplasm, which is in contrast to RP58 that was localized in the nucleus. Interestingly, co-transfection with simiRP58 and RP58 induced changes in the localization patterns of both proteins.
  Biochemical and Biophysical Research Communications 05/2008; 368(3):637-42. · 2.48 Impact Factor
• Article: Ca2+-permeable AMPA receptors regulate growth of human glioblastoma via Akt activation.

  Shogo Ishiuchi, Yukari Yoshida, Kenichi Sugawara, Masanori Aihara, Toshiyuki Ohtani, Takashi Watanabe, Nobuhito Saito, Keisuke Tsuzuki, Haruo Okado, Akiko Miwa, Yoichi Nakazato, Seiji Ozawa
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  ABSTRACT: Evidence has been accumulated that glioblastoma cells release and exploit glutamate for proliferation and migration by autocrine or paracrine loops through Ca2+-permeable AMPA-type glutamate receptors. Here, we show that Ca2+ signaling mediated by AMPA receptor regulates the growth and motility of glioblastoma cells via activation of Akt. Ca2+ supplied through Ca2+-permeable AMPA receptor phosphorylated Akt at Ser-473, thereby facilitating proliferation and mobility. A dominant-negative form of Akt inhibited cell proliferation and migration accelerated by overexpression of Ca2+-permeable AMPA receptor. In contrast, introduction of a constitutively active form of Akt rescued tumor cells from apoptosis induced by the conversion of Ca2+-permeable AMPA receptor to Ca2+-impermeable receptors by the delivery of GluR2 cDNA. Therefore, Akt functions as downstream effectors for Ca2+-signaling mediated by AMPA receptor in glioblastoma cells. The activation of the glutamate-AMPA receptor-Akt pathway may contribute to the high degree of anaplasia and invasive growth of human glioblastoma. This novel pathway might give an alternative therapeutic target.
  Journal of Neuroscience 08/2007; 27(30):7987-8001. · 7.11 Impact Factor
• Article: Spatial and temporal expression of RP58, a novel zinc finger transcriptional repressor, in mouse brain.

  Chiaki Ohtaka-Maruyama, Akiko Miwa, Hitoshi Kawano, Masataka Kasai, Haruo Okado
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  ABSTRACT: RP58, a novel zinc finger protein containing a POZ domain, is a sequence-specific transcriptional repressor. To understand the role of this protein, we examined RP58 gene expression in the developing mouse brain by quantitative polymerase chain reaction (PCR) and in situ hybridization. RP58 mRNA expression was detected at embryonic day (E) 10 in the neuroepithelium, and subsequently in the ventricular zones of the cerebral cortex in the E12 embryo. Strong expression was observed in the preplate in the cerebral cortex from this stage onward. High levels of expression continued to be detected in the cortical plate and subventricular zone of the neocortex, hippocampus, and parts of the amygdala, but not in the thalamus or striatum. These results suggest that RP58 plays a crucial role in neuronal proliferation, migration, and differentiation in the developing cerebral cortex. RP58 is also expressed in the adult mouse neocortex, hippocampus, parts of the amygdala, and granule cells in the cerebellum. Double in situ hybridization using GAD67 or VGLUT1 probes revealed that RP58 is expressed in glutamatergic excitatory neurons.
  The Journal of Comparative Neurology 07/2007; 502(6):1098-108. · 3.81 Impact Factor
• Source

  Available from: PubMed Central

  Article: Sequential exocytosis of insulin granules is associated with redistribution of SNAP25.

  Noriko Takahashi, Hiroyasu Hatakeyama, Haruo Okado, Akiko Miwa, Takuya Kishimoto, Tatsuya Kojima, Teruo Abe, Haruo Kasai
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  ABSTRACT: We have investigated sequential exocytosis in beta cells of intact pancreatic islets with the use of two-photon excitation imaging of a polar fluorescent tracer, sulforhodamine B, and a fusion protein comprising enhanced cyan fluorescent protein (ECFP) and the SNARE protein SNAP25 (synaptosome-associated protein of 25 kD) transfected with an adenoviral vector. Sequential exocytosis was found to account for <10% of exocytic events in beta cells stimulated either with glucose under various conditions or by photolysis of a caged-Ca2+ compound. Multigranular exocytosis, in which granule-to-granule fusion occurs before exocytosis, was rarely found. We detected redistribution of ECFP-SNAP25 from the plasma membrane into the membrane of the fused granule occurred in a large proportion (54%) of sequential exocytic events but in only a small fraction (5%) of solitary fusion events. Removal of cholesterol in the plasma membrane by methyl-beta-cyclodextrin facilitated both redistribution of ECFP-SNAP25 and sequential exocytosis by threefold. These observations support the hypothesis that SNAP25 is a plasma membrane factor that is responsible for sequential exocytosis.
  The Journal of Cell Biology 05/2004; 165(2):255-62. · 10.26 Impact Factor
• Article: Transfer of NMDAR2 cDNAs increases endogenous NMDAR1 protein and induces expression of functional NMDA receptors in PC12 cells.

  Yutaka Saito, Keisuke Tsuzuki, Nobuaki Yamada, Haruo Okado, Akiko Miwa, Fumio Goto, Seiji Ozawa
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  ABSTRACT: The pheochromocytoma cell line (PC12) has been used as a model system for the study of regulation of expression of NMDA receptors. PC12 cells express a substantial amount of NMDAR1 subunit (NR1) mRNA, whereas they express only a small amount of NR1 protein. The level of functional NMDA receptor expression is almost negligible. To test the possibility that NMDAR2 subunits (NR2) control expression of functional NMDA receptors as well as NR1 protein, we transferred NR2A-D cDNAs into PC12 cells using adenovirus vectors. Prominent NMDA receptor-mediated currents were recorded in PC12 cells to which NR2A or NR2B cDNA was delivered without NR1 cDNA. The amplitudes of these responses were similar to those in PC12 cells to which NR1 cDNA was delivered together with NR2A or NR2B cDNA. In cells to which either NR2C or NR2D cDNA alone was delivered, NMDA receptor-mediated currents were also detected, although to a much lesser extent. These results showed that NR2 proteins produced by gene transfer are co-assembled with the endogenous NR1 protein to form functional heteromeric receptors. The delivery of NR2A-D cDNAs also increased the amount of NR1 protein but not that of NR1 mRNA, suggesting that this protein increase is due to post-transcriptional mechanisms. The effects of NR2A-B gene transfer on expression of NR1 protein were much more efficient than those of NR2C-D gene transfer.
  Molecular Brain Research 03/2003; 110(2):159-68. · 2.00 Impact Factor
• Article: Blockage of Ca(2+)-permeable AMPA receptors suppresses migration and induces apoptosis in human glioblastoma cells.

  Shogo Ishiuchi, Keisuke Tsuzuki, Yukari Yoshida, Nobuaki Yamada, Norikazu Hagimura, Haruo Okado, Akiko Miwa, Hideyuki Kurihara, Yoichi Nakazato, Masaru Tamura, Tomio Sasaki, Seiji Ozawa
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  ABSTRACT: Glioblastoma multiforme is the most undifferentiated type of brain tumor, and its prognosis is extremely poor. Glioblastoma cells exhibit highly migratory and invasive behavior, which makes surgical intervention unsuccessful. Here, we showed that glioblastoma cells express Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors assembled from the GluR1 and/or GluR4 subunits, and that their conversion to Ca(2+)-impermeable receptors by adenovirus-mediated transfer of the GluR2 cDNA inhibited cell locomotion and induced apoptosis. In contrast, overexpression of Ca(2+)-permeable AMPA receptors facilitated migration and proliferation of the tumor cells. These findings indicate that Ca(2+)-permeable AMPA receptors have crucial roles in growth of glioblastoma. Blockage of these Ca(2+)-permeable receptors may be a useful therapeutic strategy for the prevention of glioblastoma invasion.
  Nature Medicine 10/2002; 8(9):971-8. · 22.46 Impact Factor
• Article: Two-photon excitation imaging of pancreatic islets with various fluorescent probes.

  Noriko Takahashi, Tomomi Nemoto, Ryoichi Kimura, Akira Tachikawa, Akiko Miwa, Haruo Okado, Yasushi Miyashita, Masamitsu Iino, Takashi Kadowaki, Haruo Kasai
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  ABSTRACT: Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca(2+)-sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca(2+) measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membrane-impermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution.
  Diabetes 03/2002; 51 Suppl 1:S25-8. · 8.29 Impact Factor
• Article: Continual remodeling of postsynaptic density and its regulation by synaptic activity

  Shigeo Okabe, Hong-Duck Kim, Akiko Miwa, Toshihiko Kuriu, Haruo Okado
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  ABSTRACT: A postsynaptic density (PSD) protein, PSD-95, was tagged with green fluorescent protein (GFP-PSD-95) and expressed in cultured hippocampal neurons using recombinant adenoviruses. GFP-PSD-95 was selectively localized to excitatory postsynaptic sites. Time-lapse fluorescence imaging of hippocampal neurons revealed that >20% of GFP-PSD-95 clusters turned over within 24 hours. The appearance rate of clusters was higher than the disappearance rate, and this difference accounted for the gradual increase of the cluster density observed in culture. Dynamics of PSD-95 clusters were also inhibited by blockers of excitatory synaptic transmission. Continual PSD turnover and its regulation by synaptic activity may be important in activity-dependent remodeling of neuronal connections.
  Nature Neuroscience. 08/1999; 2(9):804-811.
• Article: Postsynaptic expression of Ca2+-permeable AMPA-type glutamate receptor channels by viral-mediated gene transfer

  Makoto Sudo, Haruo Okado, Masae Iino, Keisuke Tsuzuki, Akiko Miwa, Yumi Kanegae, Izumu Saito, Seiji Ozawa
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  ABSTRACT: The ability to artificially express a particular receptor protein in the postsynaptic sites of neurons in the central nervous system (CNS) would be useful for the study of synaptic function of cloned receptor genes as well as for gene therapy of neurological disorders caused by dysfunction of postsynaptic receptors. In this study, we aimed to express the cDNA of unedited GluR2 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor that forms inwardly rectifying and Ca2+-permeable channel in CNS neurons by using adenoviral-mediated gene transfer. For this purpose, we have constructed a recombinant adenovirus bearing an expression-switching unit, where the unedited GluR2 cDNA can be activated by the Cre recombinase-mediated excisional deletion of a stuffer DNA interposed between the promotor and the coding region. When PC12 cells were infected with this recombinant adenovirus together with an adenovirus expressing Cre recombinase, the inwardly rectifying and Ca2+-permeable AMPA receptor channels were expressed in nearly 100% of infected cells. Two days after co-infection of cultured rat hippocampal neurons with these adenoviruses, fast excitatory neurotransmission in the glutamatergic synapse was mediated predominantly by the inwardly rectifying and Ca2+-permeable AMPA receptor channels. This indicates that the native AMPA receptors in the postsynaptic sites of the glutamatergic synapse are replaced rapidly with recombinant receptors newly produced by the viral-mediated gene transfer.
  Molecular Brain Research 04/1999; · 2.00 Impact Factor
• Article: Adenovirus-mediated expression of AMPA-type glutamate receptor channels in PC12 cells

  Makoto Sudo, Keisuke Tsuzuki, Haruo Okado, Akiko Miwa, Seiji Ozawa
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  ABSTRACT: The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor channels are expressed ubiquitously in brain neurons and mediate fast excitatory neurotransmission. They are composed of four subunits, GluRl, GluR2, GluR3 and/or GluR4. We constructed recombinant adenoviruses encoding rat AMPA receptor subunit cDNAs, GluRl (AxCAGluRl) and silently mutated GluR2 (AxCAGluR2X) with modified chicken β-actin promoter and cytomegalovirus immediate-early enhancer. Using these adenoviral vectors, we transferred the GluRl and GluR2 genes into PC12 cells that possess no functional AMPA receptor channels. PC12 cells infected with these viruses expressed GluRl and GluR2 RNAs. Immunoblot analysis indicated that the expressed GluRl and GluR2 proteins were equivalent to those of the rat brain. Functional expression of the AMPA receptor channels was examined using the whole-cell patch clamp technique. In AxCAGluRl-infected cells, the current–voltage (I–V) relationship of response to kainate, a non-desensitizing agonist of AMPA receptors, exhibited a strong inward rectification, indicating the formation of functional GluRl-homo-meric channels. In cells infected with both AxCAGluRl and AxCAGluR2X, the I–V relationship of kainate responses exhibited an outward rectification, indicating the formation of heteromeric GluRl/R2 channels. Immunocytochemical analysis revealed that the AMPA receptor subunit genes were transferred in more than 95% of the infected PCl2 cells. © l997 Elsevier Science B.V.
  Molecular Brain Research 11/1997; · 2.00 Impact Factor
• Article: Retrograde and anterograde labeling of cerebellar afferent projection by the injection of recombinant adenoviral vectors into the mouse cerebellar cortex

  T. Terashima, Akiko Miwa, Yumi Kanegae, Izumu Saito, Haruo Okado
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  ABSTRACT:  Adenoviral vectors have recently been recognized as highly efficient systems for gene delivery into various tissues. We show that a reporter gene introduced into nerve terminals via an adenovirus can be used to label cell bodies retrogradely and then label the axons and nerve terminals of the infected neurons anterogradely in vivo. We injected a replication-defective recombinant adenovirus carrying the E. coli β-galactosidase gene (lacZ) into the cerebellar cortex of the adult mouse. The first evidence of retrograde labeling was obtained at 2 days after the infection when neurons in the pontine nuclei and the reticulotegmental nucleus of the pons weakly expressed β-galactosidase, and at 3 days post-infection when neurons in all precerebellar nuclei, known to project to the cerebellar cortex, were strongly stained with X-gal in a Golgi-like manner. Anterograde transport of lacZ gene products was recognized at 3 days post-infection; β-galactosidase-positive axons arose from somata or dendrites of retrogradely labeled neurons, passed through the middle or inferior cerebellar peduncles, and entered the cerebellum. Anterogradely labeled mossy terminals were recognized on the injection side at 8 days post-infection, and on the contralateral side at 14 days post-infection. β-Galactosidase expression persisted for up to two months, with a decrease in the total number of labeled cells over time. We could not find any signs of anterograde or retrograde transsynaptic labeling in the nuclei synaptically linked to the cerebellar cortex at any time point after injection up to 58 days post-infection.
  Anatomy and Embryology 09/1997; 196(5):363-382. · 1.42 Impact Factor
• Article: Ambiguus nucleus neurons innervating the abdominal esophagus are malpositioned in the reeler mouse

  Yoko Fujimoto, Tomiyoshi Setsu, Yayoi Ikeda, Akiko Miwa, Haruo Okado, Toshio Terashima
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  ABSTRACT: To examine whether the migration of ambiguus nucleus (NA) neurons is affected in the reeler mouse, recombinant replication-deficient adenoviral vector carrying E. coli-galactosidase gene (lacZ) was injected into the abdominal esophagus of the reeler mouse and normal control at two months of age prior to 5 days of sacrifice of the animals. In the normal control, lacZ-positive neurons were found in the compact formation of the NA, whereas, in the reeler, they were scattered from the base of the fourth ventricle to the ventro-lateral margin of the medulla. The present study confirmed that NA neurons are malpositioned in the reeler mouse, suggesting that the migration of NA neurons is guided by the reelin-related protein (Reelin).
  Brain Research.