Updated on 2022/01/19


Faculty of Human Sciences, School of Human Sciences
Job title

Concurrent Post

  • Faculty of Human Sciences   Graduate School of Human Sciences

  • Affiliated organization   Global Education Center


  • Tokyo University   ph.D

Professional Memberships




    Society for Neuroscience


Research Areas

  • Neuroscience-general

  • Neuroscience-general

  • Anatomy

Research Interests

  • Neuroanatomy, neural stem cell, neurogenesis, glia


  • Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

    Seiya Yamada, Ayaka Sato, Hiroki Akiyama, Shin-ichi Sakakibara


     View Summary

    <title>ABSTRACT</title>Brain development is a highly orchestrated process requiring spatiotemporally regulated mitochondrial dynamics. Drp1, a key molecule in the mitochondrial fission machinery, undergoes various post-translational modifications including conjugation to the small ubiquitin-like modifier (SUMO). However, the functional significance of SUMOylation/deSUMOylation on Drp1 remains controversial. SUMO-specific protease 5 (Senp5L) catalyzes the deSUMOylation of Drp1. We revealed that a splicing variant of Senp5L, Senp5S, which lacks peptidase activity, prevents deSUMOylation of Drp1 by competing against other Senps. The altered SUMOylation level of Drp1 induced by Senp5L/5S affects Drp1 ubiquitination and tubulation of the endoplasmic reticulum (ER), thereby influencing mitochondrial morphology. A dynamic SUMOylation/deSUMOylation balance controls neuronal polarization and migration during the development of the cerebral cortex. These findings suggest a novel role of post translational modification, in which a deSUMOylation enzyme isoform competitively regulates mitochondrial dynamics and ER tubulation via Drp1 SUMOylation levels in a tightly controlled process of neuronal differentiation and corticogenesis.


  • Minocycline Alleviates Cluster Formation of Activated Microglia and Age-dependent Dopaminergic Cell Death in the Substantia Nigra of Zitter Mutant Rat.

    Daisuke Taguchi, Ayuka Ehara, Taro Kadowaki, Shin-Ichi Sakakibara, Kazuhiko Nakadate, Koichi Hirata, Shuichi Ueda

    Acta histochemica et cytochemica   53 ( 6 ) 139 - 146  2020.12  [Refereed]  [Domestic journal]

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    Microglial activation is a component of neurodegenerative pathology. Here, we examine whether activated microglia participate in age-related dopaminergic (DA) cell death in the substantia nigra pars compacta (SNc) of the zitter (zi/zi) rat, a mutant characterized by deletion of the attractin gene. Confocal microscopy with double-immunohistochemical staining revealed activated microglia-formed cell-clusters surrounding DA neurons in the SNc from 2 weeks after birth. An immunoelectron microscopic study showed that the cytoplasm of activated microglia usually contains phagosome-like vacuoles and lamellar inclusions. Expression levels of the pro-inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were increased in the midbrain of 2-month-old zi/zi rats. Chronic treatment with the anti-inflammatory agent minocycline altered the morphology of the microglia, reduced cluster formation by the microglia, and attenuated DA cell death in the SNc, and reduced the expression of IL-1β in the midbrain. These results indicate that activated microglia, at least in part and especially at the initial phase, contribute to DA cell death in the SNc of the zi/zi rat.

    DOI PubMed

  • Control of cell migration by the novel protein phosphatase-2A interacting protein inka2.

    Hiroki Akiyama, Yumi Iwasaki, Seiya Yamada, Hiroyuki Kamiguchi, Shin-Ichi Sakakibara

    Cell and tissue research   380 ( 3 ) 527 - 537  2020.06  [Refereed]  [International journal]

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    Cell migration is essential for many physiological and pathological processes, including embryonic development, wound healing, immune response and cancer metastasis. Inka2 transcripts are observed in migrating cells during embryonic development, suggesting the involvement of inka2 in cell migration. However, its precise role remains unclear. Here, we found that inka2 controlled focal adhesion dynamics and cell migration, likely by regulating protein phosphatase-2A (PP2A) function. A scratch assay revealed that inka2 shRNA-transfected NIH3T3 cells showed rapid wound closure, indicating an inhibitory effect by inka2 on cell migration. Live-cell imaging of NIH3T3 cells expressing EGFP-paxillin using total internal reflection fluorescence microscopy revealed that inka2 knockdown increased the turnover rate of focal adhesions. Given that PP2A, which consists of catalytic (C), regulatory (B) and scaffolding (A) subunits, is known to regulate focal adhesions, we examined the inka2-PP2A interaction. Immunoprecipitation revealed an association between inka2 and the PP2A C subunit. Binding of Inka2 to the C subunit prevented the association between the A and C subunits, suggesting that inka2 can inhibit PP2A function. Furthermore, both inka2 expression and PP2A inhibition decreased focal adhesion kinase-paxillin interaction, resulting in reduced formation of focal adhesions. We assessed the effect of pharmacological PP2A inhibition on the inka2 knockdown-induced increase in cell migration speed and found that treatment with a PP2A inhibitor negated the accelerated migration of inka2 knockdown cells. These results suggest that inka2 knockdown exerts its effects through PP2A-dependent regulation of focal adhesions. Our findings contribute to a better understanding of the molecular mechanisms underlying cell migration.

    DOI PubMed

  • Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

    Seiya Yamada, Ayaka Sato, Shin-Ichi Sakakibara

    iScience   23 ( 5 ) 101058 - 101058  2020.05  [Refereed]  [International journal]

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    Engagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called "purinosome." However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

    DOI PubMed

  • 女性研究者セッション〜組織細胞化学研究の魅力を語る〜 中枢神経系におけるアトラクチンの機能を見る

    江原 鮎香, 中舘 和彦, 山口 剛, 榊原 伸一, 上田 秀一

    日本組織細胞化学会総会・学術集会講演プログラム・予稿集   60回   47 - 47  2019.09

  • Zitterラットのドーパミンニューロン変性におけるミクログリアの"負の作用"

    江原 鮎香, 門脇 太郎, 中舘 和彦, 榊原 伸一, 平田 幸一, 上田 秀一

    日本組織細胞化学会総会・学術集会講演プログラム・予稿集   59回   71 - 71  2018.09

  • Expression profile of the STAND protein Nwd1 in the developing and mature mouse central nervous system.

    Seiya Yamada, Shin-Ichi Sakakibara

    The Journal of comparative neurology   526 ( 13 ) 2099 - 2114  2018.09  [Refereed]  [International journal]

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    The orchestrated events required during brain development, as well as the maintenance of adult neuronal plasticity, highly depend on the accurate responses of neuronal cells to various cellular stress or environmental stimuli. Recent studies have defined a previously unrecognized, broad class of multidomain proteins, designated as signal transduction ATPases with numerous domains (STAND), which comprises a large number of proteins, including the apoptotic peptidase activating factor 1 (Apaf1) and nucleotide-binding oligomerization domain-like receptors (NLRs), central players in cell death and innate immune responses, respectively. Although the involvement of STANDs in the central nervous system (CNS) has been postulated in terms of neuronal development and function, it remains largely unclear. Here, we identified Nwd1 (NACHT and WD repeat domain-containing protein 1), as a novel STAND protein, expressed in neural stem/progenitor cells (NSPCs). Structurally, Nwd1 was most analogous to the apoptosis regulator Apaf1, also involved in mitosis and axonal outgrowth regulation in the CNS. Using a specific antibody, we show that, during the embryonic and postnatal period, Nwd1 is expressed in nestin-positive NSPCs in vivo and in vitro, while postnatally it is found in terminally differentiated neurons and blood vessels. At the subcellular level, we demonstrate that Nwd1 is preferentially located in the cytosolic compartment of cultured NSPCs, partially overlapping with cytochrome c. These observations imply that Nwd1 might be involved in the neuronal lineage as a new STAND gene, including having a pro-apoptotic or nonapoptotic role, similar to Apaf1.

    DOI PubMed

  • Synaptic localization of the SUMOylation-regulating protease SENP5 in the adult mouse brain.

    Hiroki Akiyama, Kazuhiko Nakadate, Shin-Ichi Sakakibara

    The Journal of comparative neurology   526 ( 6 ) 990 - 1005  2018.04  [Refereed]  [International journal]

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    Covalent conjugation of small ubiquitin-like modifiers (SUMOs) or SUMOylation is a reversible post-translational modification that regulates the stability and function of target proteins. SUMOs are removed from substrate proteins by sentrin/SUMO-specific proteases (SENPs). Numerous studies have implicated SUMOylation in various physiological and pathological processes in neurons. To understand the functional roles of SUMOylation, it is necessary to determine the distribution of enzymes regulating SUMO conjugation and deconjugation; yet, the localization of SENPs has not been described in detail in intact brain tissue. Here, we report the distribution and subcellular localization of SENP3 and 5 in the adult murine brain. Immunohistochemical analyses revealed the ubiquitous distribution of both SENPs across different brain regions. Within individual cells, SENP3 was confined to the nucleus, consistent with the conventional view that SENPs regulate nuclear events. In contrast, SENP5 was detected in the neuropil but not in cell bodies. Moreover, strong SENP5 immunoreactivity was observed in regions with high numbers of synapses such as the cerebellar glomeruli, suggesting that SENP5 localizes to pre- and/or postsynaptic structures. We performed double immunolabeling in cultured neurons and found that SENP5 co-localized with pre- and post-synaptic markers, as well as a mitochondrial marker. Immunoelectron microscopy confirmed this finding and revealed that SENP5 was localized to presynaptic terminals, postsynaptic spines, and mitochondria in axon terminals. These findings advance the current understanding of the functional roles of SUMOylation in neurons, especially in synaptic regulation, and have implications for future therapeutic strategies in neurodegenerative disorders mediated by mitochondrial dysfunction.

    DOI PubMed

  • 脱SUMO化酵素SENP5の新規アイソフォームの機能解析

    秋山 博紀, 中舘 和彦, 榊原 伸一

    生命科学系学会合同年次大会   2017年度   [2P - 1210]  2017.12

  • Cytoskeletons in neuronal development

    Hiroki Akiyama, Shin-ichi Sakakibara

    The Journal of Physical Fitness and Sports Medicine   5 ( 2 ) 131  2016  [Refereed]


  • Expression profiles of inka2 in the murine nervous system

    Yumi Iwasaki, Takahito Yumoto, Shin-ichi Sakakibara

    GENE EXPRESSION PATTERNS   19 ( 1-2 ) 83 - 97  2015.09  [Refereed]

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    Dynamic rearrangement of the actin cytoskeleton impacts many cellular characteristics in both the developing and adult central nervous systems (CNS), including the migration and adhesion of highly motile neural progenitor cells, axon guidance of immature neurons, and reconstruction of synaptic structures in the adult brain. Inka1, a known regulator of actin cytoskeleton reconstruction, is predominantly expressed by the neural crest cell lineage and regulates the migration and differentiation of these cells. In the present study, we identified a novel gene, designated as inka2, which is related to inka1. Inka2/fam212b is an evolutionarily conserved gene found in different vertebrate species and constitutes a novel gene family together with inka1. Northern blot analysis showed that inka2 mRNA was highly enriched in the nervous system. The spatiotemporal propagation cell profiles of those cells that expressed inka2 transcripts were compatible with those of Olig2-positive oligodendrocyte progenitor cells, which originate in the ventral ventricular zone during embryogenesis. Intense expression of inka2 was also noted in the proliferative neuronal progenitors in the developing cerebellum. On the other hand, immature newborn neurons in the embryonic brain showed no expression of inka2, except for the cells residing in the marginal zone of the embryonic telencephalon, which is known to contain transient cells including the non-subplate pioneer neurons and CajaleRetzius cells. As brain development proceeds during the postnatal stage, inka2 expression emerged in some populations of immature neurons, including the neocortical pyramidal neurons, hippocampal pyramidal neurons, and granule cells migrating in the cerebellar cortex. In the adult brain, the expression of inka2 was interestingly confined in terminally differentiated neurons in the restricted forebrain regions. Taken together, as a novel regulator of actin cytoskeletons in the CNS, inka2 may be involved in multiple actin-driven processes, including cell migration and establishment of neuronal polarity. (C) 2015 Elsevier B.V. All rights reserved.

    DOI PubMed

  • Mutations in CKAP2L, the Human Homo log of the Mouse Radmis Gene, Cause Filippi Syndrome

    Muhammad Sajid Hussain, Agatino Battaglia, Sandra Szczepanski, Emrah Kaygusuz, Mohammad Reza Toliat, Shin-ichi Sakakibara, Janine Altmueller, Holger Thiele, Gudrun Nuernberg, Shahida Moosa, Goekhan Yigit, Filippo Beleggia, Sigrid Tinschert, Jill Clayton-Smith, Pradeep Vasudevan, Jill E. Urquhart, Dian Donnai, Alan Fryer, Ferda Percin, Francesco Brancati, Angus Dobbie, Robert Smigiel, Gabriele Gillessen-Kaesbach, Bernd Wollnik, Angelika Anna Noegel, William G. Newman, Peter Nuernberg

    AMERICAN JOURNAL OF HUMAN GENETICS   95 ( 5 ) 622 - 632  2014.11  [Refereed]

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    Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs*6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.


  • Spatiotemporal distribution of SUMOylation components during mouse brain development

    Yuta Hasegawa, Daisuke Yoshida, Yuki Nakamura, Shin-Ichi Sakakibara

    Journal of Comparative Neurology   522 ( 13 ) 3020 - 3036  2014.09  [Refereed]

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    Posttranslational modification of proteins might play an important role in brain cellular dynamics via the rapid turnover or functional change of critical proteins controlling neuronal differentiation or synaptic transmission. Small ubiquitin-like modifier protein (SUMO) is a family of ubiquitin-like small proteins that are covalently attached to target proteins to modify their function posttranslationally. Many cellular processes, such as transcription and protein trafficking, are regulated by SUMOylation, but its functional significance in the brain remains unclear. Although developmental regulation of SUMOylation levels in rat brain was recently demonstrated, no comparative immunohistochemical analysis of the cellular distribution profiles of SUMOylation components, including SUMO1, SUMO2/3, and Ubc9, has been undertaken so far. The present study used immunohistochemical and immunoblot analysis with the different developmental stages of mice and demonstrated the developmentally regulated distribution of SUMO1, SUMO2/3, and Ubc9 in the brain. During embryonic development, SUMOylation by SUMO1 and SUMO2/3 occurred in the nucleoplasm of nestin-positive neural stem cells. Although the total amount of SUMO-modified proteins decreased during postnatal brain development, intense and persistent accumulation of SUMO2/3 was detected throughout life in neural progenitor populations in neurogenic regions, including the subventricular zone and the hippocampal subgranular zone. In contrast, many neurons in the adult brain accumulated SUMO1 rather than SUMO2/3. Heavy immunoreactivity of SUMO1 was found in large projection neurons in the brainstem, whereas SUMO2/3 was almost absent from these areas. This heterogeneous distribution implies that both proteins play a specific and unique role in the brain. © 2014 Wiley Periodicals, Inc.

    DOI PubMed

  • Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

    Takahito Yumoto, Kazuhiko Nakadate, Yuki Nakamura, Yoshinobu Sugitani, Reiko Sugitani-Yoshida, Shuichi Ueda, Shin-ichi Sakakibara

    PLOS ONE   8 ( 11 ) e79895  2013.11  [Refereed]

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    Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. By differential subtractive screening with NSPCs versus their differentiated progenies, we identified the radmis (radial fiber and mitotic spindle)/ckap2l gene, a novel microtubule-associated protein (MAP) enriched in NSPCs. Radmis is a putative substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome (APC/C), and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life, and its distribution was dynamically regulated during NSPC division. In embryonic and perinatal brains, radmis localized to bipolar mitotic spindles and radial fibers (basal processes) of dividing NSPCs. As central nervous system development proceeded, radmis expression was lost in most brain regions, except for several neurogenic regions. In adult brain, radmis expression persisted in the mitotic spindles of both slowly-dividing stem cells and rapid amplifying progenitors. Overexpression of radmis in vitro induced hyperstabilization of microtubules, severe defects in mitotic spindle formation, and mitotic arrest. In vivo gain-of-function using in utero electroporation revealed that radmis directed a reduction in NSPC proliferation and a concomitant increase in cell cycle exit, causing a reduction in the Tbr2-positive basal progenitor population and shrinkage of the embryonic subventricular zone. Besides, radmis loss-of-function by shRNAs induced the multipolar mitotic spindle structure, accompanied with the catastrophe of chromosome segregation including the long chromosome bridge between two separating daughter nuclei. These findings uncover the indispensable role of radmis in mitotic spindle formation and cell-cycle progression of NSPCs.

    DOI PubMed

  • S2-02 哺乳類中枢神経発生過程に機能する新規遺伝子群の同定(S2 神経系の発生・分化の分子細胞化学,シンポジウム,第54回日本組織細胞化学会総会・学術集会)

    榊原 伸一

    日本組織細胞化学会総会プログラムおよび抄録集   ( 54 )  2013


  • Neuroprotective effects of melatonin on the nigrostriatal dopamine system in the zitter rat

    Ken-ichi Hashimoto, Shuichi Ueda, Ayuka Ehara, Shin-ichi Sakakibara, Kanji Yoshimoto, Koichi Hirata

    NEUROSCIENCE LETTERS   506 ( 1 ) 79 - 83  2012.01  [Refereed]

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    Melatonin has ubiquitous actions, both as a direct free-radical scavenger and as an indirect anti-oxidant. The present study examined in vivo neuroprotective effects of melatonin on the nigrostriatal dopaminergic system in zitter (zi/zi) rat, which displays abnormal metabolism of superoxide leading to age-related degeneration of the dopaminergic system. For up to 9 months after weaning, zi/zi rats had ad libitum access to drinking water containing melatonin. Chronic treatment with melatonin attenuated the decreases of dopamine and its metabolite in zi/zi rat caudate-putamen (CPU). Immunohistochemistry for tyrosine hydroxylase (TH) was consistent with neurochemical data in the CPU and demonstrated substantial sparing of the reduction of TH-immunoreactive neurons in the substantia nigra pars compacta. Quantitative polymerase chain reaction (qPCR) was performed to analyze mRNA expressions of pro-inflammatory cytokines (IL-1 beta and TNF-alpha) and the anti-oxidant enzymes (catalase (CAT). superoxide dismutase (SOD) 1 and 2, and glutathione peroxidase (GPx1)) in the striatum and midbrain in order to examine the neuroprotective effect of melatonin. IL-1 beta and TNF-alpha mRNA expressions were significantly increased in both areas of 3-month-old zi/zi rats, whereas there was a significant decrease in CAT mRNA expression in the striatum of 6-month-old zi/zi rat as compared to age-matched controls. With the exception of the high TNF-alpha mRNA expression in 3-month-old zi/zi midbrains, chronic treatment of melatonin attenuated expressional changes of IL-1 beta, CAT, SOD1, and GPx1. These results suggest that besides its direct scavenger effects, chronic melatonin treatment provides a neuroprotective effect against dopaminergic degeneration by suppressing pro-inflammatory cytokines and up-regulating anti-oxidant enzyme expression. Crown Copyright (C) 2011 Published by Elsevier Ireland Ltd. All rights reserved.

    DOI PubMed

  • Neural RNA-Binding Protein Musashi1 Controls Midline Crossing of Precerebellar Neurons through Posttranscriptional Regulation of Robo3/Rig-1 Expression

    Ken-ichiro Kuwako, Kyoko Kakumoto, Takao Imai, Mana Igarashi, Takao Hamakubo, Shin-ichi Sakakibara, Marc Tessier-Lavigne, Hirotaka James Okano, Hideyuki Okano

    NEURON   67 ( 3 ) 407 - 421  2010.08  [Refereed]

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    Precisely regulated spatiotemporal gene expression is essential for the establishment of neural circuits. In contrast to the increasing evidence for transcriptional regulation of axon guidance cues and receptors, the role of posttranscriptional regulation in axon guidance, especially in vivo, remains poorly characterized. Here, we demonstrate that the expression of Slit receptor Robo3/Rig-1, which plays crucial roles in axonal midline crossing, is regulated by a neural RNA-binding protein Musashi1 (Msi1). Msi1 binds to Robo3 mRNA through RNA recognition motifs and increases the protein level of Robo3 without affecting its mRNA level. In Msi1-deficient precerebellar neurons, Robo3 protein, but not its mRNA, is dramatically reduced. Moreover, similar to defects in Robo3-deficient mice, axonal midline crossing and neuronal migration of precerebellar neurons are severely impaired in Msi1-deficient mice. Together, these findings indicate that Msi1-mediated posttranscriptional regulation of Robo3 controls midline crossing of precerebellar neurons.

    DOI PubMed

  • げっ歯類脳神経核におけるattractin分布の解析

    中舘 和彦, 榊原 伸一, 大桃 秀樹, 江原 鮎香, 上田 秀一

    解剖学雑誌   85 ( Suppl. ) 190 - 190  2010.03

  • Regeneration of 5-HT fibers in hippocampal heterotopia of methylazoxymethanol-induced micrencephalic rats after neonatal 5,7-DHT injection

    Arata Nakamura, Taro Kadowaki, Shin-ichi Sakakibara, Kanji Yoshimoto, Koichi Hirata, Shuichi Ueda

    ANATOMICAL SCIENCE INTERNATIONAL   85 ( 1 ) 38 - 45  2010.03  [Refereed]

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    In order to elucidate the regeneration properties of serotonergic fibers in the hippocampus of methylazoxymethanol acetate (MAM)-induced micrencephalic rats (MAM rats), we examined serotonergic regeneration in the hippocampus following neonatal intracisternal 5,7-dihydroxytryptamine (5,7-DHT) injection. Prenatal exposure to MAM resulted in the formation of hippocampal heterotopia in the dorsal hippocampus. Immunohistochemical and neurochemical analyses revealed hyperinnervation of serotonergic fibers in the hippocampus of MAM rats. After neonatal 5,7-DHT injection, most serotonergic fibers in the hippocampus of 2-week-old MAM rats had degenerated, while a small number of serotonergic fibers in the stratum lacunosum-moleculare (SLM) of the hippocampus and in the hilus adjacent to the granular cell layer of the dentate gyrus (DG) had not. Regenerating serotonergic fibers from the SLM first extended terminals into the hippocampal heterotopia, then fibers from the hilus reinnervated the DG and some fibers extended to the heterotopia. These findings suggest that the hippocampal heterotopia exerts trophic target effects for regenerating serotonergic fibers in the developmental period in micrencephalic rats.


  • Improved learning in microencephalic rats

    Shuichi Ueda, Kanji Yoshimoto, Taro Kadowaki, Koichi Hirata, Shin-ichi Sakakibara

    CONGENITAL ANOMALIES   50 ( 1 ) 58 - 63  2010.03  [Refereed]

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    Environmental enrichment (EE) facilitates recovery from behavioral abnormalities and spatial memory disabilities in several neurological disease models. Exposure to EE improves spatial memory acquisition and enhances the survival of newly generated cells in the dentate gyri of adult rodents. However, the effects of EE on spatial learning and neurogenesis in the methylazoxymethanol acetate-induced microencephalic rat have not been investigated. Depletion of serotonin in the rat hippocampus is known to influence spatial memory and adult neurogenesis, suggesting a role for serotonin in these processes. To confirm this hypothesis, male methylazoxymethanol acetate-induced microencephalic rats were exposed to EE or conventional housing after weaning; half of these rats further received intracisternal 5,7-dihydroxytryptamine on postnatal day 3, to induce long-lasting depletion of serotonin. As adults, these microencephalic rats were observed using the Morris water maze test and examined for hippocampal neurogenesis. EE alleviated the impairment of spatial memory acquisition and enhanced neurogenesis in the dentate gyri of adult microencephalic rats. Injection of 5,7-dihydroxytryptamine during the neonatal period caused pronounced reductions in hippocampal serotonin levels in these rats. Long-lasting depletion of serotonin eliminated the EE-induced alleviation of spatial memory acquisition and neurogenesis impairment in microencephalic rats. The present results suggest that EE alleviates spatial memory performance deficits in microencephalic rats and further indicate that serotonin might be involved in the underlying mechanisms through increased hippocampal neurogenesis. These data provide new insights into therapeutic interventions for individuals with human migration disorders associated with learning disabilities.


  • Touch and pain receptive network formation with RNA binding protein Musashi2 and Pleiotrophin

    Hideyuki Okano, Shin-ichi Sakakibara, Hidemasa Furue, Megumu Yoshimura, Hirotaka J. Okano, Shinsuke Shibata

    NEUROSCIENCE RESEARCH   68   E161 - E161  2010  [Refereed]


  • 選択的神経毒の新生仔期髄液内投与後における再生セロトニン線維の特性

    上田 秀一, 江原 鮎香, 大桃 秀樹, 中舘 和彦, 榊原 伸一, 吉本 寛司

    日本組織細胞化学会総会・学術集会講演プログラム・予稿集   50回   86 - 86  2009.09

  • Zitterラットの進行性黒質ドーパミン神経変性におけるミクログリアの関与

    中舘 和彦, 大桃 秀樹, 江原 鮎香, 榊原 伸一, 上田 秀一

    日本組織細胞化学会総会・学術集会講演プログラム・予稿集   50回   87 - 87  2009.09

  • 進行性神経変性疾患モデル動物(Zitterラット)黒質におけるミクログリアの解析

    中舘 和彦, 門脇 太郎, 中村 新, 榊原 伸一, 大桃 秀樹, 江原 鮎香, 上田 秀一

    解剖学雑誌   84 ( Suppl. ) 185 - 185  2009.03

  • ラット主嗅球における小胞性グルタミン酸輸送体の発達変化

    大桃 秀樹, 江原 鮎香, 中舘 和彦, 榊原 伸一, 久野 節二, 上田 秀一

    解剖学雑誌   84 ( Suppl. ) 187 - 187  2009.03

  • Sensory network formation of RNA binding protein Musashi2

    Shibata, Shinsuke, Sakakibara, Shin-ichi, Furue, Hidemasa, Yoshimura, Megumu, Kuwako, Ken-ichiro, Okano, James Hirotaka, Okano, Hideyuki

    NEUROSCIENCE RESEARCH   65   S96 - S96  2009



    Shibata, Shinsuke, Sakakibara, Shin-Ichi, Furue, Hidemasa, Yoshimura, Megumu, Takagi, Takehiko, Ohkuma, Rika, Kuwako, Ken-Ichiro, Okano, Hirotaka J, Okano, Hideyuki

    JOURNAL OF PHYSIOLOGICAL SCIENCES   59   337 - 337  2009

  • Chronic treatment with melatonin attenuates serotonergic degeneration in the striatum and olfactory tubercle of zitter mutant rats

    Shiuchi Ueda, Shin-ichi Sakakibara, Taro Kadowaki, Takuya Naitoh, Koichi Hirata, Kanji Yoshimoto

    NEUROSCIENCE LETTERS   448 ( 2 ) 212 - 216  2008.12  [Refereed]

     View Summary

    The effects of chronic treatment with the antioxidant hormone melatonin on degeneration of serotonergic fibers were Studied in the striatum and olfactory tubercle of the zitter rat. which shows a loss-of-function mutation of the glycosylated transmembrane protein attractin. In these animals, serotonergic fibers in the striatum and olfactory tubercle undergo spontaneous and progressive degeneration as a result of abnormal metabolism of reactive oxygen species. Homozygous zitter (zi/zi) rats were provided ad libitum access to drinking water containing melatonin for 9 months (M) after weaning. High-performance liquid chromatography analysis revealed that melatonin treatment significantly increased serotonin in the Caudate-putamen, (CPU), nucleus accumbens (NA) and olfactory tubercle (OT). Immunohistochemical staining for serotonin was consistent with the neurochemical data and further demonstrated substantially increased numbers of serotonergic nerve terminals in these areas. Aberrant serotonergic fibers characterized by swollen varicosities (&gt; 1 mu m in diameter) were observed in the CPU and NA of 10 M zi/zi rats. The number of these fibers decreased after melatonin treatment ended. Furthermore, hyperinnervation of serotonergic fibers was observed in the OT of melatonin-treated zi/zi rats. These results suggest that melatonin protects serotonergic fibers and terminals in zitter rats and/or promotes their neuroplasticity. (C) 2008 Elsevier Ireland Ltd. All rights reserved.


  • Developmental and spatial expression pattern of alpha-taxilin in the rat central nervous system

    Shin-Ichi Sakakibara, Kazuhiko Nakadate, Sawako Tanaka-Nakadate, Kenji Yoshida, Satoru Nogami, Hiromichi Shirataki, Shuichi Ueda

    JOURNAL OF COMPARATIVE NEUROLOGY   511 ( 1 ) 65 - 80  2008.11  [Refereed]

     View Summary

    alpha-Taxilin has been identified as a binding partner of syntaxin family members and thus has been proposed to function in syntaxin-mediated intracellular vesicle trafficking. However, the lack of detailed information concerning the cellular and subcellular localization of alpha-taxilin impedes an understanding of the role of this protein. In the present study, we characterized alpha-taxilin-expressing cells in the rat CNS with a specific antibody. During embryonic development, alpha-taxilin was prominently expressed in nestin-positive neural stem cells in vivo and in vitro. As CNS development proceeded, the alpha-taxilin expression level was rapidly down-regulated. In the postnatal CNS, a-taxilin expression was almost confined to the neuronal lineage, with the highest levels of expression in motor neurons within the brainstem nuclei and spinal cord and in primary sensory neurons in mesencephalic trigeminal nucleus. At the cellular level, alpha-taxilin was preferentially located in Nissl substance-like structures with a tigroid or globular morphology within the soma and proximal to dendrites, but it was excluded from terminals. Combined staining with propidium iodide demonstrated that a-taxilin distribution overlapped with the cytoplasmic compartment enriched in RNA species, suggesting a close association of a-taxilin with actively translating ribosomes or polysomes in neurons. In agreement with this, a recent study indicated the preferential binding of alpha-taxilin to the nascent polypeptide-associated complex (alpha NAC), a dynamic component of the ribosomal exit tunnel in eukaryotic cells. Taken together, these findings suggest that a-taxilin plays multiple roles in the generation and maintenance of neurons through modulation of the NAC-mediated translational machinary and/or the syntaxin-mediated vesicle traffic in the soma.


  • 進行性神経変性に関与するMahogunin Ring Finger 1(Mgrn1)のラット中枢神経系における分布解析

    中舘 和彦, 榊原 伸一, 大桃 秀樹, 江原 鮎香, 上田 秀一

    日本組織細胞化学会総会・学術集会講演プログラム・予稿集   49回   60 - 60  2008.10

  • Attractin/Mahogany protein expression in the rodent central nervous system

    Kazuhiko Nakadate, Shin-Ichi Sakakibara, Shuichi Ueda

    JOURNAL OF COMPARATIVE NEUROLOGY   508 ( 1 ) 94 - 111  2008.05  [Refereed]

     View Summary

    Attractin/Mahogany protein (Atrn) is known to be involved in a number of physiological and neuropathological events. Although the ubiquitous distribution of atrn mRNA has been described in neurons, lack of detailed information concerning the cellular and subcellular localization of protein product is impeding understanding of the role of Atrn. The present study immunohistochemically examined distributions of Atrn in rat and mouse central nervous systems (CNSs) by using a novel antibody for Atrn. Atrn was intensely expressed in most neurons and dendrites of large neurons, such as cortical pyramidal neurons and cerebellar Purkinje neurons. Intense Atrn expression was also observed in the neuropil of gray matter in many regions of the CNS, such as the main and accessory olfactory bulb, cerebral cortex, caudate putamen, dorsal lateral geniculate nucleus, medial eminence, superior colliculus, hippocampus, dentate gyrus, and layers 1 and 2 of the spinal cord. Furthermore, we found that astrocytes, microglia, and ependymal cells also express Atrn protein. Immunoelectron microscopy showed the subcellular distribution of Atrn in the plasma membrane of cell soma, dendrites, and spines in neurons and in the cytoplasmic membrane of Golgi apparatus, endoplasmic reticulum, and mitochondria in neurons and glial cells. These findings indicate that Atrn is more widely expressed throughout the CNS than previously reported, and expression of Atrn by various cell types suggests that Atrn may serve multiple functions in the CNS.


  • Non-cell autonomous impairment of oligodendrocyte differentiation precedes CNS degeneration in the Zitter rat: Implications of macrophage/microglial activation in the pathogenesis

    Shin-ichi Sakakibara, Kazuhiko Nakadate, Shigeo Ookawara, Shuichi Ueda

    BMC NEUROSCIENCE   9 ( 35 )  2008.04  [Refereed]

     View Summary

    Background: The zitter (zi/zi) rat, a loss-of-function mutant of the glycosylated transmembrane protein attractin (atrn), exhibits widespread age-dependent spongiform degeneration, hypomyelination, and abnormal metabolism of reactive oxygen species (ROS) in the brain. To date, the mechanisms underlying these phenotypes have remained unclear.
    Results: Here, we show differentiation defects in zi/zi oligodendrocytes, accompanied by aberrant extension of cell-processes and hypomyelination. Axonal bundles were relatively preserved during postnatal development. With increasing in age, the injured oligodendrocytes in zi/zi rats become pathological, as evidenced by the accumulation of iron in their cell bodies. Immunohistochemical analysis revealed that atrn expression was absent from an oligodendrocyte lineage, including A2B5-positive progenitors and CNPase-positive differentiated cells. The number and distribution of Olig2-positive oligodendrocyte progenitors was unchanged in the zi/zi brain. Furthermore, an in vitro differentiation assay of cultured oligodendrocyte progenitors prepared from zi/zi brains revealed their normal competence for proliferation and differentiation into mature oligodendrocytes. Interestingly, we demonstrated the accelerated recruitment of EDI-positive macrophages/microglia to the developing zi/zi brain parenchyma prior to the onset of hypomyelination. Semiquantitative RT-PCR analysis revealed a significant up-regulation of CD26 and IL1-beta in the zi/zi brain during this early postnatal stage.
    Conclusion: We demonstrated that the onset of the impairment of oligodendrocyte differentiation occurs in a non-cell autonomous manner in zi/zi rats. Hypomyelination of oligodendrocytes was not due to a failure of the intrinsic program of oligodendrocytes, but rather, was caused by extrinsic factors that interrupt oligodendrocyte development. It is likely that macrophage/microglial activation in the zi/zi CNS leads to disturbances in oligodendrocyte differentiation via deleterious extrinsic factors, such as the cytokine IL1-beta or ROS. Atrn might be involved in the activation of brain macrophages/microglia by suppressing excessive migration of monocytes into the CNS, or by accelerating the transformation of brain monocytes into resting microglia. Understanding the pathogenesis of the zi/zi rat may provide novel insights into the developmental interaction betweens macrophages/microglia and cells of an oligodendrocyte lineage.


  • ラット視床下部、正中隆起におけるAttractin発現様式 Zitterラットとの比較

    中舘 和彦, 榊原 伸一, 大桃 秀樹, 上田 秀一

    解剖学雑誌   83 ( Suppl. ) 246 - 246  2008.03

  • zitterラット脳におけるオリゴデンドロサイトの分化異常とマクロファージ/ミクログリア活性化

    榊原 伸一, 中舘 和彦, 上田 秀一

    解剖学雑誌   83 ( Suppl. ) 249 - 249  2008.03

  • Zitterラットの遅発性ドーパミン細胞変性とミクログリア変化

    上田 秀一, 大桃 秀樹, 中舘 和彦, 榊原 伸一

    解剖学雑誌   83 ( Suppl. ) 250 - 250  2008.03

  • 【攻撃性・暴力と向精神薬をめぐる問題】攻撃性の神経回路 セロトニンニューロン系を中心に

    上田 秀一, 榊原 伸一, 中舘 和彦, 野田 隆洋

    臨床精神薬理   11 ( 2 ) 219 - 226  2008.02

     View Summary


  • Analysis of RNA binding protein Musashi2 in mammalian nervous system development

    Shibata, Shinsuke, Sakakibara, Shin-Ichi, Okano, Hirotaka J, Okano, Hideyuki

    NEUROSCIENCE RESEARCH   61   S89 - S89  2008

  • Zitterラット黒質における進行性ドーパミン神経変性

    中舘 和彦, 榊原 伸一, 熊本 賢三, 松浦 忠夫, 上田 秀一

    解剖学雑誌   82 ( Suppl. ) 194 - 194  2007.03

  • 中枢神経系前駆細胞における新規遺伝子Radmisの発現

    榊原 伸一, 中舘 和彦, 大桃 秀樹, 小川 哲郎, 上田 秀一

    解剖学雑誌   82 ( Suppl. ) 210 - 210  2007.03

  • Expression of Iba1 protein in microglial cells of zitter mutant rat

    Taro Kadowaki, Kazuhiko Nakadate, Shin-ichi Sakakibara, Koichi Hirata, Shuichi Ueda

    NEUROSCIENCE LETTERS   411 ( 1 ) 26 - 31  2007.01  [Refereed]

     View Summary

    Microglial activation has been associated with the pathogenesis of neurodegenerative disease. To characterize microglial responses in the zitter mutant rat, which shows progressive spongy degeneration, the development of microglial cells was investigated using ionized calcium-binding adaptor molecule (Iba1) antibody as a specific marker of microglial cells. Neurochemical analysis showed transiently increased Iba1 protein levels in the brains of developing Sprague-Dawley (SD) rats. However, high Iba1 protein readings continued in aged zitter rats. Immunohistochemical analysis revealed time-course differences in the transformation of microglia between SD and zitter rats and prolonged activation of microglial cells in the zitter rat. In the zitter rat, activated microglial cells characterized by swollen cell bodies and shorter, thicker processes were distributed throughout the brain from 2-weeks- to 2-months-old. After 2-months-old, numbers of activated microglial cells gradually decreased. However, these cells were not observed in SD rats. Iba1-immunoreactive cell-clusters organized by at least five activated microglial cells were also prominent in the zitter brain. These differences reflect the neuropathology of this mutant rat triggered by deletion of the attractin gene. The present data may thus suggest that microglial cells directly or indirectly contribute to progressive spongy degeneration in zitter mutant rats. (c) 2006 Elsevier Ireland Ltd. All rights reserved.


  • Progressive dopaminergic neurodegeneration of substantia nigra in the zitter mutant rat

    K Nakadate, T Noda, S Sakakibara, K Kumamoto, T Matsuura, JN Joyce, S Ueda

    ACTA NEUROPATHOLOGICA   112 ( 1 ) 64 - 73  2006.07  [Refereed]

     View Summary

    Zitter mutant rats exhibit abnormal metabolism of superoxide species and demonstrate progressive degeneration of dopamine (DA) neurons in the substantia nigra (SN). Furthermore, long-term intake of vitamin E, an effective free radical scavenger, prevents the loss of DA neurons caused by free radicals. However, it is unclear how this degeneration progresses. In this study, we ultrastructurally examined cell death in the zitter mutant rat SN. Conventional electron-microscopic examination revealed two different types of neurons in the SN pars compacta. In zitter mutant rats, although the first type (clear neurons) exhibited no obvious ultrastructural changes with aging, the second type (dark neurons) demonstrated age-related damage from 2 months. Immunoelectron-microscopic analysis clarified that the second-type neurons were dopaminergic neurons. In the dopaminergic neuronal somata, many lipofuscin granules and abnormal endoplasmic reticula were observed from 2 months of age, and these dopaminergic neurons showed progressive degeneration with age. Moreover, in zitter mutant rats, abnormally enlarged myelinated axons with dense bodies and splitting myelin with dense material were observed in the SN at 2, 4, and 12 months, and oligodendrocytes with numerous lipofuscin, multivesicular bodies, multilamellar bodies, and dense bodies were frequently observed at 4 and 12 months. These findings clarified that dopaminergic neurons in zitter mutant rats had degenerated with age, and that myelinated axons also exhibited age-related injury. Moreover, ubiquitin-immunohistochemical analysis indicated that the accumulation of products of the endosomal-lysosomal system may be involved in this degeneration.


  • In vivo and in vitro analysis of Musashi family in mammalian CNS and PNS development

    Shibata, Shinsuke, Sakakibara, Shin-ichi, Okano, Hirotaka J, Okano, Hideyuki

    NEUROSCIENCE RESEARCH   55   S182 - S182  2006

  • Degeneration of dopaminergic neurons in the substantia nigra of zitter mutant rat and protection by chronic intake of Vitamin E

    S Ueda, A Sakakibara, K Nakadate, T Noda, M Shinoda, JN Joyce

    NEUROSCIENCE LETTERS   380 ( 3 ) 252 - 256  2005.06  [Refereed]

     View Summary

    Dopaminergic cell death in the ventral and dorsal tiers of substantia nigra pars copmacta (SNc) and their prevention by anti-oxidant diet was immunohistochemically studied in the zitter mutant rats, which are characterized by abnormal metabolism of superoxide. Similar to previous reports, the number of SNc neurons in Nissl-stained section decreased with age. Tyrosine hydroxylase (TH) immunohistochemistry demonstrated that the dopaminergic neurons in the ventral tier of SNc degenerated early, whereas the dorsal tier gradually degenerated with age. Thus, the ventral tier dopaminergic neurons are affected first, but the dorsal tier neurons do become impact by the zi/zi mutation. Following 9-month period after weaning, zitter rats supplemented with 500 mg D,L-alpha-tocophenol (VE(+))/kg diet exhibited a significant increased of surviving TH-immunoreactive neurons in both the tiers of SNc as compared with the zi/zi rats with control and VE(-) diets. These results suggest that VE supplement may slow the dopaminergic cell loss in zitter mutant rat, and further support that degeneration of the dopaminergic neurons in this mutant rat is caused by oxidant stress. Thus, the zitter rat may represent a good model for studying the dopaminergic cell death by superoxide species. (c) 2004 Elsevier Ireland Ltd. All rights reserved.


  • zitter(zi/zi)ラット海馬における苔状線維の加齢変化

    野田 隆洋, 中舘 和彦, 榊原 伸一, 上田 秀一

    解剖学雑誌   80 ( Suppl. ) 234 - 234  2005.03

  • 成熟ラット中枢神経系におけるAttractinの分布

    中舘 和彦, 榊原 伸一, 野田 隆洋, 上田 秀一

    解剖学雑誌   80 ( Suppl. ) 236 - 236  2005.03

  • 成熟ラット中枢神経系におけるAttractinの分布

    中舘 和彦, 榊原 伸一, 野田 隆洋, 上田 秀一

    解剖学雑誌   80 ( Suppl. ) 260 - 260  2005.03

  • The RNA-binding protein HuD regulates neuronal cell identity and maturation

    W Akamatsu, H Fujihara, T Mitsuhashi, M Yano, S Shibata, Y Hayakawa, HJ Okano, S Sakakibara, H Takano, T Takano, T Takahashi, T Noda, H Okano


     View Summary

    Neural Hu proteins (HuB/C/D) are RNA-binding proteins that have been shown to induce neuronal differentiation activity when overexpressed in immature neural progenitor cells or undifferentiated neuronal tumors. Newly generated HuD-deficient mice exhibited a transient impaired-cranial-nerve-development phenotype at an early embryonic stage. Adult HuD-deficient mice exhibited an abnormal hind-limb reflex and poor rotarod performance. Analysis of neurosphere formation revealed that the number and self-renewal capacity of the neural stem/progenitor cells were increased in HuD-deficient mice. HuD-deficient primary neurospheres also generated a smaller number of neurons. Cohort analysis of the cellular proliferative activity by using BrdUrd and iododeoxuridine labeling revealed that the number of differentiating quiescent cells in the embryonic cerebral wall was decreased. Long-term administration of BrdUrd revealed that the number of slowly dividing stem cells in the adult subventricular zone was increased in the HuD-deficient mice. Taken together, the results suggest that HuD is required at multiple points during neuronal development, including negative regulation of proliferative activity and neuronal cell-fate acquisition of neural stem/progenitor cells.


  • Effect of long-lasting serotonin depletion on environmental enrichment-induced neurogenesis in adult rat hippocampus and spatial learning

    S Ueda, S Sakakibara, K Yoshimoto

    NEUROSCIENCE   135 ( 2 ) 395 - 402  2005  [Refereed]

     View Summary

    The dentate gyrus of the hippocampal formation produces new neurons throughout adulthood in mammalian species. Several experimental statuses and factors regulating to neurogenesis have been identified in the adult dentate gyrus. For example, exposure to an enriched environment enhances neurogenesis in the dentate gyrus and improves hippocampus-dependent spatial learning. Furthermore, serotonin is known to influence adult neurogenesis, and learning and memory. However, the effects of long-lasting depletion of serotonin over the developing period on neurogenesis have not been investigated. Thus, we examined the influence of long-lasting serotonin depletion on environmental enrichment-induced neurogenesis and spatial memory performance. As reported previously, environmental enrichment significantly increased new neurons in the dentate gyrus. However, there was no improvement of the spatial learning test in adult rats in standard and in environmental enrichment housings. Intracisternal administration of the serotonergic neurotoxin, 5,7-dihydroxytryptamine, on postnatal day 3 apparently reduced serotonin content in the adult hippocampus without regeneration. This experimental depletion of serotonin in the hippocampus of rats housed in an enriched environment had no effect on spatial memory performance, but produced significant decreases in the number of bromodeoxyuridine-labeled new cells in the dentate gyrus. These findings indicate that newly generated cells stimulated by environmental enrichment are not critical for improvements in hippocampus-dependent learning. Furthermore, numbers of bromodeoxyuridine-labeled cells in the dentate gyrus of 5,7-dihydroxytryptamine-injected rats did not differ between 1 day and 4 weeks after bromodeoxyuridine injection. These data suggest that survival of newly generated dentate gyrus cells remains relatively constant under long-lasting serotonin depletion. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.


  • ラットCalleja島の発生

    野田 隆洋, 榊原 伸一, 中舘 和彦, 岡 敦子, 上田 秀一

    解剖学雑誌   79 ( 3 ) 103 - 103  2004.09

  • 【子どもの心のケア 温かく育むために】心の科学 キレる脳 虐待と攻撃性 実験動物からのアプローチ

    上田 秀一, 中舘 和彦, 野田 隆洋, 榊原 伸一

    小児科臨床   57 ( 増刊 ) 1257 - 1264  2004.07

  • Loss of D-3 receptors in the zitter mutant rat is not reversed by L-dopa treatment

    JN Joyce, TC Der, L Renish, T Osredkar, D Hagner, M Reploge, S Sakakibara, S Ueda

    EXPERIMENTAL NEUROLOGY   187 ( 1 ) 178 - 189  2004.05  [Refereed]

     View Summary

    In Parkinson's disease (PD) and animal models of parkinsonism the destruction of nigrostriatal (NSB) system results in a marked loss of the dopamine D-3 receptor and mRNA in the islands of Calleja (ICj) and the nucleus accumbens shell (NAS). In animal models, it has been reported that both measures are elevated by repeated intermittent administration Of L-dopa. However, a large proportion of PD cases are resistant to L-dopa-induced elevation of D-3 receptor number. The zitter mutant (Zi/Zi) rat replicates the slow progressive degeneration of the NSB observed in PD and also exhibits a loss of D-3 receptor number in the NAS or ICj. To test if this could be reversed with subchronic L-dopa treatment, injections of carbidopa (10 mg/kg ip) were followed an hour later with injection of L-dopa (100 mg/kg ip) twice a day for 10 days. In control Sprague-Dawley (SD) and zitter heterozygote (Zi/-) rats that do not show a loss of D-3 receptors with vehicle treatment, L-dopa produced no change in D-3 receptor number or in DA terminal density as measured by dopamine transporter (DAT) binding and tyrosine hydroxylase immunoautoradiography (TH-IR). There was a marked loss of DAT and TH-IR in caudate-putamen (CPU) and NA, as well as D-3 receptors in NAS and ICj in Zi/Zi rats but no further change with L-dopa treatment. To determine if the resistance to L-dopa-induced increase in D-3 receptor was due to a deficiency in expression of cortical BDNF or its receptor, TrkB, in CPU and NAS, we examined BDNF mRNA by ISHH in frontal cortex and TrkB mRNA in frontal cortex, CPU, and NA. The loss of the NSB in the Zi/Zi did not alter levels of BDNF or TrkB mRNA, nor did L-dopa administration alter levels BDNF or TrkB mRNA. Thus, unlike in 6-hydroxydopamine-treated rats, in Zi/Zi rats administered L-dopa does not reverse the loss of BDNF mRNA or lead to an elevation of D-3 receptor number. (C) 2004 Elsevier Inc. All rights reserved.


  • Thyroid hormone-upregulated expression of Musashi-1 is specific for progenitor cells of the adult epithelium during amphibian gastrointestinal remodeling

    A Ishizuya-Oka, K Shimizu, S Sakakibara, H Okano, S Ueda

    JOURNAL OF CELL SCIENCE   116 ( 15 ) 3157 - 3164  2003.08  [Refereed]

     View Summary

    In the amphibian gastrointestine during metamorphosis, the primary (larval) epithelium undergoes apoptosis. By contrast, a small number of undifferentiated cells including stem cells actively proliferate and differentiate into the secondary (adult) epithelium that resembles the mammalian counterpart. In the present study, to clarify whether Musashi-1 (Msi-1), an RNA-binding protein, serves as a marker for progenitor cells of the adult epithelium, we chronologically examined Msi-1 expression in the Xenopus laevis gastrointestine by using in situ hybridization and immunohistochemistry. Similar expression profiles of Msi-1 were observed at both mRNA and protein levels. In both the small intestine and the stomach, the transient expression of Msi-1 during metamorphosis spatio-temporally correlated well with active proliferation of the progenitor cells including stem cells of the adult epithelium but did not with apoptosis of the larval epithelium. As the adult progenitor cells differentiated into organ-specific epithelial cells after active proliferation, Msi-1 expression was rapidly downregulated. Therefore, Msi-1 is useful to identify the adult progenitor cells that actively proliferate before final differentiation in the amphibian gastrointestine. Furthermore, our culture experiments have shown that thyroid hormone (TH) organ-autonomously induces Msi-1 expression only in the adult progenitor cells of the X. laevis intestine in vitro as in vivo. However, TH could not induce Msi-1 expression in the intestinal epithelium separated from the connective tissue, where the adult epithelium never developed. These results suggest that Msi-1 expression is upregulated by TH in the adult progenitor cells under the control of the connective tissue and plays important roles in their maintenance and/or active proliferation during amphibian gastrointestinal remodeling.

  • Identification of a putative intestinal stem cell and early lineage marker; Musashi-1

    CS Potten, C Booth, GL Tudor, D Booth, G Brady, P Hurley, G Ashton, R Clarke, S Sakakibara, H Okano

    DIFFERENTIATION   71 ( 1 ) 28 - 41  2003.01  [Refereed]

     View Summary

    There are few reliable markers for adult stem cells and none for those of the intestinal epithelium. Previously, indirect experimental approaches have predicted stem cell position and numbers. The Musashi-1 (Msi-1) gene encodes an RNA binding protein associated with asymmetric divisions in neural progenitor cells. Two-day-old, adult, and 4.5 h, 1-, 2-, 4- and 12-day postirradiation samples of 1313171 mouse small intestine, together with some samples of mouse colon were stained with a rat monoclonal antibody to Musashi-1 (14 H-1). Min ( + / -) mice with small intestinal adenomas of varying sizes were also analysed. Samples of human small and large bowel were also studied but the antibody staining was weak. Musashi-1 expression was observed using immunohistochemistry in neonatal, adult, and regenerating crypts with a staining pattern consistent with the predicted number and distribution of early lineage cells including the functional stein cells in these situations. Early dysplastic crypts and adenomas were also strongly Musashi-1 positive. In situ hybridization studies showed similar expression patterns for the Musashi mRNA and real-time quantitative RT-PCR showed dramatically more Msi-1 mRNA expression in Min tumours compared with adjacent normal tissue. These observations suggest that Musashi-1 is a marker of stem and early lineage progenitor cells in murine intestinal tissue.

  • RNA-binding protein Musashi family: Roles for CNS stem cells and a subpopulation of ependymal cells revealed by targeted disruption and antisense ablation

    S Sakakibara, Y Nakamura, T Yoshida, S Shibata, M Koike, H Takano, S Ueda, Y Uchiyama, T Noda, H Okano


     View Summary

    Homologues of the Musashi family of RNA-binding proteins are evolutionarily conserved across species. in mammals, two members of this family, Musashi1 (Msi1) and Musashi2 (Msi2), are strongly coexpressed in neural precursor cells, including CNS stem cells. To address the in vivo roles of msi in neural development, we generated mice with a targeted disruption of the gene encoding Will. Homozygous newborn mice frequently developed obstructive hydrocephalus with aberrant proliferation of ependymal cells in a restricted area surrounding the Sylvius aqueduct. These observations indicate a vital role for msi1 in the normal development of this subpopulation of ependymal cells, which has been speculated to be a source of postnatal CNS stem cells. On the other hand, histological examination and an in vitro neurosphere assay showed that neither the embryonic CNS development nor the self-renewal activity of CNS stem cells in embryonic forebrains appeared to be affected by the disruption of msi1, but the diversity of the cell types produced by the stem cells was moderately reduced by the msil deficiency. Therefore, we performed antisense ablation experiments to target both msil and msi2 in embryonic neural precursor cells. Administration of the antisense peptide-nucleotides, which were designed to specifically down-regulate msi2 expression, to msi1(-/-) CNS stem cell cultures drastically suppressed the formation of neurospheres in a dose-dependent manner. Anti sense-treated msi1(-/-) CNS stem cells showed a reduced proliferative activity. These data suggest that msil and msi2 are cooperatively involved in the proliferation and maintenance of CNS stem cell populations.


  • RNA binding protein Musashi1 is expressed in Sertoli cells in the rat testis from fetal life to adulthood

    PTK Saunders, SM Maguire, S Macpherson, MC Fenelon, S Sakakibara, H Okano

    BIOLOGY OF REPRODUCTION   66 ( 2 ) 500 - 507  2002.02  [Refereed]

     View Summary

    The Musashi1 (Msi1) gene identified in mouse is a member of a subfamily of RNA binding proteins that are highly conserved across species. Msi1 expression is highly enriched in proliferative cells within the developing central nervous system. Within the testis, proliferation and differentiation of germ cells takes place within the seminiferous epithelium, where these cells are supported physically and functionally by Sertoli cells that do not themselves proliferate following the onset of puberty. RNA binding proteins expressed in testicular germ cells are essential for normal fertility. Preliminary data suggested the mRNA for Msi1 was present in ovary; therefore, we used an Msi1-specific cRNA and monoclonal antibody to investigate whether Msi1 was expressed in the testis. Msi1 mRNA was expressed in rat testis from birth until adulthood; in situ hybridization revealed. silver grains within the seminiferous epithelium. immunohistochemical. studies demonstrated that at all ages examined (from Fetal Day 14.5 until adulthood) Msi1 protein was expressed in Sertoli cells. In fetal and adult rat ovaries, Msi1 was detected in granulosa cells and their precursors. In Sertoli cells, protein was detected in both cytoplasmic and nuclear compartments; in adult testes, the immunointensity of the nuclear staining was stage dependent, with highest levels of expression in Sertoli cells at stages I-VI. In rat gonads, the RNA binding protein Msi1 is expressed in both proliferating and nonproliferating Sertoli and granulosa cells.

  • RNA-binding protein Musashi2: developmentally regulated expression in neural precursor cells and subpopulations of neurons in mammalian CNS

    S Sakakibara, Y Nakamura, H Satoh, H Okano

    JOURNAL OF NEUROSCIENCE   21 ( 20 ) 8091 - 8107  2001.10  [Refereed]

     View Summary

    Musashi1 (Msi1) is a mammalian neural RNA-binding protein highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic and postnatal CNS development. Here, we identified Musashi2 (Msi2), a novel mammalian RNA-binding protein that exhibits high sequence similarity to Msi1. The Msi2 transcript appeared to be distributed ubiquitously in a wide variety of tissues, consistent with the mRNA distribution of its Xenopus homolog, xrp1. However, the present study revealed cell type-specific and developmentally regulated expression of Msi2 in the mammalian CNS. Interestingly, Msi2 was expressed prominently in precursor cells in the ventricular zone and subventricular zone with the same pattern as Msi1 throughout CNS development. In the postnatal and adult CNS, this concurrent expression of Msi2 and Msi1 was seen in cells of the astrocyte lineage, including ependymal cells, a possible source for postnatal CNS stem cells. During neurogenesis, the expression of both Msi2 and Msi1 was lost in most postmitotic neurons, whereas Msi2 expression persisted in a subset of neuronal lineage cells, such as parvalbumin-containing GABA neurons in the neocortex and neurons in several nuclei of the basal ganglia. Msi2 may have a unique role that is required for the generation and/or maintenance of specific neuronal lineages. Furthermore, in vitro studies showed that Msi2 and Msi1 have similar RNA-binding specificity. These two RNA-binding proteins may exert common functions in neural precursor cells by regulating gene expression at the post-transcriptional level.

  • Musashi1, an evolutionarily conserved neural RNA-binding protein, is a versatile marker of human glioma cells in determining their cellular origin, malignancy, and proliferative activity

    Y Kanemura, K Mori, S Sakakibara, H Fujikawa, H Hayashi, A Nakano, T Matsumoto, K Tamura, T Imai, T Ohnishi, S Fushiki, Y Nakamura, M Yamasaki, H Okano, N Arita

    DIFFERENTIATION   68 ( 2-3 ) 141 - 152  2001.09  [Refereed]

     View Summary

    Tumor cells often express phenotypic markers that are specific to the cells from which they originated. A neural RNA-binding protein, Musashil, is an evolutionarily well-conserved marker for neural stem cells/progenitor cells. To examine the origin of gliomas, we examined the expression of the human Musashil homolog, MSI1, in human glioma tissues and in normal human adult and fetal brains. As we had seen previously in rodents, in the normal human brain, MSI1 was expressed in cells located in the ventricular and subventricular zones, in GFAP-negative glial cells, and in GFAP-positive astrocytes. In glioblastomas, MSI1 was expressed in GFAP-negative tumor cells forming foci that were clearly demarcated and surrounded by GFAP-positive cells. Tumor cells arranged in pseudopalisades were also strongly immunoreactive with MSI1 antibodies. The percentage of MSI1-labeled tumor cells increased in higher-grade astrocytomas and correlated with proliferative activity, as estimated by an MIB-1 staining index. Our results indicate that MSI1 is an excellent marker for neural progenitor cells including neural stem cells in normal human brains. Furthermore, the expression of MSI1 correlates well with the immature nature as well as the malignancy of tumor cells in human gliomas. Thus, we expect the analysis of MSI1 expression to contribute to the understanding of the cellular origin and biology of human gliomas.

  • Activation of murine cytomegalovirus immediate-early promoter in cerebral ventricular zone and glial progenitor cells in transgenic mice

    RY Li, S Baba, Kosugi, I, Y Arai, H Kawasaki, Y Shinmura, S Sakakibara, H Okano, Y Tsutsui

    GLIA   35 ( 1 ) 41 - 52  2001.07  [Refereed]

     View Summary

    Cytomegalovirus (CMV) is the most common infectious cause of congenital anomalies of the CNS in humans. We recently reported that the murine cytomegalovirus (MCMV) immediate-early (IE) gene promoter directs astrocyte-specific expression in adult transgenic mice. In the present study, we analyzed the activation of the MCMV IE promoter in developing transgenic mouse brains and compared the activation with that of the Musashi 1 (Msi1) gene, which is expressed in neural progenitor cells, including neural stem cells. During the early phase of neurogenesis, the transgene was expressed predominantly in endothelial cells of the vessels, but not in neuroepithelial cells in which Msi1 was expressed. During later stages of gestation, expression of the transgene was largely restricted to the ventricular zone (VZ) in the CNS, similar to the expression of Msi1. In neurosphere cultures from transgenic embryos in the late phase of neurogenesis, the transgene was expressed in some cells of neurospheres expressing Msi1 and nestin. In neural precursor cells induced to differentiate from stem cells, expression of the transgene was detected in glial progenitor cells, expressing GFAP, nestin, and Msi1, but not in cells expressing MAP2 or MAG. In postnatal development, persistent expression of the transgene was observed in astrocyte lineage cells as was Msi1. These spatiotemporal changes of the MCMV LE promoter activity during development of transgenic mice correlated with susceptible sites in congenital HCMV infection. Moreover, this transgenic mouse model may provide useful model for analysis of the regulation of the switching of neuronal and astrocyte differentiation, and the maintenance of the astrocyte lineage. GLIA 35:41-52, 2001. (C) 2001 Wiley-Liss, Inc.

  • Expression of mouse igf2 mRNA-binding protein 3 and its implications for the developing central nervous system

    H Mori, S Sakakibara, T Imai, Y Nakamura, T Iijima, A Suzuki, Y Yuasa, M Takeda, H Okano

    JOURNAL OF NEUROSCIENCE RESEARCH   64 ( 2 ) 132 - 143  2001.04  [Refereed]

     View Summary

    Functional analyses of neural RNA-binding proteins have focused mainly on their roles as modulators of posttranscriptional gene regulation, e.g., alternative splicing, dendritic mRNA localization, and local translation. Here we identified a mouse homologue of human IMP3, which is known to bind to and repress the translation of igf2 leader 3 mRNA. The mouse igf2 mRNA-binding protein 3 (mIMP3) is a member of the zipcode binding protein-1 (ZBP-1) family previously reported in chick fibroblast cells, mIMP3 was expressed in undifferentiated neuroepithelial cells and some postmitotic neurons at early embryonic stages (E10.5-E12.5), and its expression level decreased after the midembryonic stage (E12.5) until birth. The expression profile of mIMP3 is very similar to that of mouse igf2 leader 3 mRNA. In vitro UV crosslinking experiments showed that mIMP3 preferentially bound to igf2 leader 3 mRNA rather than igf2 leader 4 mRNA and did not bind the zipcode region of p-actin or c-myc mRNA, Furthermore, persistent expression of mIMP3 protein in an undifferentiated P19 cell line revealed that mIMP3 inhibited neuronal differentiation morphologically and immunohistochemically. Taken together, these observations raise the possibility that mIMP3 represses neuronal differentiation through the regulation of igf2 mRNA expression. J, Neurosci, Res. 64, 132-143, 2001. (C) 2001 Wiley-Liss, Inc.

  • The bHLH gene Hes1 as a repressor of the neuronal commitment of CNS stem cells

    Y Nakamura, S Sakakibara, T Miyata, M Ogawa, T Shimazaki, S Weiss, R Kageyama, H Okano

    JOURNAL OF NEUROSCIENCE   20 ( 1 ) 283 - 293  2000.01  [Refereed]

     View Summary

    Hes1 is one of the basic helix-loop-helix transcription factors that regulate mammalian CNS development, and its loss- and gain-of-function phenotypes indicate that it negatively regulates neuronal differentiation.
    Here we report that Hes1(-/-) mice expressed both early (TuJ1 and Hu) and late (MAP2 and Neurofilament) neuronal markers prematurely, and that there were approximately twice the normal number of neurons in the Hes1(-/-) brain during early neural development. However, immunochemical analyses of sections and dissociated cells using neural progenitor markers, including nestin, failed to detect any changes in Hes1(-/-) progenitor population. Therefore, further characterization of neural progenitor cells that discriminated between multipotent and monopotent cells was performed using two culture methods, low-density culture, and a neurosphere assay. We demonstrate that the self-renewal activity of multipotent progenitor cells was reduced in the Hes1(-/-) brain, and that their subsequent commitment to the neuronal lineage was accelerated. The Hes1(-/-) neuronal progenitor cells were functionally abnormal, in that they divided, on average, only once, and then generated two neurons, (instead of one progenitor cell and one neuron), whereas wild-type progenitor cells divided more. In addition, some Hes1(-/-) progenitors followed an apoptotic fate. The overproduction of neurons in the early Hes1(-/-) brains may reflect this premature and immediate generation of neurons as well as a net increase in the number of neuronal progenitor cells.
    Taken together, we conclude that Hes1 is important for maintaining the self-renewing ability of progenitors and for repressing the commitment of multipotent progenitor cells to a neuronal fate, which is critical for the correct number of neurons to be produced and for the establishment of normal neuronal function.

  • Musashi1: An evolutionally conserved marker for CNS progenitor cells including neural stem cells

    Y Kaneko, S Sakakibara, T Imai, A Suzuki, Y Nakamura, K Sawamoto, Y Ogawa, Y Toyama, T Miyata, H Okano

    DEVELOPMENTAL NEUROSCIENCE   22 ( 1-2 ) 139 - 153  2000.01  [Refereed]

     View Summary

    In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopus proteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in nostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form 'neurospheres' in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(-) cells, which included T alpha 1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(-). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ. Copyright (C) 2000 S. Karger AG, Basel.

  • Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems

    W Akamatsu, HJ Okano, N Osumi, T Inoue, S Nakamura, SI Sakakibara, M Miura, N Matsuo, RB Darnell, H Okano


     View Summary

    Hu proteins are mammalian embryonic lethal abnormal visual system (ELAV)-like neuronal RNA-binding proteins that contain three RNA recognition motifs, Although Drosophila ELAV is required for the correct differentiation and survival of neurons, the roles played by the Hu genes in the mammalian nervous system remain largely unknown, To explore the in vivo functions of mouse Hu proteins, we overexpressed them in rat pheochromocytoma PC12 cells, where they induced neuronal phenotype in the absence of nerve growth factor, We have characterized the functions of various forms of mHuB and mHuC bearing point mutations or deletions. Mutants of mHuC that had amino acid exchanges in the RNP1 domain of the first or second RNA recognition motifs (RRMs) lost biologic activity as well as RNA-binding activity. In addition, the mutants containing only the third RRM failed to induce the neuronal phenotype in PC12 cells and inhibited the biologic activity of cotransfected wild-type mHuB and mHuC, thus acting as a dominant-negative form. However, these mutants could not suppress the nerve growth factor-induced differentiation of PC12 cells. Further, we misexpressed wild-type and dominant-negative Hu in E9.5 mouse embryos, by using electroporation into the neural tube at the level of the rhombencephalon. mHuB and mHuC induced the ectopic expression of neuronal markers, whereas the dominant-negative forms of mHuB and mHuC suppressed the differentiation of central nervous system motor neurons, From these results, we suggest that Hu proteins are required for neuronal differentiation in the mammalian nervous system.

  • Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1

    T Nagata, R Kanno, Y Kurihara, S Uesugi, T Imai, S Sakakibara, H Okano, M Katahira

    JOURNAL OF MOLECULAR BIOLOGY   287 ( 2 ) 315 - 330  1999.03  [Refereed]

     View Summary

    Musashil is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashil contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashil by NMR. It folds into a compact alpha beta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a beta-bulge in beta 2 and a shallow twist of the beta-sheet. The smaller H-1-N-15 nuclear Overhauser enhancement values for the residues of loop 3 between beta 2 and beta 3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller N-15 T-2 values for the residues around the border between alpha 2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded P-sheet, loops and C-terminal region are involved in the interaction. The appearance of sidechain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of beta 2 and beta 3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2. (C) 1999 Academic Press.

  • The human Musashi homolog 1 (MSI1) gene encoding the homologue of Musashi/Nrp-1, a neural RNA-binding protein putatively expressed in CNS stem cells and neural progenitor cells

    P Good, A Yoda, S Sakakibara, A Yamamoto, T Imai, H Sawa, T Ikeuchi, S Tsuji, H Satoh, H Okano

    GENOMICS   52 ( 3 ) 382 - 384  1998.09  [Refereed]

  • Fibroblast growth factor-2 brain-derived neurotrophic factor-associated maturation of new neurons generated from adult human subependymal cells

    DW Pincus, HM Keyoung, C Harrison-Restelli, RR Goodman, RAR Fraser, M Edgar, S Sakakibara, H Okano, M Nedergaard, SA Goldman

    ANNALS OF NEUROLOGY   43 ( 5 ) 576 - 585  1998.05  [Refereed]

     View Summary

    The adult mammalian forebrain harbors neuronal precursor cells in the subependymal zone (SZ). Neuronal progenitors also persist in the adult human SZ and have been cultured from epileptic temporal lobe. In the present study, we sought to identify these neural progenitors in situ, and to direct their expansion and neuronal differentiation in vitro. We prepared explants of adult human SZ, obtained from temporal lobe resections of refractory epileptics. The resultant cultures were treated with fibroblast growth factor-2 (FGF-2) for a week, with concurrent exposure to [H-3]thymidine, then switched to media containing brain-derived neurotrophic factor (BDNF) for up to 2 months. Sporadic neuronal outgrowth, verified antigenically and physiologically, was observed from SZ cultures regardless of FGF-2/BDNF treatment; however, only FGF-2/BDNF-treated cultures exhibited profuse outgrowth, and these displayed neuronal survival as long as 9 weeks in vitro. In addition, cortical cultures derived from two brains generated microtubule-associated protein-2(+) neurons, which incorporated [H-3]thymidine and exhibited significant calcium increments to depolarization. In histological sections of the subependyma, both uncommitted and restricted progenitors, defined respectively by musashi and Hu protein expression, were identified. Thus, the adult human subependyma harbors neural progenitors, which are able to give rise to neurons whose numbers can be supported for prolonged periods in vitro.

  • Expression of neural RNA-binding proteins in the postnatal CNS: Implications of their roles in neuronal and glial cell development

    S Sakakibara, H Okano

    JOURNAL OF NEUROSCIENCE   17 ( 21 ) 8300 - 8312  1997.11  [Refereed]

     View Summary

    There is an increasing interest in the role of RNA-binding proteins during neural development. Mouse-Musashi-1 (m-Msi-1) is a mouse neural RNA-binding protein with sequence similarity to Drosophila musashi (d-msi), which is essential for neural development. m-Msi-1 is highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic CNS development. The present study characterized m-Msi-1-expressing cells in the postnatal and adult CNS. Postnatally, m-Msi-1 was expressed in proliferative neuronal precursors in the external granule cell layer of the cerebellum and in the anterior corner of the subventricular zone of the lateral ventricles. In gliogenesis, the persistent expression of m-Msi-1 was observed in cells of the astrocyte lineage ranging from proliferative glial precursors in the subventricular zone (SVZ) to differentiated astrocytes in the parenchyma. In addition, we showed that m-Msi-1 was still expressed in proliferating cells in the adult SVZ, which may contain neural precursor or stem cells. Another neural RNA-binding protein Hu (the mammalian homolog of a Drosophila neuronal RNA-binding protein Elav) was present in postmitotic neurons throughout the development of the CNS, and its pattern of expression was compared with that of m-Msi-1. These observations imply that these two RNA-binding proteins may be involved in the development of neurons and glia by regulating gene expression at the post-transcriptional level.

  • Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1

    Y Kurihara, T Nagata, T Imai, A Hiwatashi, M Horiuchi, S Sakakibara, M Katahira, H Okano, S Uesugi

    GENE   186 ( 1 ) 21 - 27  1997.02  [Refereed]

     View Summary

    mouse-Musashi-1 (m-Msi-1) is an RNA-binding protein, abundantly expressed in the developing mammalian central nervous system (CNS). m-Msi-1 contains two RNA recognition motifs (RRMs). In this study, we found that the N-terminal RRM of m-Msi-1 (MMA) binds strongly to poly(G) and weakly to poly(U) in a way similar to that of the full-length m-Msi-1 protein characterized previously. The C-terminal RRM of m-Msi-1 (MMB), however, does not bind to RNA. In addition, the circular dichroism (CD) spectra of the two RRMs showed that the alpha-helical content of MMA is significantly higher than that of MMB, indicating that some differences in the secondary structure may be responsible for the distinct RNA binding properties of MMA and MMB.

  • Intrinsic and extrinsic determinants regulating cell fate decision in developing nervous system

    M Okabe, K Sawamoto, T Imai, S Sakakibara, S Yoshikawa, H Okano

    DEVELOPMENTAL NEUROSCIENCE   19 ( 1 ) 9 - 16  1997.01  [Refereed]  [Invited]

     View Summary

    Cell fate in the nervous system is determined by environmental signals and/or heredity. They can be referred to as extrinsic and intrinsic determinants, respectively. The molecular and cellular mechanisms underlying these processes are well conserved from invertebrates to vertebrates. We have identified both types of determinants through the intensive screening of P-element-induced Drosophila mutants affected in neural development. As a member of the first category, argos gene product which regulates cell-cell interaction in the developing nervous system gives a good example. As a member of the second category, we refer to Drosophila musashi gene encoding a neural RNA-binding protein. The functions of these gene products and application to the mammalian nervous system will be discussed.

  • Mouse-Musashi-1, a neural RNA-Binding protein highly enriched in the mammalian CNS stem cell

    S Sakakibara, T Imai, K Hamaguchi, M Okabe, J Aruga, K Nakajima, D Yasutomi, T Nagata, Y Kurihara, S Uesugi, T Miyata, M Ogawa, K Mikoshiba, H Okano

    DEVELOPMENTAL BIOLOGY   176 ( 2 ) 230 - 242  1996.06  [Refereed]

     View Summary

    There is increasing interest in the role of RNA-binding proteins during neural development. Drosophila Musashi is one of the neural RNA-binding proteins essential for neural development and required for asymmetric cell divisions in the Drosophila adult sensory organ development. Here, a novel mammalian neural RNA-binding protein, mouse-Musashi-1, was identified based on the homology to Drosophila Musashi and Xenopus NRP-1. In the developing CNS, mouse-Musashi-1 protein was highly enriched in the CNS stem cell. Single-cell culture experiments indicated that mouse-Musashi-1 expression is associated with neural precursor cells that are capable of generating neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1 expression is lost. This expression pattern of mouse-Musashi-1 is complementary to that of another mammalian neural RNA-binding protein, Hu (a mammalian homologue of a Drosophila neuronal RNA-binding protein Elav), that is expressed in postmitotic neurons within the CNS. In vitro studies indicated that mouse-Musashi-1 possesses binding preferences on poly(G) RNA homopolymer, whereas Hu is known to preferentially bind to short A/U-rich regions in RNA. Based on their differential expression patterns and distinct preferential target RNA sequences, we believe that the mouse-Musashi-1 and Hu proteins may play distinct roles in neurogenesis, either through sequential regulatory mechanisms or differential sorting of mRNA populations during asymmetric division of neural precursor cells. (C) 1996 Academic Press, Inc.

  • Ataxia and epileptic seizures in mice lacking type 1 inositol 1,4,5-trisphosphate receptor

    M Matsumoto, T Nakagawa, T Inoue, E Nagata, K Tanaka, H Takano, O Minowa, J Kuno, S Sakakibara, M Yamada, H Yoneshima, A Miyawaki, Y Fukuuchi, T Furuichi, H Okano, K Mikoshiba, T Noda

    NATURE   379 ( 6561 ) 168 - 171  1996.01  [Refereed]

     View Summary

    THE inositol 1,4,5-trisphosphate (InsP(3)) receptor acts as an InsP(3)-gated Ca2+ release channel in a variety of cell types(1,2). Type 1 InsP(3) receptor (IP(3)R1) is the major neuronal member of the IP(3)R family in the central nervous system(3,4), predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex(5,6). Here we report that most IP(3)R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP(3)R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP(3)R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP(3)R1-deficient mice were not severely impaired.

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Research Projects

  • 樹状突起スパインの形成・安定性を担う新たなアクチン骨格制御機構の解明

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     View Summary

    正常な脳発達においてニューロンの樹状突起スパインの数や形態のコントロールは神経可塑性の獲得・維持に重要であり,精神発達障害や幾つかの精神疾患ではスパイン密度の変化や異常な形態変化が起きる。inka2遺伝子は脳に強く発現し細胞骨格再編を通してニューロン突起形成を促進する。本研究ではinka2の発現の異常が、遺伝性の精神発達障害・自閉症である脆弱X症候群(FXS)の病態に関係する可能性を検討する。正常な脳発達においてニューロンの樹状突起スパインの数や形態のコントロールは神経可塑性の獲得・維持に重要であり、精神発達障害や幾つかの精神疾患ではスパイン密度の変化や異常な形態変化が起きる。Inka2は神経系前駆細胞に強く発現する新規遺伝子であり、細胞内のアクチン骨格再編成を促進し、細胞のフィロポディア形成を促進する働きがあると考えられる。生後のシナプス形成期のニューロンでinka2 mRNA発現は急速に上昇し、成体では海馬や大脳皮質などの前脳領域のニューロンに限局して強いmRNA発現が観察されるが、タンパク質への翻訳は抑制されていることが示唆された。本研究課題ではスパイン形成におけるinka2の役割の分子レベルでの解明を目指す。inka2 KOマウスの大脳皮質をゴルジ染色により組織学的に解析した結果、生後での樹状突起スパインの密度低下、特に成熟型マッシュルーム型スパインが顕著に減少することが明らかになった。さらにInka2 mRNAの翻訳制御に関わる機構を明らかにするため、マウス脳や神経系培養細胞を用いたRNA 免疫沈降(RIP)解析を行ったところ、inka2 mRNAはRNA結合タンパク質Fmr1やTDP43の標的候補の一つであることが示唆された。Fmr1は神経系に発現するRNA結合タンパク質であり、スパイン形成・安定性に異常を示す遺伝性の精神発達障害・自閉症である脆弱X症候群(FXS)の原因遺伝子である。in vitro BrU標識したinka2 RNAを用いて、それに結合するRNP(リボヌクレオタンパク質)を回収するRiboTrap解析を行った結果、Fmr1はinka2 CDS と3’UTRに存在するG4 RNA配列に結合しすること、TDP43はGU塩基に富む配列領域に結合することを明らかとした。当初の予定通りinka2 mRNAにFmr1 およびTDP43が強く結合することを示した。しかしinka2タンパク質をwestern blotで認識する抗体の作製が遅れており、inka2翻訳制御機構の解明には至っていない。Inka2のスパイン形成における役割と翻訳調節機構を検討する。胎児期後期から出生後~成体期の各発達時期でinka2 KOマウスの脳を採取しゴルジ染色を行い、大脳皮質V層錐体細胞および海馬錐体細胞を中心に樹状突起スパインの形態、密度をさらに詳細に観察し定量化する。スパインの形態異常・密度減少が発達早期から起きるのか、あるいは成熟後にスパイン変性が亢進するのか調べる。さらにシナプスマーカーやPAK4、CRMP等アクチン骨格関連分子の変化を調べる。またルシフェラーゼcDNAの下流にinka2 mRNA G4 RNA部位を連結したコンストラクトを作成し、fmr1発現ベクターと共に培養細胞に導入する。fmr1発現量に比例しルシフェラーゼの活性の低下、すなわち翻訳抑制が起こるか検討する

  • Control of neurite development by a novel SUMOylation-regulating mechanism

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  • Analysis of functional exchange of the transcriptional factors involved in neurodegenerative diseases by SUMO modification.

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     View Summary

    PARIS is identified as a substrate of the ubiquitin ligase, parkin, a gene associated with Parkinson's disease. PARIS represses the expression of the transcriptional co-activator PGC-1α, although the mechanism that controls its repressive activity and function are largely unknown. We have shown that PARIS can be modified by SUMO and that SUMOylation of PARIS regulates its transcriptional activity. We have also shown that SUMOylation of PARIS also controls its ubiquitination. The proteasome inhibitor treatment accumulated SUMO-2/3 conjugates of PARIS. The SUMOylated PARIS was more effectively ubiquitinated than the non-SUMOylated form of PARIS. The SUMO-targeted ubiquitin ligase RNF4 promoted the ubiquitination of SUMOylated PARIS. These results suggest that RNF4-mediated ubiquitination of PARIS regulates its transcription function.We believe the present study results may improve our understanding of the role of SUMOylation in neurodegeneration in Parkinson's disease

  • Connectomics analysis of the synapse in the primary visual cortex layer IV in the ocular dominant plasticity

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     View Summary

    To clarify the mechanism of the neural circuit reorganization, we have established connectomics analysis of visual cortex. After monocular deprivation, the neurons in the visual cortex were more received from open eye input. As a result of having immunohistochemically analyzed using c-Fos, FosB and CREB, these activity dependence inducted proteins were resulted to detect the changes of ocular dominance plasticity. Using connectomics analysis, synaptic connections were cyclopedically analyzed in layer IV of visual cortex. As a result, we detected that first the synapses received from deprived eye input were reduced, next the synapses received from opened eye input were increased

  • Identification of novel genes that function in cell division and migration of neural progenitors

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     View Summary

    Developmental dynamics of neural stem/progenitor cells (NSPCs) are crucial for embryonic and adult neurogenesis, but its regulatory factors are not fully understood. We identified the radmis gene, a novel microtubule associated protein highly enriched in NSPCs. Radmis is a substrate for the E3-ubiquitin ligase, anaphase promoting complex/cyclosome, and is degraded via the KEN box. Radmis was highly expressed in regions of active neurogenesis throughout life. In addition, we identified another NSPC gene, designated as inka2, a putative regulator of actin cytoskeleton reconstruction. Inka2 transcripts were detected in Olig2-positive oligodendrocyte progenitor cells during embryogenesis. In the adult brain, the expression of inka2 was interestingly confined in terminally differentiated neurons in the restricted forebrain regions. Inka2 may be involved in multiple actin-driven processes, including cell migration and establishment of neuronal polarity

  • Regulation of the transcriptional factors involved in Parkinson's disease by SUMO modification.

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     View Summary

    PARIS/ZNF746, a member of the family of KRAB zinc-finger proteins transcriptional factors, is recently identified as a substrate of the ubiquitin E3 ligase, parkin, a gene associated with autosomal recessive juvenile parkinsonism. PARIS represses the expression of the transcriptional co-activator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the mechanism that controls its repressive activity and function are largely unknown. In this study, we show that PARIS can be modified by the small ubiquitin-related modifier (SUMO) both in vivo and in vitro. Furthermore, mutational analysis reveals that two lysine residues (K189 and K286) are the major sumoylation sites on human PARIS protein. Replacement of sumoylation site lysines with arginine decreases the repression ability of the native PGC-1αgene promoter in neuroblastoma SH-SY5Y cells. These results strongly suggest a correlation between PARIS transcriptional activity and its sumoylation

  • Role of neurotransmitters and environmental enrichment in the critical period of brain development

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    ABSTRACTThe dentate gyrus(DG) of the hippocampal formation produces new neurons throughout adulthood in mammalian species. Several experimental statuses and factors regulating neurogenesis have been identified in the adult DG. For example, exposure to an enriched environment enhances neurogenesis in the DG and improves hippocampus-dependent spatial learning. Furthermore, serotonin is known to influence adult neurogenesis, and learning and memory. However, the effects of long-lasting depletion of serotonin over the developing period on the neurogenesis have not been investigated. Thus, we examined the influence of long-lasting serotonin depletion on the environmental enrichment-induced neurogenesis and spatial memory performance. As reported previously, environmental enrichment significantly increased new neurons in the DG. However, there was no improvement of spatial learning test in adult rats in standard and in environmental enrichment housings.Intracisternal administration of the serotonergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), on postnatal day 3 apparently reduced serotonin content in the adult hippocampus without regeneration. This experimental depletion of serotonin in the hippocampus of rats housed in an enriched environment had no effect on spatial memory performance, but produced significant decreases in the number of bromodeoxyuridine (BrdU)-labeled new cells in the DG. These findings indicate that newly generated cells stimulated by environmental enrichment are not critical for improvements in hippocampus-dependent learning. Furthermore, numbers of BrdU-labeled cells in the DG of 5,7-DHT-injected rats did not differ between 1 day and 4 weeks after BrdU injection. These data suggest that survival of newly generated DG cells remains relatively constant under long-lasting serotonin depletion

  • Identfication of novel genes highly expreseed in the mitotic neural progenitor/stem cells

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    Identification of the genes regulating neural progenitor neural stem cell functions is critical to understand the mechanisms of the adult neurogenesis and neurodegenerative disease. We compared the gene expression profile of proliferating neural stem cell culture with those of differentiated cells, and identified a novel gene named "Radmis (radial fiber associated mitotic spindle protein)". Northern blot analysis indicated radmis mRNA is expressed in the neural stem cells in vitro. In the developing and matured CNS, radmis protein was highly enriched in the mitotic neural progenitors. At E11 and E13 developing cerebral cortex, radmis expression was observed in the mitotic spindle, and radial fiber of the nestin-positive neuroepithelial cells, neural stem cells, located at the ventricular surface. At early postnatal CNS, radmis is expressed in positive neural progenitor cells in the SVZ of lateral ventricles, and 4th ventricle. In the developing cerebellum at P6, radmis is expressed in the mitotic spindle of the proliferating neuronal progenitor cells within the EGL layer, the superficial layer of cerebellum, but it was absent from the NeuN-positive post-mitotic neurons in both EGL arid IGL, the inner layer of the cerebellum. Radmis expression is drastically decreased as the CNS development proceeds, but a small number of cells in SVZ neurogenic region remained to express radmis protein. In the adult SVZ surrounding the lateral ventricles, radmis is expressed in the dividing neural stem cells (type B cells) that are positive for GFAP, and also in the proliferating neuronal progenitors (rapid amplifying cells, type C cells) that are positive for nestin. During the cell cycle, radmis protein did not expressed in the microtubules in interphase cells. At prometaphase, metaphase and anaphase, radmis colocalized with mitotic spindle poles and spindle microtubules proximal to the centrosomes, suggesting that radmis may control the spindle structure and function during the embryonic and adult neurogenesis

  • Identification of novel genes involving in the cell-division of neural progenitor cells.

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Specific Research

  • Inka2によるシナプス形成制御のメカニズム


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    ヒトを含め哺乳類の神経幹細胞は発生期~成体期まで継続的に脳内に維持され,放射状細胞突起や多分化能を特徴とする。一方,神経幹細胞から産生される増殖性 のニューロン・グリア前駆細胞は系譜がより制限されるが,目的の部位に向かう高い遊走能を持ち,神経発生過程だけでなく,神経変性病態での髄鞘再形成など 脳機能再生の観点からも重要な細胞である。しかし神経前駆細胞(NSPC)の形態維持や遊走メカニズムの分子的基盤には未解明な部分が多い。我々が神経系前駆細胞に発現する新規遺伝子として同定したinka2 はInkaドメイン(神経堤細胞の移動に機能すると考えられているInka1と類似の機能不明ドメイン)を持ち、細胞移動などに関与することが推定される。inka2KOマウスを作製し大脳皮質のシナプス構築を各発達時期でゴルジ染色を行い解析した結果、ニューロン、特に大脳皮質V層錐体細胞の樹状突起スパインの形態、密度の低下、異常シナプスの増加が出生後早期から見られた。

  • 神経前駆細の形態形成におけるradmisの機能


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    &nbsp;哺乳類の胎生期中枢神経系では,幾つかのタイプの形態・分化能を備える神経幹細胞・神経前駆細胞(NSPCと略)が存在し,それらが連続的に現れ協調的に機能することで脳が形成される。これらの細胞の分裂のタイミングや回数は時空間的にコントロールされており,その細胞分裂周期の変調や破綻は脳の発生異常・小頭症などの脳奇形に至る。ヒトの遺伝性小頭症Filippi症候群の原因遺伝子radmisは,胎児期~成体脳の神経前駆細胞の分裂紡錘体極・中心体に局在し,中心体複製や紡錘体形成を制御することで神経前駆細胞の正常分裂進行に必須の機能を担うと推定される。radmis蛋白質は神経幹細胞系譜の放射状突起と分裂時の紡錘体極中心体に局在する。放射状突起中での発現分布を二重免疫蛍光染色法により比較した結果、radmisの分布は中間径フィラメントnestin やvimentinの分布と一致していた。さらにRadmis強制発現により細胞内vimentin局在が変化し、異常な中間径フィラメント形成が誘導されることが示唆された。

  • 哺乳類脳形成における中心体関連タンパク質の機能解析


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    ヒトの遺伝性小頭症Filippi症候群の原因遺伝子radmisは,胎児期~成体脳の神経前駆細胞の分裂紡錘体極・中心体に局在し,中心体複製や紡錘体形成を制御することで神経前駆細胞の正常分裂進行に必須の機能を担うと推定される。radmis蛋白質は神経幹細胞系譜の放射状突起と分裂時の紡錘体極中心体に局在する。放射状突起中での発現分布を二重免疫蛍光染色法により比較した結果、radmisの分布は中間径フィラメントnestin やvimentinの分布と一致していた。さらに抗radmis抗体と抗vimentin抗体による金コロイド二重染色により免疫電子顕微鏡法で解析したところ、放射状突起内でradmisはvimentinの極めて近傍に存在し相互作用していることが示唆された。

  • 脳形成における中心体関連タンパク質Radmisの機能解明


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  • アクチン骨格制御タンパク質Inka2の神経系における機能解析


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    ヒトを含め哺乳類の神経幹細胞は発生期~成体期まで継続的に脳内に維持され,放射状細胞突起や多分化能を特徴とする。一方,神経幹細胞から産生される増殖性 のニューロン・グリア前駆細胞は系譜がより制限されるが,目的の部位に向かう高い遊走能を持ち,神経発生過程だけでなく,神経変性病態での髄鞘再形成など 脳機能再生の観点からも重要な細胞である。しかし神経前駆細胞(NSPC)の形態維持や遊走メカニズムの分子的基盤には未解明な部分が多い。我々が神経系前駆細胞に発現する新規遺伝子として同定したinka2(MG46) はInkaドメイン(神経堤細胞の移動に機能すると考えられているInka1と類似の機能不明ドメイン)を持つことから、細胞移動などに関与することが推定される。inka2 mRNAは胎生期延髄,脊髄の腹側部脳室周囲のオリゴデンドロサイト前駆細胞に発現し,移動を終えたオリゴデンドロサイトでは発現が消失する。培養細胞にinka2を強制発現させると,アクチン線維の細胞内配置の異常が起こり,細胞形態が球状に変化し細胞接着の阻害や,過剰な数のフィロポディアが形成される。逆に,培養細胞でshRNAによるinka2発現抑制を行うと,細胞の遊走能は有意に増大し,細胞移動が促進されることから,Inka2はアクチン骨格の再編成を調節し,細胞形態変化や細胞移動の推進力を発生させるのに必要な新たな分子であると推定している。inka2と相互作用するタンパク質を解析するために、培養細胞及びマウスの脳タンパク抽出液からGSTプルダウンによりinka2結合タンパク質を単離プロテオーム解析したところ、細胞周期、増殖、分化などの多様なシグナル伝達に必要とされセリン/スレオニンホスファターゼが単離された。さらにshRNAによってinka2を抑制するとフォーカルアドヒージョン分子の一つであるパキシリンのライフタイムが減少することから、inka2はフォーカルアドヒージョン動態の変化を介して細胞移動を調節する新たな分子であることが示唆された。

  • 生理活性ペプチドプロセッシングによる脳炎症制御機構


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    炎症は、細菌やウイルスなどの異物を排除して生体の恒常性を維持するための防御反応である。しかし最近の研究から、体の中にもともと存在する細胞成分により引き起こされる非感染性の「自然炎症」と呼ばれる慢性炎症が多くの疾患の病態に深く関わっていることが明らかになりつつある。加齢と共に増加するガン、動脈硬化、肥満、虚血性脳疾患、アルツハイマー病などの種々の神経変性疾患、さらには老化そのものも、慢性的な自然炎症性の変化によって症状が進行する可能性が示唆されている。Attractin(Atrn)はヒトを含む哺乳類で保存された遺伝子で、脳神経細胞や活性化T細胞などに広く発現する。atrn遺伝子に変異を有するラット(zitter rat)は髄鞘の低形成、ニューロン変性、酸化ストレス上昇、ミクログリアの増殖・活性化、血管から脳実質へのマクロファージの浸潤など加齢に伴う脳実質の種々の炎症様変化を示すことから、Atrnが炎症発症・慢性的な炎症持続機構に深く関わっていることが予測される。タンパク質の一次構造情報から、AtrnがDPP-4(DipeptidylPeptidase-4)様のペプチダーゼ酵素活性を持つ可能性がある。神経細胞膜上および分泌型Atrnが、脳実質内に拡散・蓄積する炎症性サイトカイン、ケモカイン、神経ペプチドを基質としてDPP-4様にプロッセッシングすることで、その活性を変化・不活化し脳の炎症反応を抑制しているという可能性を検証することを目的として、まず膜結合型、および分泌型の組換えAtrnタンパク質を発現させるためのバキュロウイルスベクターの構築を行った。今後精製標品を用いて生理活性の測定、in vitroの標的ペプチドを同定する予定である。

  • 神経前駆細の形態・移動を制御する新規分子群の機能解明


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    ヒトを含め哺乳類の神経幹細胞は発生期~成体期まで継続的に脳内に維持され,放射状細胞突起や多分化能を特徴とする。一方,神経幹細胞から産生される増殖性のニューロン・グリア前駆細胞は系譜がより制限されるが,目的の部位に向かう高い遊走能を持ち,神経発生過程だけでなく,神経変性病態での髄鞘再形成など 脳機能再生の観点からも重要な細胞である。しかし神経前駆細胞(NSPC)の形態維持や遊走メカニズムの分子的基盤には未解明な部分が多い。我々が神経系前駆細胞に発現する新規遺伝子として同定したinka2(MG46) はInkaドメイン(神経堤細胞の移動に機能すると考えられているInka1と類似の機能不明ドメイン)を持つことから、細胞移動などに関与することが推定される。inka2 mRNAは胎生期延髄,脊髄の腹側部脳室周囲のオリゴデンドロサイト前駆細胞に発現し,移動を終えたオリゴデンドロサイトでは発現が消失する。培養細胞にinka2を強制発現させると,アクチン線維の細胞内配置の異常が起こり,細胞形態が球状に変化し細胞接着の阻害や,過剰な数のフィロポディアが形成される。逆に,培養細胞でshRNAによるinka2発現抑制を行うと,細胞の遊走能は有意に増大し,細胞移動が促進されることから,Inka2はアクチン骨格の再編成を調節し,細胞形態変化や細胞移動の推進力を発生させるのに必要な新たな分子であると推定している。さらに,生後のシナプス形成期のニューロンでinka2 mRNA発現は急速に上昇し,我々が作製したinka2抗体による観察からinka2蛋白質は成熟期の大脳皮質,海馬領域ニューロンの樹状突起のシナプス部位に局在していることが示唆された。以上の結果からMG46はNSPCの遊走を制御する新たな分子であると推定された。さらに個体レベルの脳形成,シナプス形成においてinka2遺伝子が果たす役割を明確にすることを目的とし,inka2遺伝子欠損マウスの作製を試みた。Inka2遺伝子の相同組換えES細胞を作製し、キメラマウスオス2匹を誕生させた。現在野生型CL57BL/6メスとの交配によりinka2ヘテロ変異マウスの繁殖が進行中である。

  • 神経前駆細の形態・移動を制御する新規分子群の機能解明


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     ヒトを含め哺乳類の神経幹細胞は発生期~成体期まで継続的に脳内に維持され,放射状細胞突起や多分化能を特徴とする。一方,神経幹細胞から産生される増殖性のニューロン・グリア前駆細胞は系譜がより制限されるが,目的の部位に向かう高い遊走能を持ち,神経発生過程だけでなく,神経変性病態での髄鞘再形成など脳機能再生の観点からも重要な細胞である。しかし神経前駆細胞(NSPC)の形態維持や遊走メカニズムの分子的基盤には未解明な部分が多い。我々が神経系前駆細胞に発現する新規遺伝子として同定したMG46 はInkaドメイン(神経堤細胞の移動に機能すると考えられているInka1と類似の機能不明ドメイン)を持つことから、細胞移動などに関与することが推定される。insitu hybridizationの結果から,NSPCの形態や遊走に関わると考えられる。MG46 mRNAは胎生期延髄,脊髄の腹側部脳室周囲のオリゴデンドロサイト前駆細胞に発現し,移動を終えたオリゴデンドロサイトやニューロンでは発現が消失する。EGFP-MG46をin vitroでNSPCに強制発現させると、EGFP-MG46は細胞膜直下に集積し,過剰な数の糸状仮足(filopodia)の形成を促進した。またinvivo強制発現によりNSPCはアクチン骨格の変化により細胞形態が球状に変化させ,細胞移動に異常が見られた。以上の結果からMG46はNSPCの遊走を制御する新たな分子であると推定された。さらにMG46タンパク質を組換えタンパクとして発現、精製し、培養細胞およびマウス脳抽出液を用いて、相互作用する分子をプロテオーム解析により同定したところ、セマフォリンシグナル伝達に関係するタンパクが複数同定された。現在、MG46がどのようにこれらの分子と相互作用し、アクチン再編成系を制御しているのかを検討している。

  • SUMO化と脱SUMO化による神経分化制御


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    中枢神経系の幹細胞(前駆細胞)は発生期から成体脳に至るまで存在し、そのニューロン産生頻度・細胞周期は、発生段階や外部環境などに依存してダイナミックに制御されている。しかし、この迅速な幹細胞の機能調節機構には不明な点も多い。我々は神経幹細胞の維持機構において、SUMO化・脱SUMO化によるタンパク分子(群)の拮抗的で動的な翻訳後修飾が重要な役割を担っていると推定している。SUMO(Small Ubiquitin-related Modifier)は標的タンパク質との結合によりその機能を修飾する事で、細胞周期進行、核-細胞質輸送、転写調節など多岐の生命現象に関わっている事が報告されている。哺乳類SUMOには3種類のアイソフォームSUMO-1, -2, -3が存在し、SUMO-2とSUMO-3は非常に高い相同性(95%)を示すサブグループを形成し、SUMO-1とは機能的相違が示唆されている。哺乳類の神経分化におけるSUMO化・脱SUMO化の役割を解明するためにSUMO-1、SUMO-2/3、およびSUMO化酵素のひとつであるUBC9の発現パターンをマウス脳の発生過程を追って詳細に解析した。胎生10日目のマウス脳胞を抗体により免疫染色した結果、SUMO-1、SUMO-2/3、UBC9はともに神経幹細胞である神経上皮の核質に発現局在が認められた。神経幹細胞におけるこれらSUMO関連分子の発現は神経幹細胞の初代培養系を用いた免疫染色により確認された。さらにwestern blot解析の結果、培養神経幹細胞のSUMO化レベルはニューロン分化に伴い低下していくことが示された。この神経前駆細胞、幹細胞におけるSUMO関連タンパク質の局在は生後から成体期にわたり持続していた。特にSUMO-2/3化は生涯に渡り神経前駆細胞において強く起こっていることが示唆された。すなわち生後1-7日においては小脳外顆粒細胞EGL、成体期の側脳室周囲のnestin陽性SVZ細胞や海馬歯状回のGFAP陽性神経前駆細胞では強いSUMO-2/3化が起きていた。一方、生後の脳発達に伴い、様々な領域ではSUMO-1とSUMO-2/3の局在の相違が顕著となる。大脳皮質では全層の分化ニューロンで高いSUMO-1局在が認められるのに対して、SUMO-2/3化のレベルは総じて低下しており、特に第4-6層のニューロンにおいてはSUMO-2/3化の顕著な低下が認められた。他の領域においてもSUMO-2/3とSUMO-1の特徴的な分布の差が認められた。例えば脳幹部においては、顔面神経核、舌下神経核などの大型の運動神経核の細胞体に顕著なSUMO-1蓄積があるのに対して、SUMO-2/3の蓄積はこれらの神経核には見られなかった。以上の詳細な組織学的な検索から、神経系前駆細胞ではSUMO-2/3化が豊富に起きており、標的タンパク質のSUMO-2/3が重要な役割を担っていることが推定された。すでに我々はプロテオーム解析により神経前駆細胞内でSUMO-2/3化されるタンパク質を同定しており、SUMO化部位を改変したミュータントタンパク質の作製を進めている。現在この変異体を神経前駆細胞など培養系に導入し、標的タンパク質の機能変化、細胞内局在の変化を解析中である。これらの解析により神経前駆細胞の維持におけるSUMO化の意義が明らかになると考えられる。

  • 神経発生の新たな基盤分子群の探求


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    哺乳動物の神経幹細胞は発生期においては神経上皮と呼ばれる脳原基の中で,特有のapicalとbasal面の極性を持つ放射状上皮様細胞の形態で存在し,特有の核の上下運動をしながら,速い速度で自己複製的分裂とニューロン,グリア細胞産生を伴う分裂を行う。大部分の神経幹細胞はニューロンの産生を終える時期に消失するが,一部は生涯に渡って海馬歯状回や側脳室,第3脳室周囲の脳室下帯(SVZ)に残り,新たなニューロンを生み出し続けることで,大人になった後のある種の記憶消去や自律神経機能調節などにも関係している事が示唆されている。我々はマウス胎児および成体SVZを用いたsuppression PCR-subtraction法により胎児期・成体脳の神経幹細胞にニューロン新生が起きる部位に発現する6個の新規遺伝子(radmis,MG46,MB14,MB61,SD35,ME55)を同定した。radmis (radial fiber associated mitotic spindle protein) は特徴的なモチーフ構造を持たず,機能的に未知の遺伝子である。特異的抗体を作製し免疫染色を行ったところ、radmisは胎児期の脳室周囲のventricular zone (VZ) および成体脳の側脳室周囲 SVZに少数存在するtype-B 細胞神経幹細胞の放射状細胞突起と分裂の時に一過性に現われる分裂紡錘体に局在することを明らかとなった。またradmisタンパク質は細胞周期のM期に強く発現することから細胞周期依存的な翻訳後制御を受ける可能性が考えられた。実際Radmisには細胞周期依存的ユビキチン化複合体APC/C-cdh1の基質にみられるコンセンサス配列KEN boxがみられた。そこでKEN boxにアミノ酸置換を導入し、ユビキチン化を受けないように改変した変異型radmis遺伝子を作製し、子宮内電気穿孔法によりマウス胎児の脳室内へ遺伝子導入を行った結果、正常では幹細胞分裂期の終了時には発現が消失していたradmisタンパク質の半減期が劇的に伸びるとともに、脳室周囲でPH3神経陽性の前駆細胞の分裂の亢進が起きた。この際、TBR2陽性のSVZ細胞数の増加が一過性にみられた後、急速にSVZ細胞数が減少することから、radmisを発現し続けた前駆細胞は一時的にM期でarrestしてしまい、再び細胞周期に戻れなくなると推定された。RadmisのKEN boxを標的としたユビキチン化が起きていることを示すために、我々はさらに培養細胞に、野生型Radmis、KEN box変異型Radmisとcdh1あるいはその類似アダプターcdc20をco-transfectionしてimmunoblotによりradmisの細胞内での半減期の変化を解析した。その結果、野生型Radmisはcdh1と共発現した場合細胞内のタンパク量著しく低下するのに対して、KEN box変異型Radmisをcdh1と共発現した場合にはタンパク量の低下がみられないこと、またcdc20にはradmisタンパク量変化に影響しないことが示された。以上のデータよりradmisタンパクは神経幹細胞の分裂後は速やかにAPC/C・cdh1によるユビキチン化を受けて分解され,幹細胞内から除去されること、またその除去が神経前駆細胞の細胞周期進行に必要であることが示された。さらに胎児脳室においてradmis shRNAによる機能抑制を行ったところ、神経前駆細胞の分裂際に正常な染色体分離が阻害されることが示された。培養細胞において同様の実験を行ったところ、染色体の分離の異常を伴う分裂紡錘体の多極化を高頻度に観察された。以上の結果からradmisは神経前駆細胞分裂過程で必須の因子であり、radmiの適切なタイミングでの発現と消失が正常な神経前駆細胞の維持に必要であることが明らかとなった。一方,MG46遺伝子を子宮内電気穿孔法によりマウス胎児脳で強制発現させたところ、神経前駆細胞の形態が異常な球形に変化することが示された。MG46遺伝子産物は細胞骨格系アクチン繊維を類似の細胞内分布を示すことから、神経前駆細胞のアクチン骨格の再編成を制御してその形態変化に関わっている可能性が示唆された。

  • 神経発生過程におけるタンパク質SUMO化の役割


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    中枢神経系の幹細胞(前駆細胞)は発生期から成体脳に至るまで存在し、そのニューロン産生頻度・細胞周期は、発生段階や外部環境などに依存してダイナミックに制御されている。しかし、この迅速な幹細胞の機能調節機構には不明な点も多い。我々は神経幹細胞の維持機構において、SUMO化・脱SUMO化によるタンパク分子(群)の拮抗的で動的な翻訳後修飾が重要な役割を担っていると推定している。SUMO(Small Ubiquitin-related Modifier)は標的タンパク質との結合によりその機能を修飾する事で、細胞周期進行、核-細胞質輸送、転写調節など多岐の生命現象に関わっている事が報告されている。哺乳類SUMOには3種類のアイソフォームSUMO-1, -2, -3が存在し、SUMO-2とSUMO-3は非常に高い相同性(95%)を示すサブグループを形成し、SUMO-1とは機能的相違が示唆されている。哺乳類の神経分化におけるSUMO化・脱SUMO化の役割を解明することを目的としてSUMO-1、SUMO-2/3、およびSUMO化酵素のひとつであるUBC9の発現パターンをマウス脳の発生過程を追って詳細に解析した結果、神経前駆細胞においてはSUMO-2/3によるSUMO化が豊富に起きていることが示された。一方、分化したニューロンではSUMO-2/3化のレベルが急速に低下することから、神経前駆細胞の維持に標的タンパク質のSUMO-2/3によるSUMO化の関与が予想された。そこで神経系前駆細胞でSUMO-2/3化されている標的タンパク質を同定するために、成体脳のSVZ領域を剖出しタンパク質を抽出し、SIMペプチドを固定化したカラムを用いてSUMO化タンパク質を精製した。精製標品はSDS-PAGEで展開後、バンドを切り出しLC-MS/MSプロテオーム解析により網羅的に同定した。その結果、NotchやWntシグナルカスケードなどに関わることが知られている細胞内シグナル分子や、機能未知のタンパク質など興味深いタンパク質群が同定された。現在これらのタンパク質群から幹細胞維持に関与している可能性が高い分子を選定し、実際に生体内でのSUMO化の有無を検討している。さらに同定した標的タンパク質のSUMO化部位を改変したミュータントタンパク質を作製し、子宮内胎児脳室への電気穿孔法により強制発現させることにより、神経前駆細胞での標的タンパク質のSUMO化の意義を明らかにする予定である。これらの解析により神経前駆細胞の維持におけるSUMO化の意義が明らかになると考えられる。

  • 成体脳で神経幹細胞が維持されるための分子基盤の解明


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    哺乳動物の神経幹細胞は発生期においては神経上皮と呼ばれる脳原基の中で,特有のapicalとbasal面の極性を持つ放射状上皮様細胞の形態で存在し,特有の核の上下運動をしながら,速い速度で自己複製的分裂とニューロン,グリア細胞産生を伴う分裂を行う。大部分の神経幹細胞はニューロンの産生を終える時期に消失するが,一部は生涯に渡って海馬歯状回や側脳室,第3脳室周囲の脳室下帯(SVZ)に残り,新たなニューロンを生み出し続けることで,大人になった後のある種の記憶消去や自律神経機能調節などにも関係している事が示唆されている。我々はマウス胎児および成体SVZを用いたsuppression PCR-subtraction法により胎児期・成体脳の神経幹細胞にニューロン新生が起きる部位に発現する6個の新規遺伝子(radmis,MG46,MB14,MB61,SD35,ME55)を同定した。radmis (radial fiber associated mitotic spindle protein) は特徴的なモチーフ構造を持たず,機能的に未知の遺伝子である。radmisは胎児期の脳室周囲のventricular zone (VZ) および成体脳の側脳室周囲 SVZに少数存在するtype-B 細胞神経幹細胞の放射状細胞突起と分裂の時に一過性に現われる分裂紡錘体に局在することを明らかとした。radmisタンパクは神経幹細胞の分裂後は速やかにユビキチン化を受けて分解され,幹細胞内から除去されることが示された。そこでユビキチン化を受けないよう改変した変異型radmis遺伝子を作製し,子宮内電気穿孔法を用いてマウス胎児脳に遺伝子導入すると,脳室周囲で神経前駆細胞の分裂の亢進が起きた。現在、胎児脳室においてradmis shRNAによる機能抑制を行い、radmisの神経幹細胞分裂過程での機能をより詳細に解析している。一方,MG46遺伝子はapical側細胞膜への局在化シグナルを持ち,EGFPとの融合タンパク質の培養細胞での強制発現の結果から,その産物は神経幹細胞の放射状形態が形成される際,一部の細胞膜直下に限局して発現する事が判明した。神経幹細胞の細胞形態の維持や極性の形成にMG49が関わっている可能性を検討するため、子宮内電気穿孔法によるマウス胎児脳でのMG49遺伝子導入を行っている。その結果,MG49発現細胞の形態以上が示唆された。現在さらにMG49タンパクの局在を検討するため、特異的抗体を作製中である。

  • 中枢神経系発生におけるタンパク質の脱SUMO化の役割


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    哺乳動物の脳が形成されるプロセスにおいては、多数の神経幹細胞・前駆細胞が速い細胞周期で同期的に増殖する過程と並行して、幹細胞は適切な時期に適切なタイプのニューロンを適切な数だけ産生する。このようなダイナミックで精巧な細胞分化のプロセスを遂行するためには、神経幹細胞で起きる転写レベルでの遺伝子発現調節に加えて、タンパク質の翻訳後修飾が重要な役割を担っていると考えられる。SUMO(Small Ubiquitin-related Modifier)は標的タンパク質との結合によりその機能を修飾する事で、細胞周期進行、核-細胞質輸送、転写調節など多岐の生命現象に関わっている事が報告されている。哺乳類SUMOには3種類のアイソフォームSUMO-1, -2, -3が存在し、SUMO-2とSUMO-3は非常に高い相同性(95%)を示すサブグループを形成し、SUMO-1とは機能的相違が示唆されている。一方、タンパク質からSUMOを切り離す脱SUMO化反応はSUMO特異的プロテアーゼ(SENP)により行われる。これまでに6つの異なるファミリー遺伝子SENP-1,- 2, -3, -5, -6, -7が発見されているが、その生理的役割は明確でない。哺乳類の神経分化におけるSUMO化・脱SUMO化の役割を解明することを目的としてSUMO-1、SUMO-2/3、およびSENP-2, -3, -5, -7の各タンパク質の発現パターンをマウス脳の発生過程を追って詳細に解析した。その結果、胎児期では脳室周囲に位置する神経前駆細胞および幼弱なニューロンにSUMO-2/3の発現が認められた。神経前駆細胞でのSUMO-2/3発現は出生後も維持され、成体では側脳室周囲の脳室下帯SVZに存在する神経幹細胞・前駆細胞に強い発現があった。一方、分化したニューロンではSUMO-2/3は急速に発現が消失する。また神経前駆細胞内ではSUMO-1発現は低レベルであることが示された。脱SUMO化酵素群で脳での発現が認められたのはSENP3とSENP5であるが、SENP3は前駆細胞を含めユビキタスに発現しているのに対し、SENP5は胎児期の神経前駆細胞と幼弱ニューロンで発現するが、生後になり神経発生が進むと前駆細胞での発現が消失し、ニューロンの樹状突起、特にシナプスpost側にのみ発現が局在するようになる。以上の結果から、胎児期から成体に至るまで前駆細胞の維持に未知の標的タンパク質(群)のSUMO-2/3によるSUMO化の関与が予想された。

  • 神経幹細胞に発現する遺伝子群の同定と機能解析


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    ヒトを含め哺乳動物の成体脳には、多分化能や自己複製能を特徴とする神経幹細胞が限られた部位に存在し続ける事が報告されている。しかし神経幹細胞の脳内での分布や、幹細胞を維持するための遺伝子発現の制御機構については依然不明な点が多い。我々は神経幹細胞に強く発現する新規遺伝子群を同定し、その発現部位、機能を明らかにすることにより神経幹細胞の維持、制御機構の解明を目指している。 本特定課題研究では、我々が既に単離同定している神経幹細胞に発現する遺伝子群について解析を進めた。MB14およびME55遺伝子はNotchシグナル関連分子に弱い相同性を示し、SD35は細胞周期にブレーキをかけるタンパク質との弱い相同性が認められた。またMG49遺伝子は糖鎖転移酵素の活性中心と類似のドメインを持つ新規タンパク質であった。in situ hybridization解析により、MB14、ME55 mRNA発現は胎生期の脳室周囲層、および生後や成体脳の側脳室の脳室下層といった神経幹細胞が局在すると考えられる部位での発現が認められた。また解析が先行しているradmis遺伝子については、神経幹細胞の放射状細胞突起と分裂期紡錘体微小管に一過性に局在し、分裂後は速やかに後期促進複合体APC/Cdh1によるユビキチン化を受けてタンパクが分解され、幹細胞内から除去される可能性が示された。実際の個体レベルでのradmis遺伝子の機能を検討するため、ユビキチン化を受けないよう改変した変異型radmis遺伝子を作製し、子宮内電気穿孔法によりマウス胎児の脳室内へ遺伝子導入を行った。その結果、radmis遺伝子を強制発現した神経幹細胞では分裂の亢進と細胞移動の異常が起きる事が明らかとなった。以上のデータからradmisが神経幹細胞の分裂制御に重要な働きを持つことが示唆された。

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