Updated on 2024/12/07

写真a

 
OHSHIMA, Toshio
 
Affiliation
Faculty of Science and Engineering, School of Advanced Science and Engineering
Job title
Professor
Degree
MD, PhD ( Yamanashi Medical University )

Research Areas

  • Anatomy and histopathology of nervous system / Neuroscience-general

Awards

  • 日本神経化学会奨励賞

    2001  

 

Papers

  • Drug Treatment Attenuates Retinal Ganglion Cell Death by Inhibiting Collapsin Response Mediator Protein 2 Phosphorylation in Mouse Models of Normal Tension Glaucoma.

    Yuebing Wang, Musukha Mala Brahma, Kazuya Takahashi, Alessandra Nolia Blanco Hernandez, Koki Ichikawa, Syuntaro Minami, Yoshio Goshima, Takayuki Harada, Toshio Ohshima

    Neuromolecular medicine   26 ( 1 ) 13 - 13  2024.04  [International journal]

     View Summary

    Normal tension glaucoma (NTG) is a progressive neurodegenerative disease in glaucoma families. Typical glaucoma develops because of increased intraocular pressure (IOP), whereas NTG develops despite normal IOP. As a subtype of open-angle glaucoma, NTG is characterized by retinal ganglion cell (RGC) degeneration, gradual loss of axons, and injury to the optic nerve. The relationship between glutamate excitotoxicity and oxidative stress has elicited great interest in NTG studies. We recently reported that suppressing collapsin response mediator protein 2 (CRMP2) phosphorylation in S522A CRMP2 mutant (CRMP2 KIKI) mice inhibited RGC death in NTG mouse models. This study evaluated the impact of the natural compounds huperzine A (HupA) and naringenin (NAR), which have therapeutic effects against glutamate excitotoxicity and oxidative stress, on inhibiting CMRP2 phosphorylation in mice intravitreally injected with N-methyl-D-aspartate (NMDA) and GLAST mutant mice. Results of the study demonstrated that HupA and NAR significantly reduced RGC degeneration and thinning of the inner retinal layer, and inhibited the elevated CRMP2 phosphorylation. These treatments protected against glutamate excitotoxicity and suppressed oxidative stress, which could provide insight into developing new effective therapeutic strategies for NTG.

    DOI PubMed

  • Phosphorylated CRMP1, axon guidance protein, is a component of spheroids and is involved in axonal pathology in amyotrophic lateral sclerosis.

    Yuko Kawamoto, Mikiko Tada, Tetsuya Asano, Haruko Nakamura, Aoi Jitsuki-Takahashi, Hiroko Makihara, Shun Kubota, Shunta Hashiguchi, Misako Kunii, Toshio Ohshima, Yoshio Goshima, Hideyuki Takeuchi, Hiroshi Doi, Fumio Nakamura, Fumiaki Tanaka

    Frontiers in neurology   13   994676 - 994676  2022.09  [International journal]

     View Summary

    In amyotrophic lateral sclerosis (ALS), neurodegeneration is characterized by distal axonopathy that begins at the distal axons, including the neuromuscular junctions, and progresses proximally in a “dying back” manner prior to the degeneration of cell bodies. However, the molecular mechanism for distal axonopathy in ALS has not been fully addressed. Semaphorin 3A (Sema3A), a repulsive axon guidance molecule that phosphorylates collapsin response mediator proteins (CRMPs), is known to be highly expressed in Schwann cells near distal axons in a mouse model of ALS. To clarify the involvement of Sema3A–CRMP signaling in the axonal pathogenesis of ALS, we investigated the expression of phosphorylated CRMP1 (pCRMP1) in the spinal cords of 35 patients with sporadic ALS and seven disease controls. In ALS patients, we found that pCRMP1 accumulated in the proximal axons and co-localized with phosphorylated neurofilaments (pNFs), which are a major protein constituent of spheroids. Interestingly, the pCRMP1:pNF ratio of the fluorescence signal in spheroid immunostaining was inversely correlated with disease duration in 18 evaluable ALS patients, indicating that the accumulation of pCRMP1 may precede that of pNFs in spheroids or promote ALS progression. In addition, overexpression of a phospho-mimicking CRMP1 mutant inhibited axonal outgrowth in Neuro2A cells. Taken together, these results indicate that pCRMP1 may be involved in the pathogenesis of axonopathy in ALS, leading to spheroid formation through the proximal progression of axonopathy.

    DOI PubMed

  • Genetic inhibition of collapsin response mediator protein-2 phosphorylation ameliorates retinal ganglion cell death in normal-tension glaucoma models.

    Musukha Mala Brahma, Kazuya Takahashi, Kazuhiko Namekata, Takayuki Harada, Yoshio Goshima, Toshio Ohshima

    Genes to cells : devoted to molecular & cellular mechanisms   27 ( 8 ) 526 - 536  2022.08  [International journal]

     View Summary

    Glaucoma is a neurodegenerative disorder caused by the death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is a cause of glaucoma. However, glaucoma often develops with normal IOP and is known as normal-tension glaucoma (NTG). Glutamate neurotoxicity is considered as one of the significant causes of NTG, resulting in excessive stimulation of retinal neurons via the N-methyl-D-aspartate (NMDA) receptors. The present study examined the phosphorylation of collapsin response mediator protein-2 (CRMP2), a protein that is abundantly expressed in neurons and involved in their development. In two mouse models, NMDA-injection and glutamate/aspartate transporter (GLAST) mutant, CRMP2 phosphorylation at the cyclin-dependent kinase-5 (Cdk5) site was elevated in RGCs. We confirmed that the decrease in the number of RGCs and thickness of the inner retinal layer (IRL) could be suppressed after NMDA administration in CRMP2KI/KI mice with genetically inhibited CRMP2 phosphorylation. Next, we investigated GLAST heterozygotes (GLAST+/-) with CRMP2KI/KI (GLAST+/-;CRMP2KI/KI) and GLAST knockout (GLAST-/-) mice with CRMP2KI/KI (GLAST-/-;CRMP2KI/KI) mice and compared them with GLAST+/- and GLAST-/- mice. pCRMP2 (S522) inhibition significantly reduced RGC loss and IRL thinning. These results suggest that the inhibition of CRMP2 phosphorylation could be a novel strategy for treating NTG.

    DOI PubMed

  • Valproic Acid-Induced Anxiety and Depression Behaviors are Ameliorated in p39 Cdk5 Activator-Deficient Mice

    Miyuki Takahashi, Toshiyuki Takasugi, Arisa Kawakami, Ran Wei, Kanae Ando, Toshio Ohshima, Shin-ichi Hisanaga

    Neurochemical Research   47 ( 9 ) 2773 - 2779  2022.07  [Refereed]  [International journal]

     View Summary

    Valproic acid (VPA) is a drug used for the treatment of epilepsy, seizures, migraines, and bipolar disorders. Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase activated by p35 or p39 in neurons and plays a role in a variety of neuronal functions, including psychiatric behaviors. We previously reported that VPA suppressed Cdk5 activity by reducing the expression of p35 in cultured cortical neurons, leaving p39 unchanged. In this study, we asked for the role of Cdk5 in VPA-induced anxiety and depression behaviors. Wild-type (WT) mice displayed increased anxiety and depression after chronic administration of VPA for 14 days, when the expression of p35 was decreased. To clarify their relationship, we used p39 knockout (KO) mice, in which p35 is the only Cdk5 activator. When p39 KO mice were treated chronically with VPA, unexpectedly, they exhibited fewer anxiety and depression behaviors than WT mice. The effects were p39 cdk5r2 gene-dosage dependent. Together, these results indicate that Cdk5-p39 plays a specific role in VPA-induced anxiety and depression behaviors.

    DOI PubMed

  • An isogenic panel of App knock-in mouse models: Profiling β-secretase inhibition and endosomal abnormalities.

    Naoto Watamura, Kaori Sato, Gen Shiihashi, Ayami Iwasaki, Naoko Kamano, Mika Takahashi, Misaki Sekiguchi, Naomi Mihira, Ryo Fujioka, Kenichi Nagata, Shoko Hashimoto, Takashi Saito, Toshio Ohshima, Takaomi C Saido, Hiroki Sasaguri

    Science advances   8 ( 23 ) eabm6155  2022.06  [Refereed]  [International journal]

     View Summary

    We previously developed single App knock-in mouse models of Alzheimer's disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and evaluated its characteristics. Amyloid β peptide (Aβ) pathology was exhibited by AppG-F mice from 6 to 8 months of age and was accompanied by neuroinflammation. Aβ-secretase inhibitor, verubecestat, attenuated Aβ production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of β-secretase inhibition given that most patients with AD do not carry the Swedish mutations. Comparison of isogenic App knock-in lines revealed that multiple factors, including elevated C-terminal fragment β (CTF-β) and humanization of Aβ might influence endosomal alterations in vivo. Thus, experimental comparisons between different isogenic App, knock-in mouse lines will provide previously unidentified insights into our understanding of the etiology of AD.

    DOI PubMed

  • Involvement of Cdk5 activating subunit p35 in synaptic plasticity in excitatory and inhibitory neurons

    Miyuki Takahashi, Takeru Nakabayashi, Naoki Mita, Xiaohua Jin, Yuta Aikawa, Kodai Sasamoto, Goichi Miyoshi, Mariko Miyata, Takafumi Inoue, Toshio Ohshima

    MOLECULAR BRAIN   15 ( 1 )  2022.04  [Refereed]

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    Cyclin-dependent kinase 5 (Cdk5) /p35 is involved in many developmental processes of the central nervous system. Cdk5/p35 is also implicated in synaptic plasticity, learning and memory. Several lines of conditional Cdk5 knockout mice (KO) have been generated and have shown different outcomes for learning and memory. Here, we present our analysis of p35 conditional KO mice (p35cKO) in hippocampal pyramidal neurons or forebrain GABAergic neurons using electrophysiological and behavioral methods. In the fear conditioning task, CamKII-p35cKO mice showed impaired memory retention. Furthermore, NMDAR-dependent long-term depression (LTD) induction by low-frequency stimuli in hippocampal slices from CamkII-p35cKO mice was impaired compared to that in control mice. In contrast, Dlx-p35cKO mice showed no abnormalities in behavioral tasks and electrophysiological analysis in their hippocampal slices. These results indicated that Cdk5/p35 in excitatory neurons is important for the hippocampal synaptic plasticity and associative memory retention.

    DOI

  • Requirement of CRMP2 Phosphorylation in Neuronal Migration of Developing Mouse Cerebral Cortex and Hippocampus and Redundant Roles of CRMP1 and CRMP4

    Yuki Yamazaki, Maho Moizumi, Jun Nagai, Yoshiki Hatashita, Tianhong Cai, Papachan Kolattukudy, Takafumi Inoue, Yoshio Goshima, Toshio Ohshima

    Cerebral cortex (New York, N.Y. : 1991)   32 ( 3 ) 520 - 527  2022.01  [Refereed]

     View Summary

    The mammalian cerebral cortex is characterized by a 6-layer structure, and proper neuronal migration is critical for its formation. Cyclin-dependent kinase 5 (Cdk5) has been shown to be a critical kinase for neuronal migration. Several Cdk5 substrates have been suggested to be involved in ordered neuronal migration. However, in vivo loss-of-function studies on the function of Cdk5 phosphorylation substrates in neuronal migration in the developing cerebral cortex have not been reported. In this study, we demonstrated that Cdk5-mediated phosphorylation of collapsing mediator protein (CRMP) 2 is critical for neuronal migration in the developing cerebral cortex with redundant functions of CRMP1 and CRMP4. The cerebral cortices of triple-mutant CRMP1 knock-out (KO); CRMP2 knock-in (KI)/KI; and CRMP4 KO mice showed disturbed positioning of layers II-V neurons in the cerebral cortex. Further experiments using bromodeoxyuridine birthdate-labeling and in utero electroporation implicated radial migration defects in cortical neurons. Ectopic neurons were detected around the CA1 region and dentate gyrus in CRMP1 KO; CRMP2 KI/KI; and CRMP4 KO mice. These results suggest the importance of CRMP2 phosphorylation by Cdk5 and redundancy of CRMP1 and CRMP4 in proper neuronal migration in the developing cerebral cortex and hippocampus.

    DOI PubMed

  • Recent Advances in the Modeling of Alzheimer's Disease.

    Hiroki Sasaguri, Shoko Hashimoto, Naoto Watamura, Kaori Sato, Risa Takamura, Kenichi Nagata, Satoshi Tsubuki, Toshio Ohshima, Atsushi Yoshiki, Kenya Sato, Wakako Kumita, Erika Sasaki, Shinobu Kitazume, Per Nilsson, Bengt Winblad, Takashi Saito, Nobuhisa Iwata, Takaomi C Saido

    Frontiers in neuroscience   16   807473 - 807473  2022  [Refereed]  [International journal]

     View Summary

    Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer's disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed ( 1st generation models ). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid β peptide (Aβ) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice) were developed ( 2nd generation models ). While these models are interesting given that they exhibit Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aβ metabolism and clearance because the Aβ in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aβ antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aβ pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aβ metabolism, and to identifying somatostatin receptor subtypes involved in Aβ degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the AppNL-F and Psen1 P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the AppNL-G-F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aβ. Furthermore, a derivative AppG-F line devoid of Swedish mutations which can be utilized for preclinical studies of β-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aβ immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-K ATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aβ deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

    DOI PubMed

  • A 3rd generation mouse model of Alzheimer's disease shows early and increased cored plaque pathology composed of wild-type human amyloid β peptide.

    Kaori Sato, Naoto Watamura, Ryo Fujioka, Naomi Mihira, Misaki Sekiguchi, Kenichi Nagata, Toshio Ohshima, Takashi Saito, Takaomi C Saido, Hiroki Sasaguri

    The Journal of biological chemistry     101004 - 101004  2021.07  [International journal]

     View Summary

    We previously developed single App knock-in mouse models of Alzheimer's disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice, respectively). These models showed Aβ pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aβ metabolism and clearance because the Arctic mutation renders Aβ resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aβ antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer's disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aβ by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new 3rd generation mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice, and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

    DOI PubMed

  • Cortical transcriptome analysis after spinal cord injury reveals the regenerative mechanism of central nervous system in CRMP2 knock-in mice.

    Ayaka Sugeno, Wenhui Piao, Miki Yamazaki, Kiyofumi Takahashi, Koji Arikawa, Hiroko Matsunaga, Masahito Hosokawa, Daisuke Tominaga, Yoshio Goshima, Haruko Takeyama, Toshio Ohshima

    Neural regeneration research   16 ( 7 ) 1258 - 1265  2021.07  [Refereed]  [International journal]

     View Summary

    Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

    DOI PubMed

  • Modality-Specific Impairment of Hippocampal CA1 Neurons of Alzheimer's Disease Model Mice.

    Risa Takamura, Kotaro Mizuta, Yukiko Sekine, Tanvir Islam, Takashi Saito, Masaaki Sato, Masamichi Ohkura, Junichi Nakai, Toshio Ohshima, Takaomi C Saido, Yasunori Hayashi

    The Journal of neuroscience : the official journal of the Society for Neuroscience   41 ( 24 ) 5315 - 5329  2021.06  [Refereed]  [International journal]

     View Summary

    Impairment of episodic memory, a class of memory for spatiotemporal context of an event, is an early symptom of Alzheimer's disease. Both spatial and temporal information are encoded and represented in the hippocampal neurons, but how these representations are impaired under amyloid β (Aβ) pathology remains elusive. We performed chronic imaging of the hippocampus in awake male amyloid precursor protein (App) knock-in mice behaving in a virtual reality environment to simultaneously monitor spatiotemporal representations and the progression of Aβ depositions. We found that temporal representation is preserved, whereas spatial representation is significantly impaired in the App knock-in mice. This is because of the overall reduction of active place cells, but not time cells, and compensatory hyperactivation of remaining place cells near Aβ aggregates. These results indicate the differential impact of Aβ aggregates on two major modalities of episodic memory, suggesting different mechanisms for forming and maintaining these two representations in the hippocampus.

    DOI PubMed

  • Phosphorylation of Collapsin Response Mediator Protein 1 (CRMP1) at Tyrosine 504 residue regulates Semaphorin 3A‐induced cortical dendritic growth

    Takeshi Kawashima, Aoi Jitsuki‐Takahashi, Kohtaro Takizawa, Susumu Jitsuki, Takuya Takahashi, Toshio Ohshima, Yoshio Goshima, Fumio Nakamura

    Journal of Neurochemistry    2021.01

    DOI

  • Loss of Collapsin Response Mediator Protein 4 Attenuates 6-Hydroxydopamine-Induced Impairments in a Mouse Model of Parkinson's Disease.

    Wenting Li, Yoshio Goshima, Toshio Ohshima

    Neurochemical research    2020.07  [Refereed]  [International journal]

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    Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by impaired motor symptoms induced by the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc). Many factors are speculated to operate in the mechanism of PD, including oxidative stress, mitochondrial dysfunction, abnormal protein handling, and PD induced apoptosis. Besides, researchers have recently shown that inflammatory secretions may engage neighboring cells such as astrocytes, which then induce autocrine and paracrine responses that amplify the inflammation, leading to neurodegeneration. In the present study, we analyzed the neuroprotective and anti-inflammatory effects of collapsin response mediator protein 4 (CRMP4) deletion in 6-hydroxydopamine (6-OHDA)-injected male mice, as well as its effects on motor impairments. Our findings indicated that the deletion of CRMP4 could maintain the TH-positive fibers in the striatum and the TH-positive cells in SNc, attenuate the inflammatory responses, and improve motor coordination and rotational behavior. Furthermore, based on our findings at the early time points, we hypothesized that primary differences between the Crmp4+/+ and Crmp4-/- mice may occur in microglia instead of neurons. Although further work should be carried out to clarify the specific role of CRMP4 in the pathogenesis of PD, our findings suggest that it could be a possible target for the treatment of PD.

    DOI PubMed

  • Phosphorylation of CRMP2 is required for migration and positioning of Purkinje cells: Redundant roles of CRMP1 and CRMP4.

    Yuki Yamazaki, Jun Nagai, Satoshi Akinaga, Yumeno Koga, Masaya Hasegawa, Miyuki Takahashi, Naoya Yamashita, Papachan Kolattukudy, Yoshio Goshima, Toshio Ohshima

    Brain research   1736   146762 - 146762  2020.06  [Refereed]  [International journal]

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    Proper migration and positioning of Purkinje cells are important for formation of the developing cerebellum. Although several cyclin-dependent kinase 5 (Cdk5) substrates are known to be critical for ordered neuronal migration, there are no reports of mutant mouse-based, in vivo studies on the function of Cdk5-phosphorylation substrates in migration of Purkinje cells. We focused on the analysis of collapsin response mediator protein 2 (CRMP2), one of the Cdk5 substrates, because a previous study reported migration defects of cortical neurons with shRNA-mediated knockdown of CRMP2. However, CRMP2 KI/KI mice, in which Cdk5-phosphorylation is inhibited, showed little defects in Purkinje cell migration and positioning. We hypothesized compensatory redundant functions of the other CRMPs, and analyzed the migration and positioning of Purkinje cells in the cerebellum in every combination of CRMP1 knockout (KO), CRMP2 KI/KI, and CRMP4 KO mice. Severe disturbance of migration and positioning of Purkinje cells were observed in the triple mutant mice. We also found motor coordination defects in the triple CRMPs mutant mice. These results suggest the importance of both, phosphorylation of CRMP2 by Cdk5 and the redundant functions of CRMP1 and CRMP4 in proper migration and positioning of Purkinje cells in developing cerebellum.

    DOI PubMed

  • Lanthionine ketimine ester improves outcome in an MPTP-induced mouse model of Parkinson's disease via suppressions of CRMP2 phosphorylation and microglial activation.

    Kentaro Togashi, Masaya Hasegawa, Jun Nagai, Ken Kotaka, Arina Yazawa, Miyuki Takahashi, Daiki Masukawa, Yoshio Goshima, Kenneth Hensley, Toshio Ohshima

    Journal of the neurological sciences   413   116802 - 116802  2020.03  [Refereed]  [International journal]

     View Summary

    Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (L-Dopa), the current main treatment for PD, reduces PD symptoms by partially replacing dopamine, but it does not slow neurodegeneration. Recent studies have evidenced that neuroinflammatory processes contribute to the degeneration of dopaminergic neurons in the SNc under cytopathic conditions, while other lines of inquiry have implicated phosphorylation of collapsin response mediator protein 2 (CRMP2) as a causal factor in axonal retraction after neural injury. We recently reported on the therapeutic effect of lanthionine ketimine ester (LKE) which associates with CRMP2 following axonal injury in the spinal cord. In the present study, we report that LKE protects SNc dopaminergic neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) challenge, a common model for PD, and reduces the number of activated microglia proximal to the damaged SNc. The results also show that MPTP-induced motor impairment was suppressed in LKE treatment. Furthermore, the results show that LKE inhibits the elevation of CRMP2 phosphorylation in dopaminergic neurons in the SNc after MPTP injection. These data suggest that modification of CRMP2 phosphorylation and suppression of microglial activation with LKE administration may represent a novel strategy for slowing progress of pathological processes in PD.

    DOI PubMed

  • Collapsin Response Mediator Proteins: Their Biological Functions and Pathophysiology in Neuronal Development and Regeneration.

    Fumio Nakamura, Toshio Ohshima, Yoshio Goshima

    Frontiers in cellular neuroscience   14   188 - 188  2020  [Refereed]  [International journal]

     View Summary

    Collapsin response mediator proteins (CRMPs), which consist of five homologous cytosolic proteins, are one of the major phosphoproteins in the developing nervous system. The prominent feature of the CRMP family proteins is a new class of microtubule-associated proteins that play important roles in the whole process of developing the nervous system, such as axon guidance, synapse maturation, cell migration, and even in adult brain function. The CRMP C-terminal region is subjected to posttranslational modifications such as phosphorylation, which, in turn, regulates the interaction between the CRMPs and various kinds of proteins including receptors, ion channels, cytoskeletal proteins, and motor proteins. The gene-knockout of the CRMP family proteins produces different phenotypes, thereby showing distinct roles of all CRMP family proteins. Also, the phenotypic analysis of a non-phosphorylated form of CRMP2-knockin mouse model, and studies of pharmacological responses to CRMP-related drugs suggest that the phosphorylation/dephosphorylation process plays a pivotal role in pathophysiology in neuronal development, regeneration, and neurodegenerative disorders, thus showing CRMPs as promising target molecules for therapeutic intervention.

    DOI PubMed

  • Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes

    Hirotaka Fujita, Ryota Oikawa, Mayu Hayakawa, Fumiaki Tomoike, Yasuaki Kimura, Hiroyuki Okuno, Yoshiki Hatashita, Carolina Fiallos Oliveros, Haruhiko Bito, Toshio Ohshima, Satoshi Tsuneda, Hiroshi Abe, Takafumi Inoue

    Journal of Biological Chemistry   in press  2020  [Refereed]  [International journal]

  • Genetic inhibition of CRMP2 phosphorylation delays Wallerian degeneration after optic nerve injury.

    Yuki Kinoshita, Syunsuke Kondo, Kazuya Takahashi, Jun Nagai, Shuji Wakatsuki, Toshiyuki Araki, Yoshio Goshima, Toshio Ohshima

    Biochem Biophys Res Commun.   514 ( 4 ) 1037 - 1039  2019.07  [Refereed]  [International journal]

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    Axonal degeneration occurs in patients with various neurological diseases and traumatic nerve injuries, and Wallerian degeneration is a phenomenon in the prototypical axonal degradation that is observed after injury. Collapsin response mediator protein 2 (CRMP2) is phosphorylated by glycogen synthase kinase 3β (GSK3β), and it is involved in Wallerian degeneration after optic nerve injury. We previously developed a CRMP2 knock-in (CRMP2 KI) mouse line, in which CRMP2 phosphorylation by GSK3β is inhibited; however, Wallerian degeneration in CRMP2 KI mice has not yet been examined. In this study, we examined whether Wallerian degeneration of the optic nerve is suppressed in CRMP2 KI mice. Using one eye removal model, we compared Wallerian degeneration of the optic nerve based on histological and biochemical analyses. Our experimental results indicated that the genetic inhibition of CRMP2 phosphorylation delays Wallerian degeneration after optic nerve injury.

    DOI PubMed

  • Genetic inhibition of CRMP2 phosphorylation at serine 522 promotes axonal regeneration after optic nerve injury.

    Kondo S, Takahashi K, Kinoshita Y, Nagai J, Wakatsuki S, Araki T, Goshima Y, Ohshima T

    Scientific reports   9 ( 1 ) 7188 - 7188  2019.05  [Refereed]  [International journal]

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    Axonal degeneration occurs in various neurological diseases and traumatic nerve injury, and axonal regeneration is restricted by inhibitory factors in the central nervous system. Cyclin-dependent kinase 5 and glycogen synthase kinase 3β (GSK3β) are activated by one of those inhibitors, and collapsin response mediator protein 2 (CRMP2) is phosphorylated by both kinases. We previously developed a CRMP2 knock-in (CRMP2 KI) mouse line, in which CRMP2 phosphorylation at Ser 522 is inhibited. Because CRMP2 KI mice showed promotion of axonal regeneration after spinal cord injury, we hypothesized that CRMP2 KI mice would show higher axonal regeneration after optic nerve injury. In this study, we first show that depolymerization of microtubules after optic nerve crush (ONC) injury was suppressed in CRMP2 KI mice. Loss of retinal ganglia cells was also reduced after ONC. We found that protein level of GAP43, a marker of regenerative axons, was higher in the optic nerve from CRMP2KI than that from wild type 4 weeks after of ONC. We further observed increased numbers of axons labeled by tracer in the optic nerve after ONC in CRMP2 KI mice. These results suggest that inhibition of phosphorylation of CRMP2 suppresses axonal degeneration and promotes axonal regeneration after optic nerve injury.

    DOI PubMed

  • Inhibition of collapsin response mediator protein-2 phosphorylation ameliorates motor phenotype of ALS model mice expressing SOD1G93A.

    Yurika Numata-Uematsu, Shuji Wakatsuki, Seiichi Nagano, Megumi Shibata, Kazuhisa Sakai, Noritaka Ichinohe, Katsuhiko Mikoshiba, Toshio Ohshima, Naoya Yamashita, Yoshiro Goshima, Toshiyuki Araki

    Neuroscience research   139   63 - 68  2019.02  [Refereed]  [International journal]

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    Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disease characterized by the selective degeneration of motor neurons leading to paralysis and immobility. Missense mutations in the gene coding for the Cu2+/Zn2+ superoxide dismutase 1 (SOD1) accounts for 15-20% of familial ALS, and mice overexpressing ALS-linked SOD1 mutants have been frequently used as an animal model for ALS. Degeneration of motor neurons in ALS progresses in a manner called "dying back", in which the degeneration of synapses and axons precedes the loss of cell bodies. Phosphorylation of collapsin response mediator protein 2 (CRMP2) is implicated in the progression of neuronal/axonal degeneration of different etiologies. To evaluate the role of CRMP2 phosphorylation in ALS pathogenesis, we utilized CRMP2 S522A knock-in (CRMP2ki/ki) mice, in which the serine residue 522 was homozygously replaced with alanine and thereby making CRMP2 no longer phosphorylatable by CDK5 or GSK3B. We found that the CRMP2ki/ki/SOD1G93A mice showed delay in the progression of the motor phenotype compared to their SOD1G93-Tg littermates. Histological analysis revealed that the CRMP2ki/ki/SOD1G93A mice retained more intact axons and NMJs than their SOD1G93A-Tg littermates. These results suggest that the phosphorylation of CRMP2 may contribute to the axonal degeneration of motor neurons in ALS.

    DOI PubMed

  • Genetic suppression of collapsin response mediator protein 2 phosphorylation improves outcome in methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's model mice.

    Togashi K, Hasegawa M, Nagai J, Tonouchi A, Masukawa D, Hensley K, Goshima Y, Ohshima T

    Genes to cells : devoted to molecular & cellular mechanisms   24 ( 1 ) 31 - 40  2018.10  [Refereed]  [International journal]

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    Parkinson's disease (PD) is a common neurodegenerative disorder characterized by slow and progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (l-Dopa), the current main treatment for PD, supplies dopamine, but it does not prevent neurodegeneration. There is thus no promising remedy for PD. Recent in vitro study showed the increase in the phosphorylation levels of Collapsin Response Mediator Protein 2 (CRMP2) is involved in dopaminergic axon degeneration. In the present study, we report elevation of CRMP2 phosphorylation in dopaminergic neurons in SNc after challenge with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a common model for PD. Genetic suppression of CRMP2 phosphorylation by mutation of the obligatory Cyclin-dependent kinase 5 (Cdk5)-targeted serine-522 site prevented axonal degradation in the nigrostriatal pathway of transgenic mice. As a result, the degree of MPTP-induced motor impairment in the rotarod test was suppressed. These results suggest that suppression of CRMP2 phosphorylation may be a novel therapeutic target for PD.

    DOI PubMed

  • Proteome and behavioral alterations in phosphorylation-deficient mutant Collapsin Response Mediator Protein2 knock-in mice.

    Haruko Nakamura, Aoi Takahashi-Jitsuki, Hiroko Makihara, Tetsuya Asano, Yayoi Kimura, Jun Nakabayashi, Naoya Yamashita, Yuko Kawamoto, Fumio Nakamura, Toshio Ohshima, Hisashi Hirano, Fumiaki Tanaka, Yoshio Goshima

    Neurochemistry international   119   207 - 217  2018.10  [Refereed]  [International journal]

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    CRMP2, alternatively designated as DPYSL2, was the first CRMP family member to be identified as an intracellular molecule mediating the signaling of the axon guidance molecule Semaphorin 3A (Sema3A). In Sema3A signaling, cyclin-dependent kinase 5 (Cdk5) primarily phosphorylates CRMP2 at Ser522. Glycogen synthase kinase-3β (GSK-3β) subsequently phosphorylates the residues of Thr509 and Thr514 of CRMP2. Previous studies showed that CRMP2 is involved in pathogenesis of neurological disorders such as Alzheimer's disease. In Alzheimer's disease, hyper-phosphorylated forms of CRMP2 are accumulated in the paired helical filaments. To get insight into the possible involvement of the phosphorylation of CRMP2 in pathogenesis of neurological disorders, we previously created CRMP2 S522A knock-in (crmp2ki/ki) mice and demonstrated that the phosphorylation of CRMP2 at Ser522 is involved in normal dendrite patterning in cortical neurons. However, the behavioral impact and in vivo signaling network of the CRMP2 phosphorylation are not fully understood. In this study, we performed behavioral and proteomics analysis of crmp2ki/ki mice. The crmp2ki/ki mice appeared healthy and showed no obvious differences in physical characteristics compared to wild-type mice, but they showed impaired emotional behavior, reduced sociality, and low sensitivity to pain stimulation. Through mass-spectrometry-based proteomic analysis, we found that 59 proteins were increased and 77 proteins were decreased in the prefrontal cortex of crmp2ki/ki mice. Notably, CRMP3, CRMP4, and CRMP5, the other CRMP family proteins, were increased in crmp2ki/ki mice. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses identified 14 pathways in increased total proteins and 13 pathways in decreased total proteins which are associated with the pathogenesis of Parkinson's, Alzheimer's, and Huntington's diseases. We also detected 20 pathways in increased phosphopeptides and 16 pathways in decreased phosphopeptides including "inflammatory mediator regulation of TRP channels" in crmp2ki/ki mice. Our study suggests that the phosphorylation of CRMP2 at Ser522 is involved in the signaling pathways that may be related to neuropsychiatric and neurodegenerative diseases and pain.

    DOI PubMed

  • Excitatory Neuronal Hubs Configure Multisensory Integration of Slow Waves in Association Cortex

    Satoshi Kuroki, Takamasa Yoshida, Hidekazu Tsutsui, Mizuho Iwama, Reiko Ando, Takayuki Michikawa, Atsushi Miyawaki, Toshio Ohshima, Shigeyoshi Itohara

    Cell Reports   22 ( 11 ) 2809 - 2817  2018.03  [Refereed]

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    Multisensory integration (MSI) is a fundamental emergent property of the mammalian brain. During MSI, perceptual information encoded in patterned activity is processed in multimodal association cortex. The systems-level neuronal dynamics that coordinate MSI, however, are unknown. Here, we demonstrate intrinsic hub-like network activity in the association cortex that regulates MSI. We engineered calcium reporter mouse lines based on the fluorescence resonance energy transfer sensor yellow cameleon (YC2.60) expressed in excitatory or inhibitory neurons. In medial and parietal association cortex, we observed spontaneous slow waves that self-organized into hubs defined by long-range excitatory and local inhibitory circuits. Unlike directional source/sink-like flows in sensory areas, medial/parietal excitatory and inhibitory hubs had net-zero balanced inputs. Remarkably, multisensory stimulation triggered rapid phase-locking mainly of excitatory hub activity persisting for seconds after the stimulus offset. Therefore, association cortex tends to form balanced excitatory networks that configure slow-wave phase-locking for MSI. Video Abstract [Figure presented] Kuroki et al. performed cell-type-specific, wide-field FRET-based calcium imaging to visualize cortical network activity induced by multisensory inputs. They observed phase-locking of cortical slow waves in excitatory neuronal hubs in association cortical areas that may underlie multisensory integration.

    DOI PubMed

  • Inducible Knockout of the Cyclin-Dependent Kinase 5 Activator p35 Alters Hippocampal Spatial Coding and Neuronal Excitability.

    Eriko Kamiki, Roman Boehringer, Denis Polygalov, Toshio Ohshima, Thomas J McHugh

    Frontiers in cellular neuroscience   12   138 - 138  2018  [Refereed]  [International journal]

     View Summary

    p35 is an activating co-factor of Cyclin-dependent kinase 5 (Cdk5), a protein whose dysfunction has been implicated in a wide-range of neurological disorders including cognitive impairment and disease. Inducible deletion of the p35 gene in adult mice results in profound deficits in hippocampal-dependent spatial learning and synaptic physiology, however the impact of the loss of p35 function on hippocampal in vivo physiology and spatial coding remains unknown. Here, we recorded CA1 pyramidal cell activity in freely behaving p35 cKO and control mice and found that place cells in the mutant mice have elevated firing rates and impaired spatial coding, accompanied by changes in the temporal organization of spiking both during exploration and rest. These data shed light on the role of p35 in maintaining cellular and network excitability and provide a physiological correlate of the spatial learning deficits in these mice.

    DOI PubMed

  • Endoplasmic reticulum stress responses in mouse models of Alzheimer's disease: Overexpression paradigm versus knockin paradigm

    Shoko Hashimoto, Ayano Ishii, Naoko Kamano, Naoto Watamura, Takashi Saito, Toshio Ohshima, Makoto Yokosuka, Takaomi C. Saido

    Journal of Biological Chemistry   293 ( 9 ) 3118 - 3125  2018

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    Endoplasmic reticulum (ER) stress is believed to play an important role in the etiology of Alzheimer's disease (AD). The accumulation of misfolded proteins and perturbation of intracellular calcium homeostasis are thought to underlie the induction of ER stress, resulting in neuronal dysfunction and cell death. Several reports have shown an increased ER stress response in amyloid precursor protein (APP) and presenilin1 (PS1) double-transgenic (Tg) AD mouse models. However, whether the ER stress observed in these mouse models is actually caused byADpathology remains unclear. APP and PS1 contain one and nine transmembrane domains, respectively, for which it has been postulated that overexpressed membrane proteins can become wedged in a misfolded configuration in ER membranes, thereby inducing nonspecific ER stress. Here, we used an App-knockin (KI) AD mouse model that accumulates amyloid-β (Aβ) peptide without overexpressing APP to investigate whether the ER stress response is heightened because ofAβ pathology. Thorough examinations indicated that no ER stress responses arose in App-KI or single APP-Tg mice. These results suggest thatPS1overexpression or mutation induced a nonspecific ER stress response that was independent of Aβ pathology in the double-Tg mice. Moreover, we observed no ER stress in a mouse model of tauopathy (P301S-Tau-Tg mice) at various ages, suggesting that ER stress is also not essential in tau pathology-induced neurodegeneration. We conclude that the role of ER stress in AD pathogenesis needs to be carefully addressed in future studies.

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  • A role for CA3 in social recognition memory

    Chiang, Ming Ching, Chiang, Ming Ching, Huang, Arthur J.Y, Wintzer, Marie E, Ohshima, Toshio, McHugh, Thomas J

    Behavioural Brain Research   354   22 - 30  2018.01  [International journal]

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    © 2018 Elsevier B.V. Social recognition memory is crucial for survival across species, underlying the need to correctly identify conspecifics, mates and potential enemies. In humans the hippocampus is engaged in social and episodic memory, however the circuit mechanisms of social memory in rodent models has only recently come under scrutiny. Work in mice has established that the dorsal CA2 and ventral CA1 regions play critical roles, however a more comprehensive comparative analyses of the circuits and mechanisms required has not been reported. Here we employ conditional genetics to examine the differential contributions of the hippocampal subfields to social memory. We find that the deletion of NMDA receptor subunit 1 gene (NR1), which abolishes NMDA receptor synaptic plasticity, in CA3 pyramidal cells led to deficits in social memory; however, mice lacking the same gene in DG granule cells performed indistinguishable from controls. Further, we use conditional pharmacogenetic inhibition to demonstrate that activity in ventral, but not dorsal, CA3 is necessary for the encoding of a social memory. These findings demonstrated CA3 pyramidal cell plasticity and transmission contribute to the encoding of social stimuli and help further identify the distinct circuits underlying the role of the hippocampus in social memory.

    DOI PubMed

  • Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners

    Bozong Xu, Ayumi Kumazawa, Shunsuke Kobayashi, Shin-ichi Hisanaga, Takafumi Inoue, Toshio Ohshima

    DEVELOPMENTAL NEUROBIOLOGY   77 ( 10 ) 1175 - 1187  2017.10  [Refereed]

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    Cyclin-dependent kinase 5 (Cdk5) is recognized as a unique member among other Cdks due to its versatile roles in many biochemical processes in the nervous system. The proper development of neuronal dendrites is required for the formation of complex neural networks providing the physiological basis of various neuronal functions. We previously reported that sparse dendrites were observed on cultured Cdk5-null Purkinje cells and Purkinje cells in Wnt1(cre)-mediated Cdk5 conditional knockout (KO) mice. In the present study, we generated L7(cre)-mediated p35; p39 double KO (L7(cre)-p35(f/f); p39(-/-)) mice whose Cdk5 activity was eliminated specifically in Purkinje cells of the developing cerebellum. Consequently, these mice exhibited defective Purkinje cell migration, motor coordination deficiency and a Purkinje dendritic abnormality similar to what we have observed before, suggesting that dendritic growth of Purkinje cells was cell-autonomous in vivo. We found that mixed and overlay cultures of WT cerebellar cells rescued the dendritic deficits in Cdk5-null Purkinje cells, however, indicating that Purkinje cell dendritic development was also supported by non-cell-autonomous factors. We then again rescued these abnormalities in vitro by applying exogenous brain-derived neurotrophic factor (BDNF). Based on the results from culture experiments, we attempted to rescue the developmental defects of Purkinje cells in L7(cre)-p35(f/f); p39(-/-) mice by using a TrkB agonist. We observed partial rescue of morphological defects of dendritic structures of Purkinje cells. These results suggest that Cdk5 activity is required for Purkinje cell dendritic growth in cell-autonomous and non-cell-autonomous manners. (c) 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1175-1187, 2017

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  • Wnt and Shh signals regulate neural stem cell proliferation and differentiation in the optic tectum of adult zebrafish

    Shiori Shitasako, Yoko Ito, Ryoichi Ito, Yuto Ueda, Yuki Shimizu, Toshio Ohshima

    DEVELOPMENTAL NEUROBIOLOGY   77 ( 10 ) 1206 - 1220  2017.10  [Refereed]

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    Adult neurogenesis occurs more commonly in teleosts, represented by zebrafish, than in mammals. Zebrafish is therefore considered a suitable model to study adult neurogenesis, for which the regulatory molecular mechanisms remain little known. Our previous study revealed that neuroepithelial-like neural stem cells (NSCs) are located at the edge of the dorsomedial region. We also showed that Notch signaling inhibits NSC proliferation in this region. In the present study, we reported the expression of Wnt and Shh signaling components in this region of the optic tectum. Moreover, inhibitors of Wnt and Shh signaling suppressed NSC proliferation, suggesting that these pathways promote NSC proliferation. Shh is particularly required for maintaining Sox2-positive NSCs. Our experimental data also indicate the involvement of these signaling pathways in neural differentiation from NSCs. (c) 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1206-1220, 2017

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  • Zebrafish Mecp2 is required for proper axonal elongation of motor neurons and synapse formation

    Keisuke Nozawa, Yanbin Lin, Ryota Kubodera, Yuki Shimizu, Hideomi Tanaka, Toshio Ohshima

    DEVELOPMENTAL NEUROBIOLOGY   77 ( 9 ) 1101 - 1113  2017.09  [Refereed]

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    Rett syndrome is a severe neurodevelopmental disorder. It is caused by a mutation in methyl-CpG binding protein 2 (MecP2), a transcriptional regulator that recruits protein complexes involved in histone modification and chromatin remodeling. However, the role of Mecp2 in Rett syndrome remains unclear. In this study, we investigated the function of Mecp2 in neuronal development using zebrafish embryos. Mecp2 expression was detected ubiquitously in the central nervous system and muscles at 28 h postfertilization (hpf). We injected an antisense morpholino oligonucleotide (AMO) to induce Mecp2 knockdown phenotype. In mecp2 morphants (embryos with Mecp2 knockdown by AMO) at 28 and 72 hpf, we found an increase in abnormal axonal branches of caudal primary motor neurons and a decrease in motor activity. In mecp2 morphants at 24 hpf, we observed an increase in the expression of an mecp2 downstream candidate gene, brain derived neurotrophic factor (bdnf). In mecp2 morphants at 72 hpf, the presynaptic area stained by an anti-SV2 antibody was increased at the neuromuscular junction (NMJ). Interestingly, the size of SV2-positive presynaptic area at the NMJ was also increased following bdnf mRNA injection, while it was normalized in a double knockdown of mecp2 and bdnf. These results imply that Mecp2 is an important functional regulator of bdnf gene expression during neural circuit formation in zebrafish embryo. (c) 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1101-1113, 2017

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  • CRMPs Function in Neurons and Glial Cells: Potential Therapeutic Targets for Neurodegenerative Diseases and CNS Injury

    Jun Nagai, Rina Baba, Toshio Ohshima

    MOLECULAR NEUROBIOLOGY   54 ( 6 ) 4243 - 4256  2017.08  [Refereed]

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    Neurodegeneration in the adult mammalian central nervous system (CNS) is fundamentally accelerated by its intrinsic neuronal mechanisms, including its poor regenerative capacity and potent extrinsic inhibitory factors. Thus, the treatment of neurodegenerative diseases faces many obstacles. The degenerative processes, consisting of axonal/dendritic structural disruption, abnormal axonal transport, release of extracellular factors, and inflammation, are often controlled by the cytoskeleton. From this perspective, regulators of the cytoskeleton could potentially be a therapeutic target for neurodegenerative diseases and CNS injury. Collapsin response mediator proteins (CRMPs) are known to regulate the assembly of cytoskeletal proteins in neurons, as well as control axonal growth and neural circuit formation. Recent studies have provided some novel insights into the roles of CRMPs in several inhibitory signaling pathways of neurodegeneration, in addition to its functions in neurological disorders and CNS repair. Here, we summarize the roles of CRMPs in axon regeneration and its emerging functions in non-neuronal cells, especially in inflammatory responses. We also discuss the direct and indirect targeting of CRMPs as a novel therapeutic strategy for neurological diseases.

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  • Protein Tyrosine Phosphatase delta Mediates the Sema3A-Induced Cortical Basal Dendritic Arborization through the Activation of Fyn Tyrosine Kinase

    Fumio Nakamura, Takako Okada, Maria Shishikura, Noriko Uetani, Masahiko Taniguchi, Takeshi Yagi, Yoichiro Iwakura, Toshio Ohshima, Yoshio Goshima, Stephen M. Strittmatter

    JOURNAL OF NEUROSCIENCE   37 ( 30 ) 7125 - 7139  2017.07  [Refereed]

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    Leukocyte common antigen-related (LAR) class protein tyrosine phosphatases (PTPs) are critical for axonal guidance; however, their relation to specific guidance cues is poorly defined. We here show that PTP-3, a LAR homolog in Caenorhabditis elegans, is involved in axon guidance regulated by Semaphorin-2A-signaling. PTP delta, one of the vertebrate LAR class PTPs, participates in the Semaphorin-3A (Sema3A)-induced growth cone collapse response of primary cultured dorsal root ganglion neurons from Mus musculus embryos. In vivo, however, the contribution of PTP delta in Sema3A-regualted axon guidance was minimal. Instead, PTP delta played a major role in Sema3A-dependent cortical dendritic growth. Ptp delta(-/-) and Sema3a(-/-) mutant mice exhibited poor arborization of basal dendrites of cortical layer V neurons. This phenotype was observed in both male and female mutants. The double-heterozygous mutants, Ptp delta(+/-); Sema3a(+/-), also showed a similar phenotype, indicating the genetic interaction. In Ptp delta(-/-) brains, Fyn and Src kinases were hyperphosphorylated at their C-terminal Tyr527 residues. Sema3A-stimulation induced dephosphorylation of Tyr527 in the dendrites of wild-type cortical neurons but not of Ptp delta(-/-) Arborization of cortical basal dendrites was reduced in Fyn(-/-) as well as in Ptp delta(+/-); Fyn(+/-) double-heterozygous mutants. Collectively, PTP? mediates Sema3A-signaling through the activation of Fyn by C-terminal dephosphorylation.

    DOI PubMed

  • Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis

    Brian T. D. Tobe, Andrew M. Crain, Alicia M. Winquist, Barbara Calabrese, Hiroko Makihara, Wen-ning Zhao, Jasmin Lalonde, Haruko Nakamura, Glenn Konopaske, Michelle Sidor, Cameron D. Pernia, Naoya Yamashita, Moyuka Wada, Yuuka Inoue, Fumio Nakamura, Steven D. Sheridan, Ryan W. Logan, Michael Brandel, Dongmei Wu, Joshua Hunsberger, Laurel Dorsett, Cordulla Duerr, Ranor C. B. Basa, Michael J. McCarthy, Namrata D. Udeshi, Philipp Mertins, Steven A. Carr, Guy A. Rouleau, Lina Mastrangelo, Jianxue Li, Gustavo J. Gutierrez, LaurenceM. Brill, Nikolaos Venizelos, Guang Chen, Jeffrey S. Nye, Husseini Manji, Jeffrey H. Price, Colleen A. McClung, Hagop S. Akiskal, Martin Alda, De-Maw M. Chuang, Joseph T. Coyle, Yang Liu, Yang D. Teng, Toshio Ohshima, Katsuhiko Mikoshiba, Richard L. Sidman, Shelley Halpain, Stephen J. Haggarty, Yoshio Goshima, Evan Y. Snyder

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   114 ( 22 ) E4462 - E4471  2017.05  [Refereed]

     View Summary

    The molecular pathogenesis of bipolar disorder (BPD) is poorly understood. Using human-induced pluripotent stem cells (hiPSCs) to unravel such mechanisms in polygenic diseases is generally challenging. However, hiPSCs from BPD patients responsive to lithium offered unique opportunities to discern lithium's target and hence gain molecular insight into BPD. By profiling the proteomics of BDP-hiPSC-derived neurons, we found that lithium alters the phosphorylation state of collapsin response mediator protein-2 (CRMP2). Active non-phosphorylated CRMP2, which binds cytoskeleton, is present throughout the neuron; inactive phosphorylated CRMP2, which dissociates from cytoskeleton, exits dendritic spines. CRMP2 elimination yields aberrant dendritogenesis with diminished spine density and lost lithium responsiveness (LiR). The "set-point" for the ratio of pCRMP2: CRMP2 is elevated uniquely in hiPSC-derived neurons from LiR BPD patients, but not with other psychiatric (including lithium-nonresponsive BPD) and neurological disorders. Lithium (and other pathway modulators) lowers pCRMP2, increasing spine area and density. Human BPD brains show similarly elevated ratios and diminished spine densities; lithium therapy normalizes the ratios and spines. Consistent with such "spine-opathies," human LiR BPD neurons with abnormal ratios evince abnormally steep slopes for calcium flux; lithium normalizes both. Behaviorally, transgenic mice that reproduce lithium's postulated site-of-action in dephosphorylating CRMP2 emulate LiR in BPD. These data suggest that the " lithium response pathway" in BPD governs CRMP2's phosphorylation, which regulates cytoskeletal organization, particularly in spines, modulating neural networks. Aberrations in the posttranslational regulation of this developmentally critical molecule may underlie LiR BPD pathogenesis. Instructively, examining the proteomic profile in hiPSCs of a functional agent-even one whose mechanism-of-action is unknown-might reveal otherwise inscrutable intracellular pathogenic pathways.

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  • CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling

    Basem M. Abdallah, Florence Figeac, Kenneth H. Larsen, Nicholas Ditzel, Pankaj Keshari, Adiba Isa, Abbas Jafari, Thomas L. Andersen, Jean-Marie Delaisse, Yoshio Goshima, Toshio Ohshima, Moustapha Kassem

    JOURNAL OF BONE AND MINERAL RESEARCH   32 ( 5 ) 913 - 926  2017.05  [Refereed]

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    We identified the neuroprotein collapsing response mediator protein-4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1-CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain-of-function and loss-of-function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4-deficient mice (Crmp4(-/-)) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild-type controls. Increased bone mass in Crmp4(-/-) mice was associated with enhanced BMP2 signaling and BMP2-induced osteoblast differentiation in Crmp4(-/-) osteoblasts (OBs). Furthermore, Crmp4(-/-) OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4(-/-) OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin-dependent kinase inhibitor 1B, p27(Kip1) and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. (c) 2016 American Society for Bone and Mineral Research.

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  • Cdk5 is required for the positioning and survival of GABAergic neurons in developing mouse striatum.

    Kodai Sasamoto, Jun Nagai, Takeru Nakabayashi, Xiaojuan He, Toshio Ohshima

    Developmental neurobiology   77 ( 4 ) 483 - 492  2017.04  [International journal]

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    Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its activity is dependent upon an association with a neuron-specific activating subunit. It was previously reported that Cdk5-/- mice exhibit perinatal lethality and defective neuronal positioning. In this study, they focused on the analysis of neuronal positioning of GABAergic neurons in the forebrain. Defective formation of the ventral striatum, nucleus accumbens, and olfactory tubercles was found in Cdk5-/- embryos. To further study this abnormal development, we generated and analyzed Dlx5/6-Cre p35 conditional KO (cKO); p39-/- mice in which forebrain GABAergic neurons have lost their Cdk5 kinase activity. Defective formation of the nucleus accumbens and olfactory tubercles as well as neuronal loss in the striatum of Dlx5/6-Cre p35cKO; p39-/- mice was found. Elevated levels of phosphorylated JNK were observed in neonatal striatal samples from Dlx5/6-Cre p35cKO; p39-/- mice, suggestive of neuronal death. These results indicate that Cdk5 is required for the formation of the ventral striatum in a cell-autonomous manner, and loss of the kinase activity of Cdk5 causes GABAergic neuronal death in the developing mouse forebrain. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017.

    DOI PubMed

  • Optic Nerve Input-Dependent Regulation of Neural Stem Cell Proliferation in the Optic Tectum of Adult Zebrafish

    Yuki Sato, Hiroaki Yano, Yuki Shimizu, Hideomi Tanaka, Toshio Ohshima

    DEVELOPMENTAL NEUROBIOLOGY   77 ( 4 ) 474 - 482  2017.04  [Refereed]

     View Summary

    Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial-type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU-positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU-positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli-dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. (C) 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 474-482, 2017.

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  • All-trans retinoic acid improved impaired proliferation of neural stem cells and suppressed microglial activation in the hippocampus in an Alzheimer's mouse model

    Risa Takamura, Naoto Watamura, Miyu Nikkuni, Toshio Ohshima

    JOURNAL OF NEUROSCIENCE RESEARCH   95 ( 3 ) 897 - 906  2017.03  [Refereed]

     View Summary

    Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive impairment with neuronal loss. The number of patients suffering from AD has increased, but none of the present therapies stops the progressive symptoms in patients with AD. It has been reported that the activation of microglial cells induces harmful chronic inflammation, leading to neuronal death. Furthermore, the impairment of adult neurogenesis in the hippocampus has been observed earlier than amyloid plaque formation. Inflammatory response may lead to impaired adult neurogenesis in patients with AD. This study examines the relationship between adult neurogenesis and neuroinflammation using APPswe/PS1M146V/tauP301L (3xTg) mice. We observed a decline in the proliferation of neural stem cells and the occurrence of severe inflammation in the hippocampus of 3xTg mouse brains at 12 months of age. Previously, our research had shown an anti-inflammatory effect of all-trans retinoic acid (ATRA) in the 3xTg mouse brain. We found that ATRA has effects on the recovery of proliferative cells along with suppression of activated microglia in the hippocampus. These results suggest that the inhibition of microglial activation by ATRA leads to recovery of adult neurogenesis in the hippocampus in an AD mouse model. (c) 2016 Wiley Periodicals, Inc.

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  • Lanthionine ketimine ester promotes locomotor recovery after spinal cord injury by reducing neuroinflammation and promoting axon growth

    Ken Kotaka, Jun Nagai, Kenneth Hensley, Toshio Ohshima

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   483 ( 1 ) 759 - 764  2017.01  [Refereed]

     View Summary

    The mammalian central nervous system (CNS) has limited regenerative ability after injury, largely due to scar formation and axonal growth inhibitors. Experimental suppression of neuroinflammation encourages recovery from spinal cord injury (SCI), yet practical means for pharmacologically treating SCI have remained elusive. Lanthionine ketimine (LK) is a natural brain sulfur amino acid metabolite with demonstrated anti-neuroinflammatory and neurotrophic activities. LK and its synthetic brainpenetrating ethyl ester (LKE) promote growth factor-dependent neurite extension in cultured cell and suppress microglial activation in animal models of neurodegeneration. Thus far however, LKE has not been explored as a potential therapy for SCI. The present study investigated the hypothesis that systemic LKE could improve motor functional recovery after SCI in a mouse model. Intraperitoneal administration of LKE (100 mg/kg/d) after near-complete transect of spinal cord at the T7 level significantly improved motor function over a 4-week time course. Vehicle-treated mice, in contrast, demonstrated negligible functional recovery. In terms of histology, LKE treatment reduced pro-neuroinflammatory microglia/ macrophage activation evidenced by quantitative Iba1 labeling and shifted the microglial phenotype toward a more neurotrophic M2 character evidenced by changes in the M2 marker arginase-1. This was correlated with less dense scar formation and more extensive axonal regrowth across the transection site demonstrated by 5-hydroxytryptamine (5HT) immunolabeling of raphespinal tract axons. These data provide evidence that LKE or similar compounds have potential therapeutic value for recovery after certain forms of SCI. (C) 2016 Elsevier Inc. All rights reserved.

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  • CRMP1 and CRMP4 are required for proper orientation of dendrites of cerebral pyramidal neurons in the developing mouse brain

    Ryosuke Takaya, Jun Nagai, Wenfui Piao, Emi Niisato, Takeru Nakabayashi, Yuki Yamazaki, Fumio Nakamura, Naoya Yamashita, Papachan Kolattukudy, Yoshio Goshima, Toshio Ohshima

    BRAIN RESEARCH   1655   161 - 167  2017.01  [Refereed]

     View Summary

    Neural circuit formation is a critical process in brain development. Axon guidance molecules, their receptors, and intracellular mediators are important to establish neural circuits. Collapsin response mediator proteins (CRMPs) are known intercellular mediators of a number of repulsive guidance molecules. Studies of mutant mice suggest roles of CRMPs in dendrite development. However, molecular mechanisms of CRMP-mediated dendritic development remain to elucidate. In this study, we show abnormal orientation of basal dendrites (extension to deeper side) of layer V pyramidal neurons in the cerebral cortex of CRMP4-/- mice. Moreover, we observed severe abnormality in orientation of the basal dendrites of these neurons in double knockout of CRMP1 and 4, suggesting redundant functions of these two genes. Redundant gene functions were also observed in proximal bifurcation phenotype in apical dendrites of hippocampal CA1 pyramidal neurons. These results indicate that CRMP1 and CRMP4 regulate proper orientation of the basal dendrites of layer V neurons in the cerebral cortex.

    DOI

  • Deletion of Crmp4 attenuates CSPG-induced inhibition of axonal growth and induces nociceptive recovery after spinal cord injury

    Jun Nagai, Ryosuke Takaya, Wenhui Piao, Yoshio Goshima, Toshio Ohshima

    MOLECULAR AND CELLULAR NEUROSCIENCE   74   42 - 48  2016.07  [Refereed]

     View Summary

    The capacity for regeneration in the injured adult mammalian central nervous system (CNS) is largely limited by potent inhibitory barriers. Chondroitin sulfate proteoglycans (CSPGs) are major inhibitors of axonal regeneration/sprouting and accumulate at lesion sites after CNS trauma. Despite extensive research during the two decades since their discovery, the molecular mechanisms remain elusive, including intracellular phosphorylation events. Collapsin response mediator protein 4 (CRMP4) is known to directly regulate cytoskeletal dynamics and neurite extension, while phosphorylated CRMP4 loses its binding affinity for cytoskeletal proteins. We have previously found that spinal cord injury (SCI) induces CRMP4 upregulation and phosphorylation and that CRMP4 knockout (Crmp4-/-) mice show behavioral recovery of locomotor function after SCI. However, the role of CRMP4 in the recovery of other forms of physiological function such as sensation remains largely unknown. We here have demonstrated CRMP4 involvement in CSPG-induced inhibitory signaling and nociceptive recovery in Crmp4-/- mice after SCI. We cultured dorsal root ganglion (DRG) neurons on CSPG-coated dishes; Crmp4 deletion overrode CSPG-induced inhibition of axon growth in vitro. CRMP4 levels were increased in DRGs in vivo after SCI. Crmp4-/- mice exhibited axonal growth of sensory neurons and recovery of nociceptive function after spinal transection. These results support Crmp4 deletion as a therapeutic target in the treatment of SCI. (C) 2016 Elsevier Inc. All rights reserved.

    DOI

  • Loss of collapsin response mediator protein 4 suppresses dopaminergic neuron death in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineinduced mouse model of Parkinson's disease

    Aine Tonouchi, Jun Nagai, Kentaro Togashi, Yoshio Goshima, Toshio Ohshima

    JOURNAL OF NEUROCHEMISTRY   137 ( 5 ) 795 - 805  2016.06  [Refereed]

     View Summary

    Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Several lines of evidence suggest that neurodegeneration in PD is accelerated by a vicious cycle in which apoptosis in dopaminergic neurons triggers the activation of microglia and harmful inflammatory processes that further amplify neuronal death. Recently, we demonstrated that the deletion of collapsin response mediator protein 4 (CRMP4) suppresses inflammatory responses and cell death in a mouse model of spinal cord injury, leading to improved functional recovery. We thus hypothesized that Crmp4-1 mice may have limited inflammatory responses and a decrease in the loss of SNc dopaminergic neurons in an 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We observed CRMP4 expression in neurons, astrocytes, and microglia/macrophages following the injection of 25 mg/kg MPTP. We compared the number of dopaminergic neurons and the inflammatory response in SNc between Crmp4+I+ and Crmp4 l mice after MPTP injection. Limited loss of SNc dopaminergic neurons and decreased activations of microglia and astrocytes were observed in Crrnp4-1 mice. These results suggest that CRMP4 is a novel therapeutic target in the treatment of PD patients.

    DOI

  • PPAR gamma agonist pioglitazone improves cerebellar dysfunction at pre-A beta deposition stage in APPswe/PS1dE9 Alzheimer's disease model mice

    Junya Toba, Miyu Nikkuni, Masato Ishizeki, Aya Yoshii, Naoto Watamura, Takafumi Inoue, Toshio Ohshima

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   473 ( 4 ) 1039 - 1044  2016.05  [Refereed]

     View Summary

    Alzheimer's disease (AD) is one of the best known neurodegenerative diseases; it causes dementia and its pathological features include accumulation of amyloid beta (A beta) and neurofibrillary tangles (NFTs) in the brain. Elevated Cdk5 activity and CRMP2 phosphorylation have been reported in the brains of AD model mice at the early stage of the disease, but the significance thereof in human AD remains unelucidated. We have recently reported that A beta accumulation in the cerebellum of AD model APPswe/PS1dE9 (APP/PS1) mice, and cerebellar dysfunctions, such as impairment of motor coordination ability and long-term depression (LTD) induction, at the pre-A beta accumulation stage.
    In the present study, we found increased phosphorylation levels of CRMP2 as well as increased p35 protein levels in the cerebellum of APP/PS1 mice. Interestingly, we show that pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma, normalized the p35 protein and CRMP2 phosphorylation levels in the cerebellum. Impaired motor coordination ability and LTD in APP/PS1 mice were ameliorated by pioglitazone treatment at the pre-A beta accumulation stage. These results suggest a correlation between CRMP2 phosphorylation and AD pathophysiology, and indicate the effectiveness of pioglitazone treatment at the pre-A beta accumulation stage in AD model mice. (C) 2016 Elsevier Inc. All rights reserved.

    DOI

  • Cyclin-Dependent Kinase 5 Regulates Dendritic Spine Formation and Maintenance of Cortical Neuron in the Mouse Brain

    Naoki Mita, Xiaojuan He, Kodai Sasamoto, Tomohide Mishiba, Toshio Ohshima

    CEREBRAL CORTEX   26 ( 3 ) 967 - 976  2016.03  [Refereed]

     View Summary

    Cyclin-dependent kinase 5 (Cdk5) activity is dependent on its association with 1 of 2 neuron-specific activators, p35 or p39. Cdk5 and its activators play an important role in brain development as well as higher functions like synaptic plasticity, learning, and memory. Reduction in p35 was reported in postmortem schizophrenia brain, in which reduced dendritic spine density was observed. Previous in vitro experiments have shown that Cdk5 is involved in dendritic spine formation, although in vivo evidence is limited. We examined dendritic spine formation in inducible-p35 conditional knockout (p35 cKO); p39 KO mice. When we deleted the p35 gene either during early postnatal days or at adult stage, we observed reduced spine densities of layer V neurons in the cerebral cortex and CA1 pyramidal neurons in the hippocampus. We further generated CA1-specific p35 conditional knockout (CA1-p35 cKO) mice and also CA1-p35 cKO; p39 KO mice in which have specific deletion of p35 in the CA1 region of hippocampus. We found a greater reduction in spine densities in CA1 pyramidal neurons in CA1-p35 cKO; p39 KO mice than in CA1-p35 cKO mice. These results indicate that dendritic spine formation and neuronal maintenance are dependent on Cdk5 activity.

    DOI

  • Inhibition of CRMP2 phosphorylation repairs CNS by regulating neurotrophic and inhibitory responses

    Jun Nagai, Kazuki Owada, Yoshiteru Kitamura, Yoshio Goshima, Toshio Ohshima

    EXPERIMENTAL NEUROLOGY   277   283 - 295  2016.03  [Refereed]

     View Summary

    Central nervous system (CNS) regeneration is restricted by both the lack of neurotrophic responses and the presence of inhibitory factors. As of yet, a common mediator of these two pathways has not been identified. Microtubule dynamics is responsible for several key processes after CNS injuries: intracellular trafficking of receptors for neurotrophic factors, axonal retraction by inhibitory factors, and secondary tissue damages by inflammation and scarring. Kinases regulating microtubule organization, such as Cdk5 or GSK3 beta, may play pivotal roles during CNS recovery, but the molecular mechanisms remain to be elucidated. Collapsin response mediator protein 2 (CRMP2) stabilizes cytoskeletal polymerization, while CRMP2 phosphorylation by Cdk5 and GSK3 beta loses its affinity for cytoskeleton proteins, leading to the inhibition of axonal growth. Here, we characterized CRMP2 phosphorylation as the first crucial factor regulating neurotrophic and inhibitory responses after spinal cord injury (SCI). We found that pharmacological inhibition of GSK3 beta enhanced brain-derived neurotrophic factor (BDNF)-induced axonal growth response in cultured dorsal root ganglion (DRG) neurons. DRG neurons from CRMP2 knock-in (Crmp2KI/KI) mice, where CRMP2 phosphorylation was eliminated, showed elevated sensitivity to BDNF as well. Additionally, cultured Crmp2KI/KI neurons exhibited suppressed axonal growth inhibition by chondroitin sulfate proteoglycan (CSPG). These data suggest a couple of new molecular insights: the BDNF/GSK3 beta/CRMP2 and CSPG/GSK3 beta/CRMP2 pathways. Next we tested the significance of CRMP2 phosphorylation after CNS injury in vivo. The phosphorylation level of CRMP2 was enhanced in the injured spinal cord. Crmp2KI/KI mice exhibited prominent recovery of locomotive and nociceptive functions after SCI, which correlated with the enhanced axonal growth of the motor and sensory neurons. Neuroprotective effects against SCI, such as microtubule stabilization, reduced inflammation, and suppressed scarring were also observed by inhibiting CRMP2 phosphorylation. Therefore, inhibition of CRMP2 phosphorylation demonstrates the unique potential to repair SCI by both enhancing sensitivity to BDNF and reducing inhibitory responses. (C) 2016 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Colocalization of Phosphorylated Forms of WAVE1, CRMP2, and Tau in Alzheimer's Disease Model Mice: Involvement of Cdk5 Phosphorylation and the Effect of ATRA Treatment

    Naoto Watamura, Junya Toba, Aya Yoshii, Miyu Nikkuni, Toshio Ohshima

    JOURNAL OF NEUROSCIENCE RESEARCH   94 ( 1 ) 15 - 26  2016.01  [Refereed]

     View Summary

    Alzheimer's disease (AD) is the most common type of dementia among the elderly. Neurofibrillary tangles (NFTs), a major pathological hallmark of AD, are composed of tau protein that is hyperphosphorylated by cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 beta (GSK3 beta). NFTs also contain Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) and collapsin response-mediator protein 2 (CRMP2). Although Cdk5 is known to phosphorylate tau, WAVE1, and CRMP2, the significance of this with respect to NFT formation remains to be elucidated. This study examines the involvement of phosphorylated (p-) CRMP2 and WAVE1 in p-tau aggregates using a triple-transgenic (3xTg; APPswe/PS1M146V/tauP301L) AD mouse model. First, we verified the colocalization of p- WAVE1 and p-CRMP2 with aggregated hyperphosphorylated tau in the hippocampus at 23 months of age. Biochemical analysis revealed the inclusion of p-WAVE1, p-CRMP2, and tau in the sarkosyl-insoluble fractions of hippocampal homogenates. To test the significance of phosphorylation of these proteins further, we administered all-trans-retinoic acid (ATRA) to the 3xTg mice, which downregulates Cdk5 and GSK3b activity. In ATRA-treated mice, fewer and smaller tau aggregates were observed compared with non-ATRA-treated mice. These results suggest the possibility of novel therapeutic target molecules for preventing tau pathology. (C) 2015 Wiley Periodicals, Inc.

    DOI

  • Phosphorylation of CRMP2 by Cdk5 Regulates Dendritic Spine Development of Cortical Neuron in the Mouse Hippocampus

    Xiaohua Jin, Kodai Sasamoto, Jun Nagai, Yuki Yamazaki, Kenta Saito, Yoshio Goshima, Takafumi Inoue, Toshio Ohshima

    NEURAL PLASTICITY   2016  2016  [Refereed]

     View Summary

    Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5) is required for the development and maintenance of dendritic spines of cortical neurons in the mouse brain. Previous in vitro studies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been tested in vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2) in the dendritic spines of cultured hippocampal neurons and in vivo in the mouse brain. When we eliminated CRMP2 phosphorylation in CRMP2(KI/KI) mice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus.

    DOI

  • Colocalization of Phosphorylated Forms of WAVE1, CRMP2, and Tau in Alzheimer's Disease Model Mice: Involvement of Cdk5 Phosphorylation and the Effect of ATRA Treatment

    Naoto Watamura, Junya Toba, Aya Yoshii, Miyu Nikkuni, Toshio Ohshima

    JOURNAL OF NEUROSCIENCE RESEARCH   94 ( 1 ) 15 - 26  2016.01

     View Summary

    Alzheimer's disease (AD) is the most common type of dementia among the elderly. Neurofibrillary tangles (NFTs), a major pathological hallmark of AD, are composed of tau protein that is hyperphosphorylated by cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 beta (GSK3 beta). NFTs also contain Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) and collapsin response-mediator protein 2 (CRMP2). Although Cdk5 is known to phosphorylate tau, WAVE1, and CRMP2, the significance of this with respect to NFT formation remains to be elucidated. This study examines the involvement of phosphorylated (p-) CRMP2 and WAVE1 in p-tau aggregates using a triple-transgenic (3xTg; APPswe/PS1M146V/tauP301L) AD mouse model. First, we verified the colocalization of p- WAVE1 and p-CRMP2 with aggregated hyperphosphorylated tau in the hippocampus at 23 months of age. Biochemical analysis revealed the inclusion of p-WAVE1, p-CRMP2, and tau in the sarkosyl-insoluble fractions of hippocampal homogenates. To test the significance of phosphorylation of these proteins further, we administered all-trans-retinoic acid (ATRA) to the 3xTg mice, which downregulates Cdk5 and GSK3b activity. In ATRA-treated mice, fewer and smaller tau aggregates were observed compared with non-ATRA-treated mice. These results suggest the possibility of novel therapeutic target molecules for preventing tau pathology. (C) 2015 Wiley Periodicals, Inc.

    DOI PubMed

  • Radial glial cell-specific ablation in the adult Zebrafish brain

    Yuki Shimizu, Yoko Ito, Hideomi Tanaka, Toshio Ohshima

    Genesis   53 ( 7 ) 431 - 439  2015.07

     View Summary

    The zebrafish brain can continue to produce new neurons in widespread neurogenic brain regions throughout life. In contrast, neurogenesis in the adult mammalian brain is restricted to the subventricular zone (SVZ) and dentate gyrus (DG). In neurogenic regions in the adult brain, radial glial cells (RGCs) are considered to function as neural stem cells (NSCs). We generated a Tg(gfap:Gal4FF) transgenic zebrafish line, which enabled us to express specific genes in RGCs. To study the function of RGCs in neurogenesis in the adult zebrafish brain, we also generated a Tg(gfap: Gal4FF
    UAS:nfsB-mcherry) transgenic zebrafish line, which allowed us to induce cell death exclusively within RGCs upon addition of metronidazole (Mtz) to the media. RGCs expressing nitroreductase were specifically ablated by the Mtz treatment, decreasing the number of proliferative RGCs. Using the Tg(gfap:Gal4FF
    UAS:nfsB-mcherry) transgenic zebrafish line, we found that RGCs were specifically ablated in the adult zebrafish telencephalon. The Tg(gfap:Gal4FF) line could be useful to study the function of RGCs.

    DOI PubMed

  • Crmp4 deletion promotes recovery from spinal cord injury by neuroprotection and limited scar formation

    Jun Nagai, Yoshiteru Kitamura, Kazuki Owada, Naoya Yamashita, Kohtaro Takei, Yoshio Goshima, Toshio Ohshima

    Scientific Reports   5  2015.02  [Refereed]

     View Summary

    Axonal outgrowth inhibitors and scar formation are two major obstacles to central nervous system (CNS) repair. No target molecule that regulates both axonal growth and scarring has been identified. Here we identified collapsin response mediator protein 4 (CRMP4), a common mediator of inhibitory signals after neural injury, as a crucial factor that contributes to both axonal growth inhibition and scarring after spinal cord injury (SCI). We found increases in the inhibitory and toxic forms of CRMP4 in injured spinal cord. Notably, CRMP4 expression was evident in inflammatory cells as well as in neurons after spinal cord transection. Crmp4(-/-) mice displayed neuroprotection against SCI and reductions in inflammatory response and scar formation. This permissive environment for axonal growth due to CRMP4 deletion restored locomotor activity at an unusually early phase of healing. These results suggest that deletion of CRMP4 is a unique therapeutic strategy that overcomes two obstacles to CNS repair after SCI.

    DOI

  • Neuronal migration and protein kinases

    Toshio Ohshima

    FRONTIERS IN NEUROSCIENCE   8   458  2015.01  [Refereed]

     View Summary

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulatecy to skeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration.

    DOI

  • Genetic deletion of Crmp4 promotes axonal regrowth after spinal cord injury by reducing microtubule destabilization and inflammatory responses.

    Nagai J, Kitamura Y, Owada K, Yamashita N, Takei K, Yoshio Goshima Y, Ohshima T

    Sci. Rep   5   8269  2015

  • Cdk5/p35 functions as a crucial regulator of spatial learning and memory

    Tomohide Mishiba, Mika Tanaka, Naoki Mita, Xiaojuan He, Kodai Sasamoto, Shigeyoshi Itohara, Toshio Ohshima

    MOLECULAR BRAIN   7  2014.11  [Refereed]

     View Summary

    Background: Cyclin-dependent kinase 5 (Cdk5), which is activated by binding to p35 or p39, is involved in synaptic plasticity and affects learning and memory formation. In Cdk5 knockout (KO) mice and p35 KO mice, brain development is severely impaired because neuronal migration is impaired and lamination is disrupted. To avoid these developmental confounders, we generated inducible CreER-p35 conditional (cKO) mice to study the role of Cdk5/p35 in higher brain function.
    Results: CreER-p35 cKO mice exhibited spatial learning and memory impairments and reduced anxiety-like behavior. These phenotypes resulted from a decrease in the dendritic spine density of CA1 pyramidal neurons and defective long-term depression induction in the hippocampus.
    Conclusions: Taken together, our findings reveal that Cdk5/p35 regulates spatial learning and memory, implicating Cdk5/p35 as a therapeutic target in neurological disorders.

    DOI

  • Valproic acid, a histone deacetylase inhibitor, regulates cell proliferation in the adult zebrafish optic tectum

    Miki Dozawa, Hiromitsu Kono, Yuki Sato, Yoko Ito, Hideomi Tanaka, Toshio Ohshima

    Developmental Dynamics   243 ( 11 ) 1401 - 1415  2014.11

     View Summary

    Background: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Several reports have demonstrated that VPA functions as a histone deacetylase (HDAC) inhibitor. While VPA is known to cause teratogenic changes in the embryonic zebrafish brain, its effects on neural stem cells (NSCs) in both the embryonic and adult zebrafish are not well understood. Results: In this study, we observed a proliferative effect of VPA on NSCs in the embryonic hindbrain. In contrast, VPA reduced cell proliferation in the adult zebrafish optic tectum. Treatment with HDAC inhibitors showed a similar inhibitory effect on cell proliferation in the adult zebrafish optic tectum, suggesting that VPA reduces cell proliferation through HDAC inhibition. Cell cycle progression was also suppressed in the optic tectum of the adult zebrafish brain because of HDAC inhibition. Recent studies have demonstrated that HDAC inhibits the Notch signaling pathway
    hence, adult zebrafish were treated with a Notch inhibitor. This increased the number of proliferating cells in the adult zebrafish optic tectum with down-regulated expression of her4, a target of Notch signaling. Conclusions: These results suggest that VPA inhibits HDAC activity and upregulates Notch signaling to reduce cell proliferation in the optic tectum of adult zebrafish.

    DOI PubMed

  • Cdk5/p35 is required for motor coordination and cerebellar plasticity

    Xiaojuan He, Masato Ishizeki, Naoki Mita, Seitaro Wada, Yoshifumi Araki, Hiroo Ogura, Manabu Abe, Maya Yamazaki, Kenji Sakimura, Katsuhiko Mikoshiba, Takafumi Inoue, Toshio Ohshima

    JOURNAL OF NEUROCHEMISTRY   131 ( 1 ) 53 - 64  2014.10  [Refereed]

     View Summary

    Previous studies have implicated the role of Purkinje cells in motor learning and the underlying mechanisms have also been identified in great detail during the last decades. Here we report that cyclin-dependent kinase 5 (Cdk5)/p35 in Purkinje cell also contributes to synaptic plasticity. We previously showed that p35(-/-) (p35 KO) mice exhibited a subtle abnormality in brain structure and impaired spatial learning and memory. Further behavioral analysis showed that p35 KO mice had a motor coordination defect, suggesting that p35, one of the activators of Cdk5, together with Cdk5 may play an important role in cerebellar motor learning. Therefore, we created Purkinje cell-specific conditional Cdk5/p35 knockout (L7-p35 cKO) mice, analyzed the cerebellar histology and Purkinje cell morphology of these mice, evaluated their performance with balance beam and rota-rod test, and performed electrophysiological recordings to assess long-term synaptic plasticity. Our analyses showed that Purkinje cell-specific deletion of Cdk5/p35 resulted in no changes in Purkinje cell morphology but severely impaired motor coordination. Furthermore, disrupted cerebellar long-term synaptic plasticity was observed at the parallel fiber-Purkinje cell synapse in L7-p35 cKO mice. These results indicate that Cdk5/p35 is required for motor learning and involved in long-term synaptic plasticity.

    DOI PubMed

  • Cdk5/p35 is required for motor coordination and cerebellar plasticity.

    Xiaojuan He, Masato Ishizeki, Naoki Mita, Seitaro Wada, Yoshifumi Araki, Hiroo Ogura, Manabu Abe, Maya Yamazaki, Kenji Sakimura, Katsuhiko Mikoshiba, Takafumi Inoue, Toshio Ohshima

    Journal of neurochemistry   131 ( 1 ) 53 - 64  2014.10  [International journal]

     View Summary

    Previous studies have implicated the role of Purkinje cells in motor learning and the underlying mechanisms have also been identified in great detail during the last decades. Here we report that cyclin-dependent kinase 5 (Cdk5)/p35 in Purkinje cell also contributes to synaptic plasticity. We previously showed that p35(-/-) (p35 KO) mice exhibited a subtle abnormality in brain structure and impaired spatial learning and memory. Further behavioral analysis showed that p35 KO mice had a motor coordination defect, suggesting that p35, one of the activators of Cdk5, together with Cdk5 may play an important role in cerebellar motor learning. Therefore, we created Purkinje cell-specific conditional Cdk5/p35 knockout (L7-p35 cKO) mice, analyzed the cerebellar histology and Purkinje cell morphology of these mice, evaluated their performance with balance beam and rota-rod test, and performed electrophysiological recordings to assess long-term synaptic plasticity. Our analyses showed that Purkinje cell-specific deletion of Cdk5/p35 resulted in no changes in Purkinje cell morphology but severely impaired motor coordination. Furthermore, disrupted cerebellar long-term synaptic plasticity was observed at the parallel fiber-Purkinje cell synapse in L7-p35 cKO mice. These results indicate that Cdk5/p35 is required for motor learning and involved in long-term synaptic plasticity.

    DOI PubMed

  • Cdk5 and its substrates, Dcx and p27kip1, regulate cytoplasmic dilation formation and nuclear elongation in migrating neurons.

    Yoshiaki V Nishimura, Mima Shikanai, Mikio Hoshino, Toshio Ohshima, Yo-ichi Nabeshima, Ken-Ichi Mizutani, Koh-Ichi Nagata, Kazunori Nakajima, Takeshi Kawauchi

    Development (Cambridge, England)   141 ( 18 ) 3540 - 50  2014.09  [Refereed]  [International journal]

     View Summary

    Neuronal migration is crucial for development of the mammalian-specific six-layered cerebral cortex. Migrating neurons are known to exhibit distinct features; they form a cytoplasmic dilation, a structure specific to migrating neurons, at the proximal region of the leading process, followed by nuclear elongation and forward movement. However, the molecular mechanisms of dilation formation and nuclear elongation remain unclear. Using ex vivo chemical inhibitor experiments, we show here that rottlerin, which is widely used as a specific inhibitor for PKCδ, suppresses the formation of a cytoplasmic dilation and nuclear elongation in cortical migrating neurons. Although our previous study showed that cortical neuronal migration depends on Jnk, another downstream target of rottlerin, Jnk inhibition disturbs only the nuclear elongation and forward movement, but not the dilation formation. We found that an unconventional cyclin-dependent kinase, Cdk5, is a novel downstream target of rottlerin, and that pharmacological or knockdown-mediated inhibition of Cdk5 suppresses both the dilation formation and nuclear elongation. We also show that Cdk5 inhibition perturbs endocytic trafficking as well as microtubule organization, both of which have been shown to be required for dilation formation. Furthermore, knockdown of Dcx, a Cdk5 substrate involved in microtubule organization and membrane trafficking, or p27(kip1), another Cdk5 substrate involved in actin and microtubule organization, disturbs the dilation formation and nuclear elongation. These data suggest that Cdk5 and its substrates, Dcx and p27(kip1), characterize migrating neuron-specific features, cytoplasmic dilation formation and nuclear elongation in the mouse cerebral cortex, possibly through the regulation of microtubule organization and an endocytic pathway.

    DOI PubMed

  • Impairments of long-term depression induction and motor coordination precede A beta accumulation in the cerebellum of APPswe/PS1dE9 double transgenic mice

    Yuki Kuwabara, Masato Ishizeki, Naoto Watamura, Junya Toba, Aya Yoshii, Takafumi Inoue, Toshio Ohshima

    JOURNAL OF NEUROCHEMISTRY   130 ( 3 ) 432 - 443  2014.08  [Refereed]

     View Summary

    Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (A beta) peptides in extracellular A beta plaques, produced from the amyloid precursor protein (APP) via sequential processing by beta- and gamma-secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that A beta peptides also impair another form of synaptic plasticity; cerebellar long-term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, A beta plaques were detected from 8 months and profound accumulation of A beta plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and A beta in the cerebellum of APPswe/PS1dE9 mice. At pre-A beta accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble A beta oligomers disturb LTD induction and cerebellar function in AD mouse model.

    DOI PubMed

  • Phosphorylation of Cyclin-dependent kinase 5 (Cdk5) at Tyr-15 is inhibited by Cdk5 activators and does not contribute to the activation of Cdk5

    Hiroyuki Kobayashi, Taro Saito, Ko Sato, Kotaro Furusawa, Tomohisa Hosokawa, Koji Tsutsumi, Akiko Asada, Shinji Kamada, Toshio Ohshima, Shin-Ichi Hisanaga

    Journal of Biological Chemistry   289 ( 28 ) 19627 - 19636  2014.07

     View Summary

    Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

    DOI PubMed

  • Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

    Hiroyuki Kobayashi, Taro Saito, Ko Sato, Kotaro Furusawa, Tomohisa Hosokawa, Koji Tsutsumi, Akiko Asada, Shinji Kamada, Toshio Ohshima, Shin-ichi Hisanaga

    JOURNAL OF BIOLOGICAL CHEMISTRY   289 ( 28 ) 19627 - 19636  2014.07  [Refereed]

     View Summary

    Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.

    DOI

  • Valproic acid downregulates Cdk5 activity via the transcription of the p35 mRNA

    Miyuki Takahashi, Manami Ishida, Taro Saito, Toshio Ohshima, Shin-Ichi Hisanaga

    Biochemical and Biophysical Research Communications   447 ( 4 ) 678 - 682  2014.05

     View Summary

    The cyclin-dependent kinase 5 (Cdk5) is a neuron-specific Ser/Thr kinase that is activated by the regulatory subunit p35. Overactivation of Cdk5, which is induced by the cleavage of p35 by calpain, is implicated in neuronal death in various neurodegenerative diseases. In contrast, depletion of the Cdk5 activity renders neurons vulnerable to stresses. Recent reports suggest the involvement of Cdk5 in mental disorders. We hypothesized that perturbation of Cdk5 activity is related to mental conditions. To verify this hypothesis, we investigated the effect of valproic acid (VPA), which is a drug of choice for psychiatric disorders, on Cdk5 activity. VPA decreased the expression of p35 at both the protein and mRNA levels in cultured neurons, resulting in a decrease of Cdk5 activity. VPA decreased the p35 mRNA via histone deacetylase inhibition. The chronic administration of VPA also downregulated p35 in mouse brains. These results indicate that VPA regulates Cdk5 activity in neurons via p35 transcription mediated by HDAC inhibition. © 2014 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Phosphorylation of Drebrin by Cyclin-Dependent Kinase 5 and Its Role in Neuronal Migration

    Kazuya Tanabe, Hiroyuki Yamazaki, Yutaka Inaguma, Akiko Asada, Taeko Kimura, Junya Takahashi, Masato Taoka, Toshio Ohshima, Teiichi Furuichi, Toshiaki Isobe, Koh-ichi Nagata, Tomoaki Shirao, Shin-ichi Hisanaga

    PLOS ONE   9 ( 3 )  2014.03  [Refereed]

     View Summary

    Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase which plays a key role in neuronal migration, neurite outgrowth, and spine formation during brain development. Dynamic remodeling of cytoskeletons is required for all of these processes. Cdk5-p35 phosphorylates many cytoskeletal proteins, but it is not fully understood how Cdk5-p35 regulates cytoskeletal reorganization associated with neuronal migration. Since actin filaments are critical for the neuronal movement and process formation, we aimed to find Cdk5 substrates among actin-binding proteins. In this study, we isolated actin gels from mouse brain extracts, which contain many actin-binding proteins, and phosphorylated them by Cdk5-p35 in vitro. Drebrin, a side binding protein of actin filaments and well known for spine formation, was identified as a phosphorylated protein. Drebrin has two isoforms, an embryonic form drebrin E and an adult type long isoform drebrin A. Ser142 was identified as a common phosphorylation site to drebrin E and A and Ser342 as a drebrin A-specific site. Phosphorylated drebrin is localized at the distal area of total drebrin in the growth cone of cultured primary neurons. By expressing nonphosphorylatable or phosphorylation mimicking mutants in developing neurons in utero, the reversible phosphorylation/dephosphorylation reaction of drebrin was shown to be involved in radial migration of cortical neurons. These results suggest that Cdk5-p35 regulates neuronal migration through phosphorylation of drebrin in growth cone processes.

    DOI

  • Suppression of neuroinflammation in forebrain-specific Cdk5 conditional knockout mice by PPAR gamma agonist improves neuronal loss and early lethality

    Elias Utreras, Ryusuke Hamada, Michaela Prochazkova, Anita Terse, Satoru Takahashi, Toshio Ohshima, Ashok B. Kulkarni

    JOURNAL OF NEUROINFLAMMATION   11   28  2014.02  [Refereed]

     View Summary

    Background: Cyclin-dependent kinase 5 (Cdk5) is essential for brain development and function, and its deregulated expression is implicated in some of neurodegenerative diseases. We reported earlier that the forebrain-specific Cdk5 conditional knockout (cKO) mice displayed an early lethality associated with neuroinflammation, increased expression of the neuronal tissue-type plasminogen activator (tPA), and neuronal migration defects.
    Methods: In order to suppress neuroinflammation in the cKO mice, we first treated these mice with pioglitazone, a PPAR gamma agonist, and analyzed its effects on neuronal loss and longevity. In a second approach, to delineate the precise role of tPA in neuroinflammation in these mice, we generated Cdk5 cKO; tPA double knockout (dKO) mice.
    Results: We found that pioglitazone treatment significantly reduced astrogliosis, microgliosis, neuronal loss and behavioral deficit in Cdk5 cKO mice. Interestingly, the dKO mice displayed a partial reversal in astrogliosis, but they still died at early age, suggesting that the increased expression of tPA in the cKO mice does not contribute significantly to the pathological process leading to neuroinflammation, neuronal loss and early lethality.
    Conclusion: The suppression of neuroinflammation in Cdk5 cKO mice ameliorates gliosis and neuronal loss, thus suggesting the potential beneficial effects of the PPAR gamma agonist pioglitazone for the treatment for neurodegenerative diseases.

    DOI

  • Cyclin-Dependent Kinase 5 Regulates Dendritic Spine Formation and Maintenance of Cortical Neuron in the Mouse Brain.

    Mita Naoki, He Xiaojuan, Sasamoto Kodai, Mishiba Tomohide, Ohshima Toshio

    Cyclin-Dependent Kinase 5 Regulates Dendritic Spine Formation and Maintenance of Cortical Neuron in the Mouse Brain.    2014

     View Summary

    :Cyclin-dependent kinase 5 (Cdk5) activity is dependent on its association with 1 of 2 neuron-specific activators, p35 or p39. Cdk5 and its activators play an important role in brain development as well as higher functions like synaptic plasticity, learning, and memory. Reduction in p35 was reported in postmortem schizophrenia brain, in which reduced dendritic spine density was observed. Previous in vitro experiments have shown that Cdk5 is involved in dendritic spine formation, although in vivo evidence is limited. We examined dendritic spine formation in inducible-p35 conditional knockout (p35 cKO); p39 KO mice. When we deleted the p35 gene either during early postnatal days or at adult stage, we observed reduced spine densities of layer V neurons in the cerebral cortex and CA1 pyramidal neurons in the hippocampus. We further generated CA1-specific p35 conditional knockout (CA1-p35 cKO) mice and also CA1-p35 cKO; p39 KO mice in which have specific deletion of p35 in the CA1 region of hippocampus. We found a greater reduction in spine densities in CA1 pyramidal neurons in CA1-p35 cKO; p39 KO mice than in CA1-p35 cKO mice. These results indicate that dendritic spine formation and neuronal maintenance are dependent on Cdk5 activity.

  • Transcription profiles of the ductus arteriosus in Brown-Norway rats with irregular elastic fiber formation

    Yi-Ting Hsieh, Norika Mengchia Liu, Eriko Ohmori, Tomohiro Yokota, Ichige Kajimura, Toru Akaike, Toshio Ohshima, Nobuhito Goda, Susumu Minamisawa

    Circulation Journal   78 ( 5 ) 1224 - 1233  2014

     View Summary

    Background: Patent ductus arteriosus (PDA) is one of the most common congenital cardiovascular defects in children. The Brown-Norway (BN) inbred rat presents a higher frequency of PDA. A previous study reported that 2 different quantitative trait loci on chromosomes 8 and 9 were significantly linked to PDA in this strain. Nevertheless, the genetic or molecular mechanisms underlying PDA phenotypes in BN rats have not been fully investigated yet. Methods and Results: It was found that the elastic fibers were abundant in the subendothelial area but scarce in the media even in the closed ductus arteriosus (DA) of full-term BN neonates. DNA microarray analysis identified 52 upregulated genes (fold difference &gt
    2.5) and 23 downregulated genes (fold difference &lt
    0.4) when compared with those of F344 control neonates. Among these genes, 8 (Tbx20, Scn3b, Stac, Sphkap, Trpm8, Rup2, Slc37a2, and RGD1561216) are located in chromosomes 8 and 9. Interestingly, it was also suggested that the significant decrease in the expression levels of the PGE2-specfic receptor, EP4, plays a critical role in elastogenesis in the DA. Conclusions: BN rats exhibited dysregulation of elastogenesis in the DA. DNA microarray analysis identified the candidate genes including EP4 involved in the DNA phenotype. Further investigation of these newly identified genes will hopefully clarify the molecular mechanisms underlying the irregular formation of elastic fibers in PDA.

    DOI PubMed CiNii

  • Impairments of long-term depression induction and motor coordination precede Aβ accumulation in the cerebellum of APPswe/PS1dE9 double transgenic mice

    Yuki Kuwabara, Masato Ishizeki, Naoto Watamura, Junya Toba, Aya Yoshii, Takafumi Inoue, Toshio Ohshima

    Journal of Neurochemistry   130 ( 3 ) 432 - 443  2014

     View Summary

    Alzheimer's disease (AD) is a neurodegenerative disorder that represents the most common type of dementia among elderly people. Amyloid beta (Aβ) peptides in extracellular Aβ plaques, produced from the amyloid precursor protein (APP) via sequential processing by β- and γ-secretases, impair hippocampal synaptic plasticity, and cause cognitive dysfunction in AD patients. Here, we report that Aβ peptides also impair another form of synaptic plasticity
    cerebellar long-term depression (LTD). In the cerebellum of commonly used AD mouse model, APPswe/PS1dE9 mice, Aβ plaques were detected from 8 months and profound accumulation of Aβ plaques was observed at 18 months of age. Biochemical analysis revealed relatively high levels of APP protein and Aβ in the cerebellum of APPswe/PS1dE9 mice. At pre-Aβ accumulation stage, LTD induction, and motor coordination are disturbed. These results indicate that soluble Aβ oligomers disturb LTD induction and cerebellar function in AD mouse model. © 2014 International Society for Neurochemistry.

    DOI PubMed

  • Cdk5/p35 functions as a crucial regulator of spatial learning and memory

    Tomohide Mishiba, Mika Tanaka, Naoki Mita, Xiaojuan He, Kodai Sasamoto, Shigeyoshi Itohara, Toshio Ohshima

    Molecular Brain   7 ( 1 ) 82  2014

     View Summary

    Background: Cyclin-dependent kinase 5 (Cdk5), which is activated by binding to p35 or p39, is involved in synaptic plasticity and affects learning and memory formation. In Cdk5 knockout (KO) mice and p35 KO mice, brain development is severely impaired because neuronal migration is impaired and lamination is disrupted. To avoid these developmental confounders, we generated inducible CreER-p35 conditional (cKO) mice to study the role of Cdk5/p35 in higher brain function. Results: CreER-p35 cKO mice exhibited spatial learning and memory impairments and reduced anxiety-like behavior. These phenotypes resulted from a decrease in the dendritic spine density of CA1 pyramidal neurons and defective long-term depression induction in the hippocampus. Conclusions: Taken together, our findings reveal that Cdk5/p35 regulates spatial learning and memory, implicating Cdk5/p35 as a therapeutic target in neurological disorders.

    DOI PubMed

  • Cyclin-dependent Kinase 5 (Cdk5) Regulates the Function of CLOCK Protein by Direct Phosphorylation

    Yongdo Kwak, Jaehoon Jeong, Saebom Lee, Young-Un Park, Seol-Ae Lee, Dong-Hee Han, Joung-Hun Kim, Toshio Ohshima, Katsuhiko Mikoshiba, Yoo-Hun Suh, Sehyung Cho, Sang Ki Park

    JOURNAL OF BIOLOGICAL CHEMISTRY   288 ( 52 ) 36878 - 36889  2013.12  [Refereed]

     View Summary

    Circadian rhythm is a biological rhythm governing physiology and behavior with a period of approximate to 24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

    DOI

  • Cyclin-dependent Kinase 5 (Cdk5) Regulates the Function of CLOCK Protein by Direct Phosphorylation

    Yongdo Kwak, Jaehoon Jeong, Saebom Lee, Young-Un Park, Seol-Ae Lee, Dong-Hee Han, Joung-Hun Kim, Toshio Ohshima, Katsuhiko Mikoshiba, Yoo-Hun Suh, Sehyung Cho, Sang Ki Park

    JOURNAL OF BIOLOGICAL CHEMISTRY   288 ( 52 ) 36878 - 36889  2013.12  [Refereed]

     View Summary

    Circadian rhythm is a biological rhythm governing physiology and behavior with a period of approximate to 24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

    DOI

  • Amyloid-beta(25-35) induces impairment of cognitive function and long-term potentiation through phosphorylation of collapsin response mediator protein 2

    Toshinari Isono, Naoya Yamashita, Masami Obara, Tomomi Araki, Fumio Nakamura, Yoshinori Kamiya, Tursun Alkam, Atsumi Nitta, Toshitaka Nabeshima, Katsuhiko Mikoshiba, Toshio Ohshima, Yoshio Goshima

    NEUROSCIENCE RESEARCH   77 ( 3 ) 180 - 185  2013.11  [Refereed]

     View Summary

    Alzheimer's disease (AD) is characterized by amyloid-beta (A beta) protein and tau deposition in the brain. Numerous studies have reported a central role of A beta in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of A beta neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of A beta(25-35) peptide, a neurotoxic fragment of A beta protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of A beta(25-35) peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of A beta(25-35) peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for A beta-induced impairment of cognitive memory and synaptic plasticity. (C) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

    DOI

  • Amyloid-beta(25-35) induces impairment of cognitive function and long-term potentiation through phosphorylation of collapsin response mediator protein 2

    Toshinari Isono, Naoya Yamashita, Masami Obara, Tomomi Araki, Fumio Nakamura, Yoshinori Kamiya, Tursun Alkam, Atsumi Nitta, Toshitaka Nabeshima, Katsuhiko Mikoshiba, Toshio Ohshima, Yoshio Goshima

    NEUROSCIENCE RESEARCH   77 ( 3 ) 180 - 185  2013.11  [Refereed]

     View Summary

    Alzheimer's disease (AD) is characterized by amyloid-beta (A beta) protein and tau deposition in the brain. Numerous studies have reported a central role of A beta in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of A beta neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of A beta(25-35) peptide, a neurotoxic fragment of A beta protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of A beta(25-35) peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of A beta(25-35) peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for A beta-induced impairment of cognitive memory and synaptic plasticity. (C) 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

    DOI

  • IP(3)R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

    Chihiro Hisatsune, Hiroyuki Miyamoto, Moritoshi Hirono, Naohide Yamaguchi, Takeyuki Sugawara, Naoko Ogawa, Etsuko Ebisui, Toshio Ohshima, Masahisa Yamada, Takao K. Hensch, Mitsuharu Hattori, Katsuhiko Mikoshiba

    FRONTIERS IN NEURAL CIRCUITS   7   156  2013.10  [Refereed]

     View Summary

    The type 1 inositol 1,4,5- trisphosphate receptor (IP(3)R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP(3)Rs. Mice lacking IP(3)R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP(3)R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP(3)R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP(3)R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway.

    DOI

  • Dual origins of the mammalian accessory olfactory bulb revealed by an evolutionarily conserved migratory stream

    Dhananjay Huilgol, Susan Udin, Tomomi Shimogori, Bhaskar Saha, Achira Roy, Shinichi Aizawa, Robert F. Hevner, Gundela Meyer, Toshio Ohshima, Samuel J. Pleasure, Yangu Zhao, Shubha Tole

    NATURE NEUROSCIENCE   16 ( 2 ) 157 - 165  2013.02  [Refereed]

     View Summary

    The accessory olfactory bulb (AOB) is a critical olfactory structure that has been implicated in mediating social behavior. It receives input from the vomeronasal organ and projects to targets in the amygdaloid complex. Its anterior and posterior components (aAOB and pAOB) display molecular, connectional and functional segregation in processing reproductive and defensive and aggressive behaviors, respectively. We observed a dichotomy in the development of the projection neurons of the aAOB and pAOB in mice. We found that they had distinct sites of origin and that different regulatory molecules were required for their specification and migration. aAOB neurons arose locally in the rostral telencephalon, similar to main olfactory bulb neurons. In contrast, pAOB neurons arose caudally, from the neuroepithelium of the diencephalic-telencephalic boundary, from which they migrated rostrally to reach their destination. This unusual origin and migration is conserved in Xenopus, providing an insight into the origin of a key component of this system in evolution.

    DOI

  • Identification and Expression Analysis of the Zebrafish Homologs of the ceramide synthase Gene Family

    Mirco Brondolin, Susanne Berger, Michael Reinke, Hideomi Tanaka, Toshio Ohshima, Bernhard Fu, Michael Hoch

    Developmental Dynamics   242 ( 2 ) 189 - 200  2013.02

     View Summary

    Background: Sphingolipids represent a major class of lipids which both serve as structural components of membranes and as bioactive molecules involved in lipid signaling. Ceramide synthases (cers) reside in the center of sphingolipid metabolism by producing ceramide through de novo synthesis or degradative pathways. While the six mammalian cers family members have been extensively studied in cell culture and in adult tissues, a systematic analysis of cers expression and function during embryogenesis is still lacking. Results: Using bioinformatic and phylogenetic analysis, we identified nine highly conserved homologs of the vertebrate cers gene family in the zebrafish genome. A systematic expression analysis throughout five developmental stages indicates that, whereas until 48 hours post fertilization most zebrafish cers homologs are expressed in distinct patterns, e.g., in the intermediate cell mass and the pronephric duct, they show a highly overlapping expression during later stages of embryonic development, mostprominently in the developing brain. Conclusions: In this study, the expression of the cers gene homologs is comprehensively analyzed for the first time during vertebrate embryogenesis. Our data indicate that each embryonic tissue has a unique profile of cers expression during zebrafish embryogenesis suggesting tissue-specific profiles of ceramides and their derivatives. © 2012 Wiley Periodicals, Inc.

    DOI PubMed

  • Dual origins of the mammalian accessory olfactory bulb revealed by an evolutionarily conserved migratory stream

    Dhananjay Huilgol, Susan Udin, Tomomi Shimogori, Bhaskar Saha, Achira Roy, Shinichi Aizawa, Robert F. Hevner, Gundela Meyer, Toshio Ohshima, Samuel J. Pleasure, Yangu Zhao, Shubha Tole

    NATURE NEUROSCIENCE   16 ( 2 ) 157 - 165  2013.02  [Refereed]

     View Summary

    The accessory olfactory bulb (AOB) is a critical olfactory structure that has been implicated in mediating social behavior. It receives input from the vomeronasal organ and projects to targets in the amygdaloid complex. Its anterior and posterior components (aAOB and pAOB) display molecular, connectional and functional segregation in processing reproductive and defensive and aggressive behaviors, respectively. We observed a dichotomy in the development of the projection neurons of the aAOB and pAOB in mice. We found that they had distinct sites of origin and that different regulatory molecules were required for their specification and migration. aAOB neurons arose locally in the rostral telencephalon, similar to main olfactory bulb neurons. In contrast, pAOB neurons arose caudally, from the neuroepithelium of the diencephalic-telencephalic boundary, from which they migrated rostrally to reach their destination. This unusual origin and migration is conserved in Xenopus, providing an insight into the origin of a key component of this system in evolution.

    DOI

  • Cyclin-dependent kinase 5 is required for normal cerebellar development

    A. Kumazawa, N. Mita, M. Hirasawa, T. Adachi, H. Suzuki, N. Shafeghat, A. B. Kulkarni, K. Mikoshiba, T. Inoue, T. Ohshima

    MOLECULAR AND CELLULAR NEUROSCIENCE   52   97 - 105  2013.01  [Refereed]

     View Summary

    Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its kinase activity is dependent upon its association with either of the activating subunits p35 or p39, which are mainly expressed in neurons. We previously reported that Cdk5 knockout (KO) mice exhibit perinatal lethality, defective neuronal migration, and abnormal positioning of neurons in the facial motor nucleus and inferior olive in the hindbrain and Purkinje cells (PCs) in the cerebellum. In this study, we focused on the analysis of the role of Cdk5 in cerebellar development. For this purpose we generated midbrain-hindbrain-specific Cdk5 conditional knockout (MHB-Cdk5 KO) mice because the cerebellum develops postnatally, whereas Cdk5 KO mice die perinatally. Histological analysis of the MHB-Cdk5 KO mice revealed a significant size reduction of the cerebellum. In addition, profound disturbance of inward migration of granule cells (GC) was observed in the developing cerebellum. A normal dendritic development of the Purkinje cells (PCs) was disturbed in MHB-Cdk5 KO mice. Cultured Cdk5-null PCs showed similar dendritic abnormalities. These results indicate that Cdk5/p35 plays an important role in neuronal migration of PCs and GCs and dendrite formation of PCs in cerebellar development. (c) 2012 Elsevier Inc. All rights reserved.

    DOI

  • Phosphorylation of CRMP2 is involved in proper bifurcation of the apical dendrite of hippocampal CA1 pyramidal neurons.

    Niisato Emi, Nagai Jun, Yamashita Naoya, Nakamura Fumio, Goshima Yoshio, Ohshima Toshio

    Phosphorylation of CRMP2 is involved in proper bifurcation of the apical dendrite of hippocampal CA1 pyramidal neurons.   73 ( 2 ) 142 - 151  2013  [International journal]

     View Summary

    :The neural circuit in the hippocampus is important for higher brain functions. Dendrites of CA1 pyramidal neurons mainly receive input from the axons of CA3 pyramidal neurons in this neural circuit. A CA1 pyramidal neuron has a single apical dendrite and multiple basal dendrites. In wild-type mice, most of CA1 pyramidal neurons extend a single trunk, or alternatively, the apical dendrite bifurcates into two daughter trunks at the stratum radiatum layer. We previously reported the proximal bifurcation phenotype in Sema3A-/-, p35-/-, and CRMP4-/- mice. Cdk5/p35 phosphorylates CRMP2 at Ser522, and inhibition of this phosphorylation suppressed Sema3A-induced growth cone collapse. In this study, we analyzed the bifurcation points of the apical dendrites of hippocampal CA1 pyramidal neurons in CRMP2KI/KI mice in which the Cdk5/p35-phosphorylation site Ser522 was mutated into an Ala residue. The proximal bifurcation phenotype was not observed in CRMP2KI/KI mice; however, severe proximal bifurcation of apical dendrites was found in CRMP2KI/KI;CRMP4-/- mice. Cultured hippocampal neurons from CRMP2KI/KI and CRMP2KI/KI;CRMP4-/- embryos showed an increased number of dendritic branching points compared to those from wild-type embryos. Sema3A increased the number of branching points and the total length of dendrites in wild-type hippocampal neurons, but these effects of Sema3A for dendrites were not observed in CRMP2KI/KI and CRMP2KI/KI;CRMP4-/-hippocampal neurons. Binding of CRMP2 to tubulin increased in both CRMP2KI/KI and CRMP2KI/KI:CRMP4-/- brain lysates. These results suggest that CRMP2 and CRMP4 synergistically regulate dendritic development, and CRMP2 phosphorylation is critical for proper bifurcation of apical dendrite of CA1 pyramidal neurons.

    DOI PubMed

  • Phosphorylation of Dpsyl2 (CRMP2) and Dpsyl3 (CRMP4) is required for positioning of caudal primary motor neurons in the zebrafish spinal cord.

    Morimura Rii, Nozawa Keisuke, Tanaka Hideomi, Ohshima Toshio

    Phosphorylation of Dpsyl2 (CRMP2) and Dpsyl3 (CRMP4) is required for positioning of caudal primary motor neurons in the zebrafish spinal cord.   73 ( 12 ) 911 - 920  2013

     View Summary

    :Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co-injection of phosphorylation-mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord.

    DOI

  • p35 deficiency accelerates HMGB-1-mediated neuronal death in the early stages of an Alzheimer's disease mouse model

    Ahram Jang, Hyunjeong Liew, Yun-Mi Kim, Heesoon Choi, Saeromi Kim, Sang Hyung Lee, Toshio Ohshima, Katsuhiko Mikoshiba, Yoo-Hun Suh

    Current Alzheimer Research   10 ( 8 ) 829 - 843  2013

     View Summary

    The activities of CDK5 and p35 are thought to be important in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). We studied the effect of p35 deletion in Tg2576 mice, which is an AD animal model. To obtain the desired mice, we crossed p35-/- with Tg2576 mice. The resulting p35-/-/Tg2576 (KO/Tg) mice displayed higher mortality rates and exhibited impaired spatial learning and memory at 6 months of age. Using immunohistochemical and biochemical approaches, we observed a reduction in the expression of pre- and post-synaptic markers such as NMDAR1, synaptophysin and GluR1. In addition, the intensity of MAP-2-positive dendrites extending from neuronal cell bodies was significantly decreased in KO/Tg mice compared with KO/WT and WT/Tg mice. We also detected increased neuronal cell death in the hippocampus, along with thinned and collapsed morphological changes in the alveus region and a dramatic increase in the number of microglial cells. Microglial infiltration in the hippocampus could result in the increased secretion of the soluble high mobility group box-1 protein (HMGB-1). The secretion of HMGB-1 is increased by Aβ, and secretion of HMGB-1 promotes neuronal cell death. Moreover, we found that HMGB-1 secretion induced by Aβ in KO/Tg mice gave rise to ER-mediated cell death. In summary, during the stages of KO/Tg mice model, the microglial infiltration and secretion of soluble HMGB-1 were significantly increased in the hippocampus. These conditions promote neuronal death, synaptic destruction and behavioral deficits. © 2013 Bentham Science Publishers.

    DOI PubMed

  • Dpysl2 (CRMP2) and Dpysl3 (CRMP4) phosphorylation by Cdk5 and DYRK2 is required for proper positioning of Rohon-Beard neurons and neural crest cells during neurulation in zebrafish

    Hideomi Tanaka, Rii Morimura, Toshio Ohshima

    Developmental Biology   370 ( 2 ) 223 - 236  2012.10

     View Summary

    Dpysl2 (CRMP2) and Dpysl3 (CRMP4) are involved in neuronal polarity and axon elongation in cultured neurons. These proteins are expressed in various regions of the developing nervous system, but their roles in vivo are largely unknown. In dpysl2 and dpysl3 double morphants, Rohon-Beard (RB) primary sensory neurons that were originally located bilaterally along the midline shifted their position to a more medial location in the dorsal-most part of spinal cord. A similar phenotype was observed in the cdk5 and dyrk2 double morphants. Dpysl2 and Dpysl3 phosphorylation mimics recovered this phenotype. Cell transplantation analysis demonstrated that this ectopic RB cell positioning was non-cell autonomous and correlated with the abnormal position of neural crest cells (NCCs), which also occupied the dorsal-most part of the spinal cord during the neural rod formation stage. The cell position of other interneuron and motor neurons within the central nervous system was normal in these morphants. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and DYRK2 is required for the proper positioning of RB neurons and NCCs during neurulation in zebrafish embryos. © 2012 Elsevier Inc.

    DOI PubMed

  • Dpysl2 (CRMP2) and Dpysl3 (CRMP4) phosphorylation by Cdk5 and DYRK2 is required for proper positioning of Rohon-Beard neurons and neural crest cells during neurulation in zebrafish

    Hideomi Tanaka, Rii Morimura, Toshio Ohshima

    DEVELOPMENTAL BIOLOGY   370 ( 2 ) 223 - 236  2012.10  [Refereed]

     View Summary

    Dpysl2 (CRMP2) and Dpysl3 (CRMP4) are involved in neuronal polarity and axon elongation in cultured neurons. These proteins are expressed in various regions of the developing nervous system, but their roles in vivo are largely unknown. In dpysl2 and dpysl3 double morphants, Rohon-Beard (RB) primary sensory neurons that were originally located bilaterally along the midline shifted their position to a more medial location in the dorsal-most part of spinal cord. A similar phenotype was observed in the cdk5 and dyrk2 double morphants. Dpysl2 and Dpysl3 phosphorylation mimics recovered this phenotype. Cell transplantation analysis demonstrated that this ectopic RB cell positioning was non-cell autonomous and correlated with the abnormal position of neural crest cells (NCCs), which also occupied the dorsal-most part of the spinal cord during the neural rod formation stage. The cell position of other interneuron and motor neurons within the central nervous system was normal in these morphants. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and DYRK2 is required for the proper positioning of RB neurons and NCCs during neurulation in zebrafish embryos. (c) 2012 Elsevier Inc. All rights reserved.

    DOI

  • CRMP4 mediates MAG-induced inhibition of axonal outgrowth and protection against Vincristine-induced axonal degeneration

    Jun Nagai, Yoshio Goshima, Toshio Ohshima

    Neuroscience Letters   519 ( 1 ) 56 - 61  2012.06

     View Summary

    Suppression of inhibition of axonal outgrowth and promotion of axonal protection from progressive axonal degeneration are both therapeutic strategies for the treatment of neuronal diseases characterized by axonal loss. Myelin-associated inhibitors (MAIs) have been shown to suppress axonal outgrowth, but a specific MAI, myelin-associated glycoprotein (MAG), has also been shown to protect neurons from axonal degeneration through activation of the small GTPase protein RhoA. Recent in vitro studies have shown that collapsin response mediator protein 4 (CRMP4) interacts with RhoA and that the CRMP4b/RhoA complex mediates MAG-induced inhibitory signaling against axonal outgrowth. However, whether CRMP4 is involved in MAG-mediated axon protection signaling remains unclear. Here, we show involvement of CRMP4 in MAG-induced inhibition of axonal outgrowth and axonal protection using the CRMP4-/- mouse model. In dorsal root ganglion (DRG) neurons, loss of CRMP4 prevents MAG-induced inhibition of axonal outgrowth and growth cone collapse and increases sensitivity to microtubule destabilizing factor Vincristine (VNC)-induced axonal degeneration. MAG-mediated axon protection against VNC is suppressed in CRMP4-/- DRG neurons. Understanding the molecular mechanism of MAG-mediated inhibition and protection via CRMP4 may provide novel opportunities to control axonal degeneration and regeneration. © 2012 Elsevier Ireland Ltd.

    DOI PubMed

  • Phosphorylation of Kif26b Promotes Its Polyubiquitination and Subsequent Proteasomal Degradation during Kidney Development

    Takeshi Terabayashi, Masaji Sakaguchi, Kaori Shinmyozu, Toshio Ohshima, Ai Johjima, Teru Ogura, Hiroaki Miki, Ryuichi Nishinakamura

    PLOS ONE   7 ( 6 )  2012.06  [Refereed]

     View Summary

    Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development.

    DOI

  • CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus.

    Niisato Emi, Nagai Jun, Yamashita Naoya, Abe Takaya, Kiyonari Hiroshi, Goshima Yoshio, Ohshima Toshio

    CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus.   72 ( 11 ) 1447 - 1457  2012  [International journal]

     View Summary

    :Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1-5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin-3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4-/- mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4-/- mice. We also observed increased dendritic branching in cultured CRMP4-/- hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4-/- hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling.

    DOI PubMed

  • Phosphorylation of CRMP2 (Collapsin Response Mediator Protein 2) Is Involved in Proper Dendritic Field Organization

    Naoya Yamashita, Toshio Ohshima, Fumio Nakamura, Papachan Kolattukudy, Jerome Honnorat, Katsuhiko Mikoshiba, Yoshio Goshima

    JOURNAL OF NEUROSCIENCE   32 ( 4 ) 1360 - 1365  2012.01  [Refereed]

     View Summary

    Collapsin response mediator proteins (CRMPs) are intracellular proteins that mediate signals for several extracellular molecules, such as Semaphorin3A and neurotrophins. The phosphorylation of CRMP1 and CRMP2 by Cdk5 at Ser522 is involved in axonal guidance and spine development. Here, we found that the Ser522-phosphorylated CRMP1 and/or CRMP2 are enriched in the dendrites of cultured cortical neurons and P7 cortical section. To determine the physiological role of CRMPs in dendritic development, we generated CRMP2 knock-in mutant mice (crmp2(ki/ki)) in which the Ser residue at 522 was replaced with Ala. Strikingly, the cortical basal dendrites of double mutant crmp2(ki/ki) and crmp1(-/-) mice exhibited severe abnormal dendritic patterning, which we defined as "curling phenotype." These findings demonstrate that the function of CRMP1 and CRMP2 synergistically control dendritic projection, and the phosphorylation of CRMP2 at Ser522 is essential for proper dendritic field organization in vivo.

    DOI

  • Phosphorylation of Kif26b promotes its polyubiquitination and subsequent proteasomal degradation during kidney development

    Terabayashi T, Sakaguchi M, Shinmyozu K, Ohshima T, Johjima A

    PLoS ONE   7 ( 6 ) e39714  2012  [Refereed]

  • Hypomyelination phenotype caused by impaired differentiation of oligodendrocytes in Emx1-cre mediated Cdk5 conditional knockout mice

    Xiaojuan He, Satoru Takahashi, Hiromi Suzuki, Tsutomu Hashikawa, Ashok B. Kulkarni, Katsuhiko Mikoshiba, Toshio Ohshima

    Neurochemical Research   36 ( 7 ) 1293 - 1303  2011.07

     View Summary

    Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in neuronal migration and differentiation, and in axonal elongation. Although many studies have been conducted to analyze neuronal functions of Cdk5, its kinase activity has also been reported during oligodendrocyte differentiation, which suggests Cdk5 may play an important role in oligodendrocytes. Here, we describe a hypomyelination phenotype observed in Emx1-cre mediated Cdk5 conditional knockout (cKO) mice (Emx1-cKO), in which the Cdk5 gene was deleted in neurons, astrocytes and oligodendrocyte -lineage cells. In contrast, the Cdk5 gene in CaMKII cKO mice was deleted only in neurons. Because the development of mature oligodendrocytes from oligodendrocyte precursor cells is a complex process, we performed in situ hybridization using markers for the oligodendrocyte precursor cell and for the differentiated oligodendrocyte. Our results indicate that hypomyelination in Emx1-cKO is due to the impaired differentiation of oligodendrocytes, rather than to the proliferation or migration of their precursors. The present study confirmed the in vivo role of Cdk5 in oligodendrocyte differentiation. © 2011 Springer Science+Business Media, LLC.

    DOI PubMed

  • Genetic manipulation of mammalian lateral and medial habenula homolog in zebrafish: Toward functional analysis of the habenula

    Ryunosuke Amo, Masakazu Agetsuma, Masae Kinoshita, Toshiyuki Shiraki, Shin-ichi Higashijima, Masaru Matsuda, Maximiliano L. Suster, Koichi Kawakami, Toshio Ohshima, Hidenori Aizawa, Hitoshi Okamoto

    NEUROSCIENCE RESEARCH   71   E271 - E271  2011  [Refereed]

    DOI

  • Disturbed Purkinje cell spike activity produces dystonia in mice lacking the type 1 inositol 1,4,5-trisphosphate receptor

    Chihiro Hisatsune, Hiroyuki Miyamoto, Moritoshi Hirono, Naohide Yamaguchi, Takeyuki Sugawara, Naoko Ogawa, Toshio Ohshima, Masahisa Yamada, Takao K. Hensch, Mitsuharu Hattori, Katsuhiko Mikoshiba

    NEUROSCIENCE RESEARCH   71   E99 - E99  2011  [Refereed]

    DOI

  • Islet1 Selectively Promotes Peripheral Axon Outgrowth in Rohon-Beard Primary Sensory Neurons

    Hideomi Tanaka, Yasuhiro Nojima, Wataru Shoji, Miki Sato, Ryoko Nakayama, Toshio Ohshima, Hitoshi Okamoto

    DEVELOPMENTAL DYNAMICS   240 ( 1 ) 9 - 22  2011.01  [Refereed]

     View Summary

    We isolated a novel zebrafish mutant, lullaby (llb), and showed that the llb locus encodes the zebrafish orthologue of isl1. Rohon-Beard (RB) primary sensory neurons are multipolar neurons that extend their central axons longitudinally within the spinal cord and also extend their peripheral axons under the skin. In llb embryos, the outgrowth of the peripheral axons of RB neurons was selectively impaired, which correlated with down-regulation of the expression of dihydropyrimidinase-like 3 (dpysl3, also known as collapsin response mediator protein 4, crmp4). Antisense morpholino oligonucleotide (AMO)-mediated knockdown of dpysl3 inhibited the outgrowth of the peripheral axons of RB neurons, and semaphorin 3d (sema3d) AMO enhanced this effect. These data indicate that Dpysl3 is cooperating with Sema3d in the peripheral axon outgrowth, and Isl1 is required for the selective outgrowth of the peripheral axons of RB neurons by maintaining the expression of dpysl3. Developmental Dynamics 240: 9-22, 2011. (C) 2010 Wiley-Liss, Inc.

    DOI

  • Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum

    Yoko Ito, Hideomi Tanaka, Hitoshi Okamoto, Toshio Ohshima

    DEVELOPMENTAL BIOLOGY   342 ( 1 ) 26 - 38  2010.06  [Refereed]

     View Summary

    In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions-the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100 beta. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish. (C) 2010 Elsevier Inc. All rights reserved.

    DOI

  • Phosphorylation of AATYK1 by Cdk5 Suppresses Its Tyrosine Phosphorylation

    Koji Tsutsumi, Tetsuya Takano, Ryo Endo, Mitsunori Fukuda, Toshio Ohshima, Mineko Tomomura, Shin-ichi Hisanaga

    PLOS ONE   5 ( 4 ) e10260  2010.04  [Refereed]

     View Summary

    Apoptosis-associated tyrosine kinase 1 (AATYK1), a novel serine/threonine kinase that is highly expressed in the brain, is involved in neurite extension and apoptosis of cerebellar granule neurons; however, its precise function remains unknown. In this study, we investigated the interaction of AATYK1A with Cyclin-dependent kinase 5 (Cdk5)/p35, a proline-directed protein kinase that is predominantly expressed in neurons. AATYK1A bound to the p35 activation subunit of Cdk5 in cultured cells and in mouse brains and colocalized with p35 on endosomes in COS-7 cells. AATYK1A was phosphorylated at Ser34 by Cdk5/p35 in vitro, in cultured neurons and in mouse brain. In PC12D cells, Ser34 phosphorylation increased after treatment with nerve growth factor and phosphorylated AATYK1A accumulated in growth cones of PC12D cells. Ser34 phosphorylation suppressed the tyrosine phosphorylation of AATYK1A by Src family kinases. These results suggest a possibility that AATYK1A plays a role in early to recycling endosomes and its function is regulated by phosphorylation with Cdk5 or Src-family kinases.

    DOI

  • Conditional deletion of neuronal cyclin-dependent kinase 5 in developing forebrain results in microglial activation and neurodegeneration

    Satoru Takahashi, Toshio Ohshima, Motoyuki Hirasawa, Tej K. Pareek, Thomas H. Bugge, Alexei Morozov, Kenji Fujieda, Roscoe O. Brady, Ashok B. Kulkarni

    American Journal of Pathology   176 ( 1 ) 320 - 329  2010

     View Summary

    Neuronal migration disorders are often identified in patients with epilepsy refractory to medical treatment. The prolonged or repeated seizures are known to cause neuronal death
    however, the mechanism underlying seizure-induced neuronal death remains to be elucidated. An essential role of cyclin-dependent kinase 5 (Cdk5) in brain development has been demonstrated in Cdk5-/- mice, which show neuronal migration defects and perinatal lethality. Here, we show the consequences of Cdk5 deficiency in the postnatal brain by generating Cdk5 conditional knockout mice, in which Cdk5 is selectively eliminated from neurons in the developing forebrain. The conditional mutant mice were viable, but exhibited complex neurological deficits including seizures, tremors, and growth retardation. The forebrain not only showed disruption of layering, but also neurodegenerative changes accompanied by neuronal loss and microglial activation. The neurodegenerative changes progressed with age and were accompanied by up-regulation of the neuronal tissue-type plasminogen activator, a serine protease known to mediate microglial activation. Thus age-dependent neurodegeneration in the Cdk5 conditional knockout mouse brain invoked a massive inflammatory reaction. These findings indicate an important role of Cdk5 in inflammation, and also provide a mouse model to examine the possible involvement of inflammation in the pathogenesis of progressive cognitive decline in patients with neuronal migration disorders. Copyright © American Society for Investigative Pathology.

    DOI

  • Conditional Deletion of Neuronal Cyclin-Dependent Kinase 5 in Developing Forebrain Results in Microglial Activation and Neurodegeneration

    Satoru Takahashi, Toshio Ohshima, Motoyuki Hirasawa, Tej K. Pareek, Thomas H. Bugge, Alexei Morozov, Kenji Fujieda, Roscoe O. Brady, Ashok B. Kulkarni

    AMERICAN JOURNAL OF PATHOLOGY   176 ( 1 ) 320 - 329  2010.01  [Refereed]

     View Summary

    Neuronal migration disorders are often identified in patients with epilepsy refractory to medical treatment. The prolonged or repeated seizures are known to cause neuronal death; however, the mechanism underlying seizure-induced neuronal death remains to be elucidated. An essential role of cyclin-dependent kinase 5 (Cdk5) in brain development has been demonstrated in Cdk5(-/-) mice, which show neuronal migration defects and perinatal lethality. Here, we show the consequences of Cdk5 deficiency in the postnatal brain by generating Cdk5 conditional knockout mice, in which Cdk5 is selectively eliminated from neurons in the developing forebrain. The conditional mutant mice were viable, but exhibited complex neurological deficits including seizures, tremors, and growth retardation. The forebrain not only showed disruption of layering, but also neurodegenerative changes accompanied by neuronal loss and microglial activation. The neurodegenerative changes progressed with age and were accompanied by up-regulation of the neuronal tissue-type plasminogen activator, a serine protease known to mediate microglial activation. Thus age-dependent neurodegeneration in the Cdk5 conditional knockout mouse brain invoked a massive inflammatory reaction. These findings indicate an important role of Cdk5 in inflammation, and also provide a mouse model to examine the possible involvement of inflammation in the pathogenesis of progressive cognitive decline in patients with neuronal migration disorders. (Am J Pathol 2010, 176:320-329; DOI: 10.2353/ajpath.2010.081158)

    DOI

  • Role of collapsin response mediator proteins for dendritic development in hippocampal CA1 pyramidal neurons

    Emi Niisato, Naoya Yamashita, Fumio Nakamura, Yoshio Goshima, Toshio Ohshima

    NEUROSCIENCE RESEARCH   68   E250 - E251  2010  [Refereed]

    DOI

  • Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum.

    Ito Yoko, Tanaka Hideomi, Okamoto Hitoshi, Ohshima Toshio

    Characterization of neural stem cells and their progeny in the adult zebrafish optic tectum.   342 ( 1 ) 26 - 38  2010

     View Summary

    :In the adult teleost brain, proliferating cells are observed in a broad area, while these cells have a restricted distribution in adult mammalian brains. In the adult teleost optic tectum, most of the proliferating cells are distributed in the caudal margin of the periventricular gray zone (PGZ). We found that the PGZ is largely divided into 3 regions: 1 mitotic region and 2 post-mitotic regions-the superficial and deep layers. These regions are distinguished by the differential expression of several marker genes: pcna, sox2, msi1, elavl3, gfap, fabp7a, and s100beta. Using transgenic zebrafish Tg (gfap:GFP), we found that the deep layer cells specifically express gfap:GFP and have a radial glial morphology. We noted that bromodeoxyuridine (BrdU)-positive cells in the mitotic region did not exhibit glial properties, but maintained neuroepithelial characteristics. Pulse chase experiments with BrdU-positive cells revealed the presence of self-renewing stem cells within the mitotic region. BrdU-positive cells differentiate into glutamatergic or GABAergic neurons and oligodendrocytes in the superficial layer and into radial glial cells in the deep layer. These results demonstrate that the proliferating cells in the PGZ contribute to neuronal and glial lineages to maintain the structure of the optic tectum in adult zebrafish.

    DOI

  • JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration.

    Jin Junghee, Suzuki Hiromi, Hirai Syu-Ichi, Mikoshiba Katsuhiko, Ohshima Toshio

    JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration.   70 ( 14 ) 929 - 942  2010

     View Summary

    :Doublecortin (DCX) is expressed in young neurons and functions as a microtubule-associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migration. Previous in vitro phosphorylation experiments revealed that cyclin-dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5-/- mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP-DCX-WT, or GFP-DCX-S332A and analyzed neurite extension and migration. Introduction of GFP-DCX-WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP-DCX-S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5-/- brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function.

    DOI

  • Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

    Yutaka Uchida, Toshio Ohshima, Naoya Yamashita, Miyuki Ogawara, Yukio Sasaki, Fumio Nakamura, Yoshio Goshima

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 40 ) 27393 - 27401  2009.10  [Refereed]

     View Summary

    Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr(32) residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr(32) of CRMP showed that Tyr(32)-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr(32) to Phe(32)) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr(32) is involved in Sema3A signaling.

    DOI

  • Commitment of 1-Methyl-4-phenylpyrinidinium Ion-induced Neuronal Cell Death by Proteasome-mediated Degradation of p35 Cyclin-dependent Kinase 5 Activator

    Ryo Endo, Taro Saito, Akiko Asada, Hiroyuki Kawahara, Toshio Ohshima, Shin-ichi Hisanaga

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 38 ) 26029 - 26039  2009.09  [Refereed]

     View Summary

    The dysfunction of proteasomes and mitochondria has been implicated in the pathogenesis of Parkinson disease. However, the mechanism by which this dysfunction causes neuronal cell death is unknown. We studied the role of cyclin-dependent kinase 5 (Cdk5)-p35 in the neuronal cell death induced by 1-methyl-4-phenylpyrinidinium ion (MPP(+)), which has been used as an in vitro model of Parkinson disease. When cultured neurons were treated with 100 mu M MPP(+), p35 was degraded by proteasomes at 3 h, much earlier than the neurons underwent cell death at 12-24 h. The degradation of p35 was accompanied by the down-regulation of Cdk5 activity. We looked for the primary target of MPP(+) that triggered the proteasome-mediated degradation of p35. MPP(+) treatment for 3 h induced the fragmentation of the mitochondria, reduced complex I activity of the respiratory chain without affecting ATP levels, and impaired the mitochondrial import system. The dysfunction of the mitochondrial import system is suggested to up-regulate proteasome activity, leading to the ubiquitin-independent degradation of p35. The overexpression of p35 attenuated MPP(+)-induced neuronal cell death. In contrast, depletion of p35 with short hairpin RNA not only induced cell death but also sensitized to MPP(+) treatment. These results indicate that a brief MPP(+) treatment triggers the delayed neuronal cell death by the down-regulation of Cdk5 activity via mitochondrial dysfunction-induced up-regulation of proteasome activity. We propose a role for Cdk5-p35 as a survival factor in countering MPP(+)-induced neuronal cell death.

    DOI

  • Increased proximal bifurcation of CA1 pyramidal apical dendrites in sema3A mutant mice.

    Nakamura F, Ugajin K, Yamashita N, Okada T, Uchida Y, Taniguchi M, Ohshima T, Goshima Y

    J Comp Neurol   516 ( 5 ) 360 - 375  2009.06

    CiNii

  • Suppression of mutant huntingtin aggregate formation by Cdk5/p35 through the effect on microtubule stability

    Sayuko Kaminosono, Taro Saito, Fumitaka Oyama, Toshio Ohshima, Akiko Asada, Yoshitaka Nagai, Nobuyuki Nukina, Shin-ichi Hisanaga

    JOURNAL OF NEUROSCIENCE   28 ( 35 ) 8747 - 8755  2008.08  [Refereed]

     View Summary

    Huntington's disease (HD) is a polyglutamine [ poly(Q)] disease with an expanded poly(Q) stretch in the N terminus of the huntingtin protein (htt). A major pathological feature of HD neurons is inclusion bodies, detergent-insoluble aggregates composed of poly(Q)expanded mutant htt (mhtt). Misfolding of mhtt is thought to confer a toxic property via formation of aggregates. Although toxic molecular species are still debated, it is important to clarify the aggregation mechanism to understand the pathogenesis of mhtt. Weshow Cdk5/p35 suppresses the formation of mhtt inclusion bodies in cell lines and primary neurons. Although we expressed the N-terminal exon 1 fragment of htt lacking phosphorylation sites for Cdk5 in COS-7 cells, the kinase activity of Cdk5 was required for the suppression. Furthermore, Cdk5/p35 suppressed inclusion formation of atrophin-1, another poly(Q) protein, raising the possibility that Cdk5/p35 generally suppresses inclusion formation of poly(Q) proteins. Microtubules (MTs) were a downstream component of Cdk5/p35 in the suppression of inclusion formation; Cdk5/p35 disrupted MTs, which were required for the formation of inclusions. Moreover, stabilization of MTs by Taxol induced inclusions even with overexpression of Cdk5/p35. The formation of inclusions was also regulated by manipulating the Cdk5/p35 activity in primary rat or mouse cortical neuron cultures. These results indicate that Cdk5-dependent regulation of MT organization is involved in the development of aggregate formation and subsequent pathogenesis of poly(Q) diseases. This Cdk5 inhibition of htt aggregates is a novel mechanism different from htt phosphorylation and interaction with Cdk5 reported previously.

    DOI

  • Unusual patch-matrix organization in the retrosplenial cortex of the reeler mouse and Shaking rat Kawasaki

    Noritaka Ichinohe, Adrian Knight, Masaharu Ogawa, Toshio Ohshima, Katsuhiko Mikoshiba, Yoshihiro Yoshihara, Toshio Terashima, Kathleen S. Rockland

    CEREBRAL CORTEX   18 ( 5 ) 1125 - 1138  2008.05  [Refereed]

     View Summary

    The rat granular retrosplenial cortex (GRS) is a simplified cortex, with distinct stratification and, in the uppermost layers, distinct modularity. Thalamic and cortical inputs are segregated by layers and in layer 1 colocalize, respectively, with apical dendritic bundles originating from neurons in layers 2 or 5. To further investigate this organization, we turned to reelin-deficient reeler mouse and Shaking rat Kawasaki. We found that the disrupted lamination, evident in Nissl stains in these rodents, is in fact a patch-matrix mosaic of segregated afferents and dendrites. Patches consist of thalamocortical connections, visualized by vesicular glutamate transporter 2 (VGluT2) or AChE. The surrounding matrix consists of corticocortical terminations, visualized by VGluT1 or zinc. Dendrites concentrate in the matrix or patches, depending on whether they are OCAM positive (matrix) or negative (patches). In wild-type rodents and, presumably, mutants, OCAM(+) structures originate from layer 5 neurons. By double labeling for dendrites (filled by Lucifer yellow in fixed slice) and OCAM immunofluorescence, we ascertained 2 populations in reeler: dendritic branches either preferred (putative layer 5 neurons) or avoided (putative supra-granular neurons) the OCAM 1 matrix. We conclude that input-target relationships are largely preserved in the mutant GRS and that dendrite-dendrite interactions involving OCAM influence the formation of the mosaic configuration.

    DOI PubMed

  • Regulation of spine development by Semaphorin3A through cyclin-dependent kinase 5 phosphorylation of collapsin response mediator protein 1

    Naoya Yamashita, Asa Morita, Yutaka Uchida, Fumio Nakamura, Hiroshi Usui, Toshio Ohshima, Masahiko Taniguchi, Jerome Honnorat, Nicole Thomasset, Kohtaro Takei, Takuya Takahashi, Pappachan Kolattukudy, Yoshio Goshima

    JOURNAL OF NEUROSCIENCE   27 ( 46 ) 12546 - 12554  2007.11  [Refereed]

     View Summary

    Collapsin response mediator protein 1(CRMP1) is one of the CRMP family members that mediates signal transduction of axonal guidance and neuronal migration. We show here evidence that CRMP1 is involved in semaphorin3A (Sema3A)-induced spine development in the cerebral cortex. In the cultured cortical neurons from crmp1(+/-) mice, Sema3A increased the density of clusters of synapsin I and postsynaptic density-95, but this increase was markedly attenuated in crmp1(-/-) mice. This attenuation was also seen in cyclin-dependent kinase 5 (cdk5)(-/-) neurons. Furthermore, the introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, (Thr 509 and Ser 522 were replaced by Ala), a mutant that cannot be phosphorylated by Cdk5, into crmp1(-/-) neurons rescued the defect in Sema3A responsiveness. The Golgi-impregnation method showed that the crmp1(-/-) layer V cortical neurons showed a lower density of synaptic bouton-like structures and that this phenotype had genetic interaction with sema3A. These findings suggest that Sema3A-induced spine development is regulated by phosphorylation of CRMP1 by Cdk5.

    DOI

  • Cyclin-dependent kinase 5 is required for control of neuroblast migration in the postnatal subventricular zone

    Yuki Hirota, Toshio Ohshima, Naoko Kaneko, Makiko Ikeda, Takuji Iwasato, Ashok B. Kulkarni, Katsuhiko Mikoshiba, Hideyuki Okano, Kazunobu Sawamoto

    JOURNAL OF NEUROSCIENCE   27 ( 47 ) 12829 - 12838  2007.11  [Refereed]

     View Summary

    At the lateral wall of the lateral ventricles in the adult rodent brain, neuroblasts form an extensive network of elongated cell aggregates called chains in the subventricular zone and migrate toward the olfactory bulb. The molecular mechanisms regulating this migration of neuroblasts are essentially unknown. Here, we report a novel role for cyclin-dependent kinase 5 (Cdk5), a neuronal protein kinase, in this process. Using in vitro and in vivo conditional knock-out experiments, we found that Cdk5 deletion impaired the chain formation, speed, directionality, and leading process extension of the neuroblasts in a cell-autonomous manner. These findings suggest that Cdk5 plays an important role in neuroblast migration in the postnatal subventricular zone.

    DOI

  • A stream of cells migrating from the caudal telencephalon reveals a link between the amygdala and neocortex

    Ryan Remedios, Dhananjay Huilgol, Bhaskar Saha, Padmanabhan Hari, Lahar Bhatnagar, Thomas Kowalczyk, Robert F. Hevner, Yoko Suda, Shinichi Aizawa, Toshio Ohshima, Anastassia Stoykova, Shubha Tole

    NATURE NEUROSCIENCE   10 ( 9 ) 1141 - 1150  2007.09  [Refereed]

     View Summary

    The amygdaloid complex consists of diverse nuclei that belong to distinct functional systems, yet many issues about its development are poorly understood. Here, we identify a stream of migrating cells that form specific amygdaloid nuclei in mice. In utero electroporation showed that this caudal amygdaloid stream ( CAS) originated in a unique domain at the caudal telencephalic pole that is contiguous with the dorsal pallium, which was previously thought to generate only neocortical cells. The CAS and the neocortex share mechanisms for specification ( transcription factors Tbr1, Lhx2 and Emx1/ 2) and migration ( reelin and Cdk5). Reelin, a critical cue for migration in the neocortex, and Cdk5, which is specifically required for migration along radial glia in the neocortex, were both selectively required for the normal migration of the CAS, but not for that of other amygdaloid nuclei. This is first evidence of a dorsal pallial contribution to the amygdala, demonstrating a developmental and mechanistic link between the amygdala and the neocortex.

    DOI

  • Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex

    Toshio Ohshima, Motoyuki Hirasawa, Hidenori Tabata, Tetsuji Mutoh, Tomoko Adachi, Hiromi Suzuki, Keiko Saruta, Takuji Iwasato, Shigeyoshi Itohara, Mistuhiro Hashimoto, Kazunori Nakajima, Masaharu Ogawa, Ashok B. Kulkarni, Katsuhiko Mikoshiba

    DEVELOPMENT   134 ( 12 ) 2273 - 2282  2007.06  [Refereed]

     View Summary

    The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.

    DOI

  • Pak1 regulates dendritic branching and spine formation

    Kanehiro Hayashi, Toshio Ohshima, Mitsuhiro Hashimoto, Katsuhiko Mikoshiba

    DEVELOPMENTAL NEUROBIOLOGY   67 ( 5 ) 655 - 669  2007.04  [Refereed]

     View Summary

    The serine/threonine kinase p21-activated kinase 1 (Pak1) modulates actin and microtubule dynamics. The neuronal functions of Pak1, despite its abundant expression in the brain, have not yet been fully delineated. Previously, we reported that Pak1 mediates initiation of dendrite formation. In the present study, the role of Pak1 in dendritogenesis, spine formation and maintenance was examined in detail. Overexpression of constitutively active-Pak1 in immature cortical neurons increased not only the number of the primary branching on apical dendrites but also the number of basal dendrites. In contrast, introduction of dominant negative-Pak caused a reduction in both of these morphological features. The length and the number of secondary apical branch points of dendrites were not significantly different in cultured neurons expressing these mutant forms, suggesting that Pak1 plays a role in dendritogenesis. Pak1 also plays a role in the formation and maintenance of spines, as evidenced by the altered spine morphology, resulting from overexpression of mutant forms of Pak1 in immature and mature hippocampal neurons. Thus, our results provide further evidence of the key role of Pak1 in the regulation of dendritogenesis, dendritic arborization, the spine formation, and maintenance. (c) 2007 Wiley Periodicals, Inc.

    DOI

  • Modulation of Reelin signaling by cyclin-dependent kinase 5

    Toshio Ohshima, Hiromi Suzuki, Toshifumi Morimura, Masaharu Ogawa, Katsuhiko Mikoshiba

    BRAIN RESEARCH   1140   84 - 95  2007.04  [Refereed]

     View Summary

    The Reelin signaling and Cyclin-dependent kinase 5 (Cdk5) both regulate neuronal positioning in the developing brain. Using double-transgenic mice, we have previously shown that these two signaling pathways lie in parallel fashion and have a genetic interaction. Disabled-1 (Dab1), an adapter protein, mediates Reelin signaling and becomes tyrosine-phosphorylated on the binding of Reelin to its receptors. Several isoforms of Dab1 are expressed in embryonic mouse brain, and p80 [Dab1(S55)] is the major protein translated. In the present study, we investigated whether CdkS-mediated phosphorylation of Dab1 modulates Reelin signaling. Cdk5 phosphorylates p80 Dabl at multiple sites in its carboxyl terminal region, and tyrosine phosphorylation of p80 Dabl by Fyn tyrosine kinase is attenuated by this Cdk5-mediated phosphorylation in vitro. Tyrosine phosphorylation. of p80 Dab1 induced by exogenous Reelin is enhanced in Cdk5-deficient neurons, corroborating the inhibitory effect of Cdk5-mediated Ser/Thr phosphorylation on tyrosine phosphorylation of p80 Dabl. Another isoform, p45 Dabl [Dabl(271)], however, is phosphorylated by CdkS at one serine residue within a unique carboxyl-terminal region, and its serine phosphorylation enhances tyrosine phosphorylation. by Fyn and results in progressive degradation of p45 Dabl. These results indicate that Cdk5 modulates Reelin signaling through the Ser/Thr phosphorylation. of Dabl differently in an isoform-specific manner. (c) 2006 Elsevier B.V. All rights reserved.

    DOI

  • Collapsin response mediator protein 1 mediates Reelin signaling in cortical neuronal migration

    Naoya Yamashita, Yutaka Uchida, Toshio Ohshima, Syu-ichi Hirai, Fumio Nakamura, Masahiko Taniguchi, Katsuhiko Mikoshiba, Jerome Honnorat, Pappachan Kolattukudy, Nicole Thomasset, Kohtaro Takei, Takuya Takahashi, Yoshio Goshima

    JOURNAL OF NEUROSCIENCE   26 ( 51 ) 13357 - 13362  2006.12  [Refereed]

     View Summary

    Collapsin response mediator protein 1 ( CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin ( Reln) signaling to regulate neuronal migration in the cerebral cortex. In crmp1(-/-) mice, radial migration of cortical neurons was retarded. This phenotype was not observed in the sema3A(-/-) and crmp1(+/+); sema3A(+/+) cortices. However, CRMP1 was colocalized with disabled- 1 ( Dab1), an adaptor protein in Reln signaling. In the Reln(rl/rl) cortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background led to the disruption of hippocampal lamination, a Reeler- like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.

    DOI

  • Phosphorylation of the tubulin-binding protein, stathmin, by Cdk5 and MAP kinases in the brain

    Kanehiro Hayashi, Yong Pan, Hongjun Shu, Toshio Ohshima, Janice W. Kansy, Charles L. White, Carol A. Tamminga, Andre Sobel, Patrick A. Curmi, Katsuhiko Mikoshiba, James A. Bibb

    JOURNAL OF NEUROCHEMISTRY   99 ( 1 ) 237 - 250  2006.10  [Refereed]

     View Summary

    Regulation of cytoskeletal dynamics is essential to neuronal plasticity during development and adulthood. Dysregulation of these mechanisms may contribute to neuropsychiatric and neurodegenerative diseases. The neuronal protein kinase, cyclin-dependent kinase 5 (Cdk5), is involved in multiple aspects of neuronal function, including regulation of cytoskeleton. A neuroproteomic search identified the tubulin-binding protein, stathmin, as a novel Cdk5 substrate. Stathmin was phosphorylated by Cdk5 in vitro at Ser25 and Ser38, previously identified as mitogen-activated protein kinase (MAPK) and p38 MAPK delta sites. Cdk5 predominantly phosphorylated Ser38, while MAPK and p38 MAPK delta predominantly phosphorylated Ser25. Stathmin was phosphorylated at both sites in mouse brain, with higher levels in cortex and striatum. Cdk5 knockout mice exhibited decreased phospho-Ser38 levels. During development, phospho-Ser25 and -Ser38 levels peaked at post-natal day 7, followed by reduction in total stathmin. Inhibition of protein phosphatases in striatal slices caused an increase in phospho-Ser25 and a decrease in total stathmin. Interestingly, the prefrontal cortex of schizophrenic patients had increased phospho-Ser25 levels. In contrast, total and phospho-Ser25 stoichiometries were decreased in the hippocampus of Alzheimer's patients. Thus, microtubule regulatory mechanisms involving the phosphorylation of stathmin may contribute to developmental synaptic pruning and structural plasticity, and may be involved in neuropsychiatric and neurodegenerative disorders.

    DOI

  • Enhanced activation of Ca2+/calmodulin-dependent kinase II upon downregulation of Cyclin-dependent kinase 5-p35.

    Hosokawa, T, Saito, T, Asada, A, Ohshima, T, Itakura, M, Takahashi, M, Fukunaga, K. Hisanaga, S

    J. Neurosci. Res.   84   747 - 754  2006.06

  • cdk5 is required for neuroblast migration in the adult mouse brain

    Yuki Hirota, Toshio Ohshima, Takuji Iwasato, Ashok B. Kulkarni, Hideyuki Okano, Kazunobu Sawamoto

    NEUROSCIENCE RESEARCH   55   S52 - S52  2006

  • Cdk5-dependent regulation of glucose-stimulated insulin secretion

    FY Wei, K Nagashima, T Ohshima, Y Saheki, YF Lu, M Matsushita, Y Yamada, K Mikoshiba, Y Seino, H Matsui, K Tomizawa

    NATURE MEDICINE   11 ( 10 ) 1104 - 1108  2005.10  [Refereed]

     View Summary

    Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications(1). Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K+ (K-ATP) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca2+ channel current and increased Ca2+ influx across the L-type voltage-dependent Ca2+ channel ( L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca2+ influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the alpha(1C) subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.

    DOI PubMed

  • Impairment of hippocampal long-term depression and defective spatial learning and memory in p35(-/-) mice

    T Ohshima, H Ogura, K Tomizawa, K Hayashi, H Suzuki, T Saito, H Kamei, A Nishi, JA Bibb, S Hisanaga, H Matsui, K Mikoshiba

    JOURNAL OF NEUROCHEMISTRY   94 ( 4 ) 917 - 925  2005.08  [Refereed]

     View Summary

    Cdk5 (cyclin-dependent kinase 5) activity is dependent upon association with one of two neuron-specific activators, p35 or p39. Genetic deletion of Cdk5 causes perinatal lethality with severe defects in corticogenesis and neuronal positioning. p35(-/-) mice are viable with milder histological abnormalities. Although substantial evidence implicates Cdk5 in synaptic plasticity, its role in learning and memory has not been evaluated using mutant mouse models. We report here that p35(-/-) mice have deficiencies in spatial learning and memory. Close examination of hippocampal circuitry revealed subtle histological defects in CA1 pyramidal cells. Furthermore, p35(-/-) mice exhibit impaired long-term depression and depotentiation of long-term potentiation in the Schaeffer collateral CA1 pathway. Moreover, the Cdk5-dependent phosphorylation state of protein phosphatase inhibitor-1 was increased in 4-week-old mice due to increased levels of p39, which co-localized with inhibitor-1 and Cdk5 in the cytoplasm. These results demonstrate that p35-dependent Cdk5 activity is important to learning and synaptic plasticity. Deletion of p35 may shift the substrate specificity of Cdk5 due to compensatory expression of p39.

    DOI PubMed

  • Control of cyclin-dependent kinase 5 (Cdk5) activity by glutamatergic regulation of p35 stability

    FY Wei, K Tomizawa, T Ohshima, A Asada, T Saito, C Nguyen, JA Bibb, K Ishiguro, AB Kulkarni, HC Pant, K Mikoshiba, H Matsui, S Hisanaga

    JOURNAL OF NEUROCHEMISTRY   93 ( 2 ) 502 - 512  2005.04  [Refereed]

     View Summary

    Although the roles of cyclin-dependent kinase 5 (Cdk5) in neurodevelopment and neurodegeneration have been studied extensively, regulation of Cdk5 activity has remained largely unexplored. We report here that glutamate, acting via NMDA or kainate receptors, can induce a transient Ca2+/calmodulin-dependent activation of Cdk5 that results in enhanced autophosphorylation and proteasome-dependent degradation of a Cdk5 activator p35, and thus ultimately down-regulation of Cdk5 activity. The relevance of this regulation to synaptic plasticity was examined in hippocampal slices using theta burst stimulation. p35(-/-) mice exhibited a lower threshold for induction of long-term potentiation. Thus excitatory glutamatergic neurotransmission regulates Cdk5 activity through p35 degradation, and this pathway may contribute to plasticity.

    DOI PubMed

  • Increased activity of cyclin-dependent kinase 5 leads to attenuation of cocaine-mediated dopamine signaling

    Satoru Takahashi, Toshio Ohshima, Andrew Cho, Taduru Sreenath, Michael J. Iadarola, Harish C. Pant, Yong Kim, Angus C. Nairn, Roscoe O. Brady, Paul Greengard, Ashok B. Kulkarni

    Proceedings of the National Academy of Sciences   102 ( 5 ) 1737 - 1742  2005.02  [Refereed]

     View Summary

    Cocaine, a drug of abuse, increases synaptic dopamine levels in the striatum by blocking dopamine reuptake at axon terminals. Cyclin-dependent kinase 5 (Cdk5) and its activator p35, proteins involved in phosphorylation of substrates in postmitotic neurons, have been found to be up-regulated after chronic exposure to cocaine. To further examine the effects of Cdk5 and p35 induction on striatal dopamine signaling, we generated two independent transgenic mouse lines in which Cdk5 or p35 was overexpressed specifically in neurons. We report here that increased Cdk5 activity, as a result of p35 but not of Cdk5 overexpression, leads to attenuation of cocaine-mediated dopamine signaling. Increased Cdk5-mediated phosphorylation of dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa (DARPP-32) at Thr-75, was accompanied by decreased phosphorylation of DARPP-32 at Thr-34. Increased Cdk5-mediated phosphorylation of extracellular signal-regulated kinase kinase 1 at Thr-286 was accompanied by decreased activation of extracellular signal-regulated kinase 1/2. These effects contributed to attenuation of cocaine-induced phosphorylation of cAMP response element-binding protein as well as a lesser induction of c-fos in the striatum. These results support the idea that Cdk5 activity is involved in altered gene expression after chronic exposure to cocaine and hence impacts the long-lasting changes in neuronal function underlying cocaine addiction.

    DOI

  • Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3 beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease

    Y Uchida, T Ohshima, Y Sasaki, H Suzuki, S Yanai, N Yamashita, F Nakamura, K Takei, Y Ihara, K Mikoshiba, P Kolattukudy, J Honnorat, Y Goshima

    GENES TO CELLS   10 ( 2 ) 165 - 179  2005.02  [Refereed]

     View Summary

    Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5(-/-) mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.

    DOI PubMed

  • Correlation between semaphorin3A-induced facilitation of axonal transport and local activation of a translation initiation factor eukaryotic translation initiation factor 4E

    CX Li, Y Sasaki, K Takei, H Yamamoto, M Shouji, Y Sugiyama, T Kawakami, F Nakamura, T Yagi, T Ohshima, Y Goshima

    JOURNAL OF NEUROSCIENCE   24 ( 27 ) 6161 - 6170  2004.07  [Refereed]

     View Summary

    An impressive body of evidence has been accumulated indicating that local protein synthesis is implicated in navigation of neurite extension induced by guidance cues, such as semaphorin3A (Sema3A). We found previously that a Src type tyrosine kinase Fyn and cyclin-dependent kinase 5 (Cdk5) mediate Sema3A-signaling. We also showed that Sema3A elicits axonal transport through neuropilin-1, a receptor for Sema3A, located at the growth cones. Here, we investigate the relationship between Sema3A-induced local signaling, protein synthesis, and axonal transport. Lavendustin A, a tyrosine kinase inhibitor, and olomoucine, a cyclin-dependent kinase inhibitor, suppressed Sema3A-induced facilitation of anterograde and retrograde axonal transport in dorsal root ganglion (DRG) neuron with and without the cell body. Sema3A-induced facilitation of axonal transport was attenuated in DRG neurons of fyn- (fyn(-/-)) and a Cdk5 activator, p35 (p35(-/-))-deficient mice when compared with those of wild-type or heterozygous mice. Inhibition of protein synthesis suppressed Sema3A-induced facilitation of axonal transport in the DRG neuron with and without the cell body. Sema3A enhanced the level of immunoreactivity of phosphorylated eukaryotic translation initiation factor 4E (eIF-4E) within 5 min in growth cones in a time course similar to that of the facilitated axonal transport. This enhanced signal for phospho-eIF4E was blocked by lavendustin A or olomoucine and was not detected in the fyn(-/-) and p35(-/-) neurons. These results provide evidence for a mutual regulatory mechanism between local protein synthesis and axonal transport.

    DOI PubMed

  • Perinatal abrogation of Cdk5 expression in brain results in neuronal migration defects

    M Hirasawa, T Ohshima, S Takahashi, G Longenecker, Y Honjo, Veeranna, HC Pant, K Mikoshiba, RO Brady, AB Kulkarni

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   101 ( 16 ) 6249 - 6254  2004.04  [Refereed]

     View Summary

    Cyclin-dependent kinase 5 (Cdk5) is essential for the proper development of the CNS, as is evident from the perinatal lethality of conventional Cdk5 knockout (Cdk5-/-) mice. Cdk5 is also implicated in numerous complex functions of the adult CNS such as synaptic transmission, synaptic plasticity, and neuronal signaling. To elucidate the molecular roles of Cdk5 in the adult CNS, we have abrogated neuronal expression of Cdk5 in perinatal mice by using a cre-loxP system. The Cdk5-loxP flanked mice were crossed with the cre-transgenic mice in which the cre expression is driven by the murine neurofilament-heavy chain promoter, resulting in generation of viable Cdk5 conditional knockout mice with the restricted deletion of the Cdk5 gene in specific neurons beginning around embryonic day 16.5. Twenty-five percent of the Cdk5 conditional knockout mice carrying the heterozygous cre allele had neuronal migration defects confined to brain areas where neuronal migration continues through the perinatal period. These results indicate that abrogation of Cdk5 expression in mature neurons results in a viable mouse model that offers further opportunities to investigate the molecular roles of Cdk5 in the adult CNS.

    DOI PubMed

  • Cophosphorylation of amphiphysin 1 and dynamin 1 by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles

    K Tomizawa, S Sunada, YF Lu, Y Oda, M Kinuta, T Ohshima, T Saito, FY Wei, M Matsushita, ST Li, K Tsutsui, S Hisanaga, K Mikoshiba, K Takei, H Matsui

    JOURNAL OF CELL BIOLOGY   163 ( 4 ) 813 - 824  2003.11  [Refereed]

     View Summary

    It has been thought that clathrin-mediated endocytosis is regulated by phosphorylation and dephosphorylation of many endocytic proteins, including amphiphysin I and dynamin I. Here, we show that Cdk5/p35-dependent cophosphorylation of amphiphysin I and dynamin I plays a critical role in such processes. Cdk5 inhibitors enhanced the electric stimulation-induced endocytosis in hippocampal neurons, and the endocytosis was also enhanced in the neurons of p35-deficient mice. Cdk5 phosphorylated the proline-rich domain of both amphiphysin I and dynamin I in vitro and in vivo. Cdk5-dependent phosphorylation of amphiphysin I inhibited the association with P-adaptin. Furthermore, the phosphorylation of dynamin I blocked its binding to amphiphysin I. The phosphorylation of each protein reduced the copolymerization into a ring formation in a cell-free system. Moreover, the phosphorylation of both proteins completely disrupted the copolymerization into a ring formation. Finally, phosphorylation of both proteins was undetectable in p35-deficient mice.

    DOI PubMed CiNii

  • Inhibition of ubiquitin ligase Siah-1A by disabled-1

    TJ Park, H Hamanaka, T Ohshima, N Watanabe, K Mikoshiba, N Nukina

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   302 ( 4 ) 671 - 678  2003.03  [Refereed]

     View Summary

    Disabled-1 (Dab I) is a cytosolic adaptor protein that plays critical roles in cortical development. However, a detailed mechanism of action has not yet been clearly defined. Through yeast two-hybrid screening, we observed that mouse Siah-1A, an E3 ubiquitin ligase containing a RING finger motif, interacts with Dab I. Co-immunoprecipitation experiments and in vitro binding experiments both indicated direct interaction between Siah and Dab I. Steady-state expression of Siah was enhanced by the presence of Dab I or lactacystin, a representative proteasomal inhibitor. Auto-ubiquitination of Siah was inhibited by the presence of Dabl, suggesting inhibition of Siah activity and subsequent increase of Siah expression by Dabl. Both Dab1-induced increase of steady-state expression of Deleted in cololectal cancer (DCC), one of the well-known substrates of Siah, and its inhibition by SiahDeltaR suggest that Dabl increases expression of DCC through inhibiting the activity of endogenous Siah. Our results suggest that Dabl inhibits the activity of Siah. (C) 2003 Elsevier Science (USA). All rights reserved.

    DOI PubMed

  • Reelin signaling and cdk5 in the control of neuronal positioning

    T Ohshima, K Mikoshiba

    MOLECULAR NEUROBIOLOGY   26 ( 2-3 ) 153 - 166  2002.10  [Refereed]

     View Summary

    Neuronal positioning is important for higher brain function because it is the architectural basis of the formation of precise synaptic circuits. Analysis of neurological mutant mice has led to dramatic progress in the identification and characterization of molecules important for neuronal positioning in the developing mammalian brain. Among these molecules, identification of signal pathways mediated by Reelin and Cdk5 kinase has provided a conceptual framework for exploring the molecular mechanisms underlying proper neuronal positioning in the developing mammalian brain. Recent evidence has implicated synergism between Reelin signaling and Cdk5 in contributing to the proper positioning of selective neuronal populations.

    DOI PubMed

  • Pak1 is involved in dendrite initiation as a downstream effector of Rac1 in cortical neurons

    K Hayashi, T Ohshima, K Mikoshiba

    MOLECULAR AND CELLULAR NEUROSCIENCE   20 ( 4 ) 579 - 594  2002.08  [Refereed]

     View Summary

    Dendrite development in neurons is one of the bases for the formation of a complex neuronal network in the nervous system, and involvement of the Rho family GTPases, including Rac1, Cdc42, and RhoA, in dendrite formation has been demonstrated. One of the effectors of Rac1 and Cdc42, p21-activated kinase 1 (Pak1), is abundant in the brain; however, the function of Pak1 in neurons remains unknown. In order to clarify the roles of Pak1 in neurons, we introduced mutant Pak1 into immature neurons by a novel gene transfer technique using in utero electroporation. Introduction of constitutive active (CA)-Pak1 led to increase the number of dendrites, whereas introduction of dominant negative (DN)-Pak1 caused a reduction, and coexpression of CA-Rac1 and DN-Pak1 also resulted in a reduction in the number of dendrites. These results suggest that Pak1 regulates dendrite initiation as a downstream effector of Rac1.

    DOI PubMed

  • Fyn and Cdk5 mediate Semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex

    Y Sasaki, C Cheng, Y Uchida, O Nakajima, T Ohshima, T Yagi, M Taniguchi, T Nakayama, R Kishida, Y Kudo, S Ohno, F Nakamura, Y Goshima

    NEURON   35 ( 5 ) 907 - 920  2002.08  [Refereed]

     View Summary

    Semaphorin-3A (Sema3A), a member of class 3 semaphorins, regulates axon and dendrite guidance in the nervous system. How Sema3A and its receptors plexin-As and neuropilins regulate neuronal guidance is unknown. We observed that in fyn- and cdk5-deficient mice, Sema3A-induced growth cone collapse responses were attenuated compared to their heterologous controls. Cdk5 is associated with plexin-A2 through the active state of Fyn. Sema3A promotes Cdk5 activity through phosphorylation of Tyr15, a phosphorylation site with Fyn. A Cdk5 mutant (Tyr15 to Ala) shows a dominant-negative effect on the Sema3A-induced collapse response. The sema3A gene shows strong interaction with fyn for apical dendrite guidance in the cerebral cortex. We propose a signal transduction pathway in which Fyn and Cdk5 mediate neuronal guidance regulated by Sema3A.

    DOI

  • Cyclin-Dependent Kinase 5/p35 Contributes Synergistically with Reelin/Dab1 to the Positioning of Facial Branchiomotor and Inferior Olive Neurons in the Developing Mouse Hindbrain

    Toshio Ohshima, Masaharu Ogawa, Kyoko Takeuchi, Satoru Takahashi, Ashok B. Kulkarni, Katsuhiko Mikoshiba

    The Journal of Neuroscience   22 ( 10 ) 4036 - 4044  2002.05  [Refereed]

    DOI

  • Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain

    Toshio Ohshima, Masaharu Ogawa, Veeranna, Motoyuki Hirasawa, Glenn Longenecker, Koichi Ishiguro, Harish C. Pant, Roscoe O. Brady, Ashok B. Kulkarni, Katsuhiko Mikoshiba

    Proceedings of the National Academy of Sciences of the United States of America   98 ( 5 ) 2764 - 2769  2001.02

     View Summary

    Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35-/- mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35-/-Cdk5+/- mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35-/- mice compared with Cdk5-/- mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35-/- causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.

    DOI PubMed

  • Phosphorylation of Drebrin by Cdk5 and its role in neuronal migration.

    Tanabe K, Yamazaki J, Inaguma H, Asada A, Kimura T, Takahashi J, Taoka M, Ohshima T, Furuichi T, Isobe T, Nagata K, Shirao T, Hisanaga SI

    PlosOne   9   3

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Books and Other Publications

  • Cyclin Dependent Kinase5

    Ohshima, T

    Spriger  2008 ISBN: 9780387788869

Presentations

  • Requirement of Cdk5 for proper cerebellar development and function

    2nd international symposium on Cdk5 Tokyo 

    Presentation date: 2009.06

Research Projects

  • Study on the role of CRMP2 phosphorylation in neural circuit formation and regeneration

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2022.04
    -
    2025.03
     

  • Cross-talk between endocrine and environmental factors in neuroimmune disorders

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2018.04
    -
    2023.03
     

  • CRMPをターゲットとした神経損傷・神経変性疾患治療戦略開発

    Project Year :

    2018.04
    -
    2021.03
     

     View Summary

    神経疾患の治療が困難である大きな理由は、神経系の乏しい再生能力にある。それは、神経再生を妨げる機構の存在に原因あり、その細胞内のシグナルに関連した因子としてCRMPが注目されている。本研究では、CRMPをターゲットとした神経変性疾患治療法開発に向けて、ヒトの病態に近いモデルマウスを用いて、CRMPの遺伝子変異の効果及びLKEなどの薬剤の効果を明らかにすることを目的とする。神経損傷のモデルとしては、脊髄損傷と視神経損傷、神経変性疾患としてはパーキンソン病を取り上げた。MPTP-induced パーキンソン病モデルにおいて、CRMP4KOでドーパミン神経細胞死が抑制されることを見出し報告した(Tonouchiら J Neurochem. 2016)が、MPTP-induced パーキンソン病モデルに比べよりヒト疾患病態に近いalpha-synuclein過剰発現によるマウスモデルを用いて検証する。alpha-synuclein過剰発現 (alpha-synuclein Tg) マウスとCRMP4KOと交配し、alpha-synuclein Tgマウスとalpha-synuclein Tg; CRMP4KOマウスの病態進行を比較することにより、CRMP4欠損のパーキンソン病態への効果を評価する。脊髄損傷と視神経損傷に関しては、野生型とCRMP変異マウスへの損傷からの回復を比較して、神経損傷からの神経再生に関するCRMPの役割を明らかにする。本年度は、alpha-synuclein Tgマウスを導入し、病態進行を組織学的、生化学的、行動学的方法により解析し、先行研究と比較した。また、alpha-synuclein TgマウスとCRMP4KOマウスの交配を行ない、alpha-synuclein Tg; CRMP4KOマウスの作成を開始した。また、視神経損傷に関して、野生型とCRMP変異マウスへの損傷からの回復を比較した。CRMP変異マウスとしては、CRMP2のSer522をAlaに置換したCRMP2KIマウスを用いて、神経損傷後の神経変性と神経再生に対するCRMP2のリン酸化抑制の効果を検討した。CRMP2KIマウスにおいては、Cdk5によるS522のリン酸化に加え、GSK3betaによるT518, T514, T509のリン酸化も起こらない。CRMP2KIマウスのおいては、視神経損傷後の視神経変性が抑制されること(Kinoshita et al., 2019)、視神経再生が促進されることを(Kondo et al., 2019)を見出して、論文発表を行なった。特に、後者の研究成果はプレスリリースを行なった。令和2年度は令和1年度に導入したパーキンソン病モデルであるalpha-synuclein Tgマウスに対するCRMP4欠損の影響を検討する。具体的には、alpha-synuclein Tgとalpha-synuclein Tg; CRMP4KOマウスの病態進行を、組織学的、生化学的、行動学的手法により比較解析して、研究成果を学会活動や学術論文等を通じて公開する

  • Elucidation of roles of CRMP in neural circuit formation and regeneration and application for the treatment of spinal cord injury

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2014.04
    -
    2017.03
     

    Ohshima Toshio, NAGAI Jun

     View Summary

    In present study, we analyzed the role of CRMP family proteins in brain development and neural regeneration by using mutant mice of CRMP genes. We reported orientation abnormality of basal dendrites of layer V neurons in cerebral cortex of CRMP4 KO mice and this phenotype became worse in CRMP 1/4 double KO mice (Takaya et al., 2017). We also reported the involvement of CRMP2 phosphorylation in dendritic spine formation (Jin et al., 2016). In neural regeneration, we found that suppression of CRMP2 phosphorylation promoted functional recovery of motor and sensory system after spinal cord injury (Nagai et al., 2016). We further showed that LKE, which modified the function of CRMP2, also promoted recovery of motor function after spinal cord injury (Kotaka et al., 2017). These results indicate the importance of CRMP family proteins in brain development and their involvements in neural regeneration

  • Functional analysis of Cdk5 by using conditional KO of its activating subunit p35

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2009
    -
    2011
     

    OHSHIMA Toshio

     View Summary

    The purpose of this study is to analyze the function of neuron-specific serine/threonine kinase Cyclin-dependent kinase 5(Cdk5) by using conditional KO mice of its activating subunit p35 trough the behavioral analysis such as spatial learning and fear conditioning test, and electrophysiological analysis and biochemical analysis. In this study we generated p35-flox mice in which two loxP sequences were inserted in p35 gene allele. We generated inducible p35 conditional KO by crossing p35-flox and CreER mice in which Cre is activated by Tamoxifen(TM) and p35 gene was destructed after brain formed. We confirmed decrease of p35 protein by p. o. administration of 2 mg TM for three days. We also generated CA1-specific p35 conditional KO mice by crossing CaMKII-Cre mice, and confirmed decreased level of p35 protein in hippocampus. We observed decreased numbers of dendritic spines in the hippocampal CA1 pyramidal neurons in CA1-p35 cKO mice. Using these p35 conditional KO mice, we currently conduct behavioral analysis of p35 cKO mice

  • The role of CRMP family proteins in the establishment of neural tissue architectures

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2005
    -
    2009
     

    GOSHIMA Yoshio, OOSHIMA Toshio

     View Summary

    In developing brain, axon and dendritic guidance are regulated by repulsive and attractive axon guidance molecules such as semaphorin3A (Sema3A) and netrin. Collpapsin response mediator protein (CRMP) has originally been identified as an intracellular protein that mediates Sema3A. We found that Sema3A elicits axoplasmic transport that may be involved in regulating the localization of AMPA type glutamate receptors in hippocampal neurons. To elucidate in vivo role of CRMPs, we generated several crmp1 and other crmp family gene-deficient mice and performed phenotypic analysis of these mice. For instance, in crmp1-deficient mice, the cell migration of cortical neurons at early embryonic stages is retarded. CRMP1 is colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In the Relnrl/rl cortex, CRMP1 and Dab1 are expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background lead to the disruption of hippocampal lamination, a Reeler-like phenotype. CRMP1 is also involved in Sema3A-induced localization of AMPA receptors and spine development in the cerebral cortex. In the cultured cortical neurons from crmp1 mice, Sema3A increases the density of clusters of synapsin I and postsynaptic density-95, but this increase is markedly attenuated in crmp1-deficient mice. In our study of C. elegans, we identified several mutant alleles which show aberrant localization of netrin/UNC-6 and axon guidance defects. We therefore conclude that the regulation of the glutamate receptor and the axon guidance molecule localization may play an important role in a wide variety of developmental processes from cell migration to neural network formation

  • Proteomics of phospho-proteins during brain development using mutant mice

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2006
    -
    2007
     

    OHSHIMA Toshio

     View Summary

    Purpose and Method : In this proposal, I attempt to understand the mechanism of brain development by analyzing protein phosphorylations which are important for such processes. Comparison of phospho-proteins between controls and mutant mice which have defective brain development will provide us new findings about molecular mechanism of brain development. For this purpose, we conducted 2D-DIGE method followed by MS analysis to identify the proteins.Results: In 2006, we analyzed the cerebral cortex samples from Cdk5 KO mice and their littermate controls at E 18.5. Cdk5 is a neuron-specific Ser/Thr protein kinase and Cdk5 KO mice exhibit defective brain development particularly in layer formation of cortical structures. We conducted protetomics of phospho-proteins by 2D-DIGE method, and identified CRMP1, 2 and 4 among 23 differential spots. We previously reported that Cdk5 phosphorylates CRMP2 at Ser522. We confirmed that Cdk5 phosphorylates CRMP1 at Thr 509 in vivo using phosphor-specific antibody for pThr509 CRMP1. However, we couldn't detect the reduction of phosphorylation of CRMP4 Ser522, indicating the redundant phosphorylation by other kinase (s). We also found the reduced phosphorylation of stathmin Ser38 (Hayashi, et. al., 2006).In 2007, we analyzed cerebella from Dab 1 mutant yotari and cerebellum-specific Cdk5 KO mice along with their littermate controls. We identified several differential spots and further analysis of these spots is now on going in our laboratory. We also reported that Cdk5 inhibits Reelin signaling through the phosphorylation of Dabl (Ohshima, et. al., 2007)

  • Identification and functional analysis of Cdk5-mediated phosphorylation of the proteins that are related to brain formation and development

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research

    Project Year :

    2003
    -
    2005
     

    OHSHIMA Toshio

     View Summary

    Purpose and Method :Cdk5 is a neuron-specific Ser/Thr protein kinase and Cdk5 KO mice exhibit defective brain development particularly in layer formation of cortical structures. Abnormalities of brain development in Cdk5 KO mice are caused by defects in the phosphorylations of brain development related-proteins. Using brains from Cdk5 KO mice, we conducted a screening to identify Cdk5 substrate(s). Functional analysis of newly identified substrate(s) is further performed. We also studied the functional significance of Cdk5-mediated phosphorylation of Dab1 which is a intracellular mediator of Reelin signaling.Results :Through the screening, we identified CRMP2 as a substrate of Cdk5. Under the collaboration with Dr.Goshima's group in Yokohama City University School of Medicine, we found Cdk5 phosphorylates CRMP2 at Ser522. This phosphorylation is required for Ser509 phosphorylation by GSK3β. This sequential phosphorylation by Cdk5 and GSK3β reduces the affinity between CRMP2 and tubulins. We also demonstrate that this sequential phosphorylation of CRMP2 is related to the production of 3F4 reactivity specifically in the brains from Alzheimer's patients. In another study, we showed that Cdk5 phosphorylates Dab1 at multiple sites in its C-terminal. Cdk5-mediated Ser/Thr phosphorylations of Dab1 inhibit tyrosine phosphorylation of Dab1 induced by Reelin. This result indicates a negative modulation of Reelin signaling by Cdk5

  • 神経特異的サイクリン依存性キナーゼ(cdk5)の個体レベルでの機能解析

     View Summary

    Cdk5の個体レベルでの機能解析のため、これまでにCdk5及び活性化subunit p35の欠損マウスを作製し、Cdk5/p35キナーゼが大脳,小脳,海馬などの皮質において神経細胞のcontrolされたmigrationに基づく層構造形成にcell-autonomousに必須であることを明らかにしてきた(Ohshima et al.,1999)。しかしセリン・スレオニンキナーゼであるCdk5/p35の欠損がなぜ神経細胞のmigrationの異常をきたすのかは不明である。標的タンパク質としてPak1や、その欠損マウスの表現型の類似性などから、Reelin/Dab1シグナリングなどへの関与が推定されている。本研究ではPak1に関してはdeletion mutantの解析などからCdk5/p35によりPak1 N-terminal(1-252)のThrがリン酸化される事が明らかになった(未発表)。またReelin/Dab1との関係を明らかにするため、p35とReelinあるいはDab1の二重欠損マウスを作製し表現型を解析した。その結果、二重欠損マウスはその神経細胞のmigrationの障害がより増悪する事からCdk5/p35はReelinシグナルの直接の下流ではないと考えた。しかし、p35欠損にReelinあるいはDab1のheterozygosityか加わる事によりmigrationの増悪が認められる事から、両者は共働的に皮質神経細胞のmigrationのcontrolに寄与している事が明らかとなった(Ohshima et al.,2001)。またNIHの研究グループとの共同研究により、Cdk5の神経細胞での機能発現はマウスのsurvivalに必須である事を明らかにした(Tanaka et al.,2001)

  • 誘導型コンディショナルKOを用いたCdk5の脳機能における役割の解明

     View Summary

    Cyclin-dependent kinase 5 (Cdk5)は、他のCdkが分裂中の細胞に活性が高いのに対して、activating subunit p35, p39が神経細胞に特異的に発現しているため、神経細胞に活性を認めるユニークなCdkである。我々のp35の欠損マウスの解析から、空間学習・記憶にCdk5が必須であり、LTDの誘導に何らかの関与がある可能性が示唆された(Ohshima, et. al., 2005)。Cdk5の誘導型コンディショナルKOのライン用いて、Cdk5の記憶・学習などの脳機能における役割を明らかにする事を計画していたが、米国のBibb博士らの研究グループから、Cdk5の誘導型コンディショナルノックアウトを解析した結果の報告がなされた(Nat. Neurosci. 10:880-886, 2007)。こうした現状を踏まえて、領域特異的なコンディショナルKOの解析を行なう事とし、Emx1Creマウスを用いたコンディショナルKO (ECKO)マウスを作成した。まず、Cdk5欠損神経細胞の放射状神経細胞の移動を解析したところ、細胞移動中に観察される双極性(bipolar)の形態から多極性(multipolar)への変換が障害されている事が明らかとなった。さらに、ECKOの解析により、こうした異常が生後脳において、結果的に大脳皮質の錐体神経細胞の形態異常を引き起こす事が明らかとなった(Development134:2273-2282, 2007)。また、共同研究により、Cdk5がamygdaloidのnLot細胞(Nat. Neurosci. 10, 1038-1047, 2007)や、RMSの細胞移動(J. Neurosci. 27:12829-12838, 2007)に必須なキナーゼである事を示した

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Misc

  • Effect of valproic acid on Cdk5 activity and mouse behavior

    M. Takahashi, M. Ishida, T. Saito, H. Kobayashi, A. Kawakami, T. Takasugi, T. Ohshima, S. Hisanaga

    JOURNAL OF NEUROCHEMISTRY   130   37 - 37  2014.08

    Research paper, summary (international conference)  

  • The role of Tyr15 phosphorylation in Cdk5 activation

    H. Kobayashi, T. Saito, K. Sato, T. Hosokawa, K. Tsutsumi, A. Asada, S. Kamada, T. Ohshima

    JOURNAL OF NEUROCHEMISTRY   123   114 - 114  2012.10

    Research paper, summary (international conference)  

  • Sema3A regulates apical dendritic growth of hippocamal CA1 pyramidal neurons

    Fumio Nakamura, Kozue Ugajin, Naoya Yamashita, Masahiko Taniguchi, Toshio Ohshima, Yoshio Goshima

    NEUROSCIENCE RESEARCH   65   S46 - S46  2009

    Research paper, summary (international conference)  

    DOI

  • Expression profiling of neural stem cell markers in dividing cells of the adult zebrafish optic tectum

    Yoko Ito, Hideomi Tanaka, Hitoshi Okamoto, Toshio Ohshima

    NEUROSCIENCE RESEARCH   61   S169 - S169  2008

    Research paper, summary (international conference)  

  • CRMP1 regulates spine maturation through mediating Semaphorin3A signaling in vivo

    Naoya Yamashita, Asa Morita, Yutaka Uchida, Toshio Ohshima, Fumio Nakamura, Jerome Honnorat, Pappachan Kolattukudy, Yoshio Goshima

    NEUROSCIENCE RESEARCH   58   S204 - S204  2007

    Research paper, summary (international conference)  

  • Phosphorylation of CRMP2 at S522 is required for proper dendritic patterning of layer V cortical neurons in vivo

    Yutaka Uchida, Toshio Ohshima, Naoya Yamashita, Fumio Nakamura, Papachan Kolattukudy, Jerome Honnorat, Yoshio Goshima

    NEUROSCIENCE RESEARCH   58   S203 - S203  2007

    Research paper, summary (international conference)  

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Syllabus

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Sub-affiliation

  • Faculty of Science and Engineering   Graduate School of Advanced Science and Engineering

  • Affiliated organization   Global Education Center

Research Institute

  • 2022
    -
    2024

    Waseda Research Institute for Science and Engineering   Concurrent Researcher

Internal Special Research Projects

  • アルツハイマー病モデルマウスにおける遺伝子発現変動の解析

    2024  

     View Summary

    アルツハイマー病は認知障害を示す神経変性疾患で、その脳病変はAPP由来のAmyloid betaの蓄積からなる老人斑と、過剰リン酸化したタウタンパク質の神経細胞内凝集体の神経原線維変化(NFT)が特徴である。APPNL-G-Fマウスは、早期にA蓄積が認められるなど患者脳の病変再現に優れている。我々は、APPNL-G-Fマウスの海馬に早期に起きる遺伝子発現変化を調べた。その結果、これまで報告されていなかった遺伝子変化を見出すことに成功した。さらに、その遺伝子変化に相応したタンパク質レベルの変化が見いだされるかを、免疫組織学的に検討している。

  • ゼブラフィッシュ神経損傷回復におけるミドカインの役割の解明

    2023  

     View Summary

    ミドカインは組織修復時に発現が上昇することが知られている分泌因子であり、ゼブラフィッシュにおいても、心臓やヒレの再生時に発現し、これら組織の修復に寄与することが知られている。中枢神経系でも網膜の再生に寄与することが報告されたが、我々が解析してきた視神経や視蓋の損傷からの再生への寄与は検討されていない。視蓋損傷時に発現の変動する遺伝子群をRNA-seqで網羅的に解析した結果、ミドカイン遺伝子mdkaの発現が上昇していた。本研究では、mdka遺伝子発現について細胞レベルで解析するとともにその役割を機能喪失の系で検討する。実験では、抗ミドカイン抗体を用いて、各種細胞マーカーとの2重染色を視蓋損傷側と非損傷側で比較検討するとともに、mdka mutant fishにおける視蓋損傷や視神経損傷における回復を野生型と比較する。こうした実験を通じて、ミドカインの損傷修復時の役割について明らかにすることを目的とした。 本研究では、視蓋損傷時において、損傷側で増殖するラジアルグリア(RG)にミドカインが発現するかを調べるため、RGにGFPが発現しているGFAP:GFPトランスジェニックフィッシュをに対し、視蓋損傷を行ない、損傷側でRGの増殖がみとめられる損傷後2日目にミドカインの抗体染色を行なった。その結果、損傷側のRGでミドカインの染色が検出された。さらに、ミドカインの阻害剤であるiMDKを用いた阻害実験の結果、RGの増殖が抑制されることが分かった。これらの結果より、損傷を受けた視蓋ではRGがミドカインを分泌して、それがRGの増殖を促進している、オートクライン的な働きが考えられた。更に、mdka mutant fishをZRICより入手したので、今後はmdka欠損ゼブラフィッシュを用いた実験を行なって行く予定である。

  • 細胞種特異的ノックアウトを用いたCRMP4の脳損傷と神経変性疾患への関与の解明

    2022  

     View Summary

    これまで脊髄損傷や薬剤誘導型PDモデルにおいてCRMP4遺伝子を欠損させることで病態進行を抑制出来ることを示してきたが、これらの知見はCRMP4の機能阻害が神経疾患治療の治療ターゲットとなる可能性を示すものである。しかしながら、神経細胞のCRMP4欠損が病態進行に対して抑制効果を引き起こすのか、アストロサイトまたはミクログリアのCRMP4欠損が引き起こすのか不明である。こうした状況から、神経細胞特異的、アストロサイト特異的、ミクログリア特異的CRMP4cKOマウスの病態モデルを作成し、組織学的、生化学的、行動学的に解析を行なうことを目的として研究を進めた。

  • ゼブラフィッシュを用いた神経再生の分子メカニズム解明

    2021  

     View Summary

    ゼブラフィッシュは脳損傷後の神経再生能力が高いことが知られているが、そのメカニズムには不明な点が多い。われわれは、視蓋においてはラジアルグリアが神経幹細胞として神経再生に寄与していることを明らかにしてきたが、加齢による影響は未解明であった。ゼブラフィッシュ3カ月齢と12カ月齢で損傷後の神経再生を視蓋で比較したところ、12カ月齢でラジアルグリアの増殖が低下していた。12カ月齢のゼブラフィッシュの視蓋損傷後にノッチシグナルの阻害剤を加えると、3か月齢と同程度にラジアルグリアの増殖が起きることを明らかとなった。今後は増殖したラジアルグリアの神経細胞への分化に加齢が関与するかを検討する。

  • ゼブラフィッシュを用いたCRMPの神経発生・神経再生への関与の解明

    2020  

     View Summary

    CRMP2はCRMP1-5からなる遺伝子ファミリーの一つであり、発生期の神経系に発現している。本研究では、CRISPR/Cas9を用いてCRMP2 knockout zebrafishの作成を行ない、その表現型を解析した。CRISPR/Cas9によるゲノム編集により1塩基挿入が起こり、結果的に短いN末のみが翻訳される変異が導入された。神経細胞をGFPで可視化するためTg(isl1:GFP)と交配して解析したところ、顔面神経核の形成不全が明らかとなった。また、この表現型は野生型CRMP2 mRNAで正常化したことから、変異導入によるCRMP2の機能喪失に特異的な表現型であることが証明された。

  • CRMPの神経発生・神経疾患への関与の解明

    2019  

     View Summary

    本研究は神経発達におけるCRMP遺伝子の機能解析を行なうことを目的としている。CRMPは神経発生期に発現が高く、CRMP1-5のfamily遺伝子があり、これらが機能的に相補的であることが示されている。そのため、単独欠損では他の代償のため表現型が軽症化するため重複欠損で解析する必要があり、今回はCRMP1KO; CRMP2KI; CRMP4KOマウスの小脳発達を野生型と比較した。その結果、プルキンエ細胞の配置異常が特に第X小葉において顕著に認められ、協調平衡運動が障害されていることが明らかとなった。以上の結果より、小脳発達特にプルキンエ細胞の位置決定にCRMPの機能が重要であることが示された。

  • CRMPタンパク質を標的とした神経損傷・神経変性疾患治療戦略開発

    2017  

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    CRMPは神経細胞の多く発現しているリン酸化タンパク質で、CRMP1-5のサブタイプがあり、4量体を形成していることが知られている。神経回路形成時に働くセマフォリン3Aの細胞内シグナル伝達に関与することが報告されたが、その後様々な軸索ガイダンス分子のシグナル伝達に関与し、神経損傷の修復や神経疾患への関与が示唆されている。我々は、CRMP4欠損が脊髄損傷からの回復に有利に働くことを報告している。CRMP2を介して機能すると考えられている薬剤LKEが脊髄損傷や神経変性疾患の治療に行こうであるか検討を続けている。また、共同研究により、CRMP2のリン酸化が躁うつ病の治療薬であるリチウムの作用点であることを明らかにした(Tobeet al., 2017)。

  • ゼブラフィッシュ成魚を用いた神経新生の制御メカニズムの解明

    2017  

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    ヒトなどの哺乳類では成体脳での神経新生が限られた脳部位でのみ起きるのに対して、ゼブラフィッシュなどの硬骨魚類では、多くの脳部位で神経新生が観察される。本研究では、ゼブラフィッシュ成魚脳の損傷時に、どの様な分子メカニズムで神経新生が促されて、損傷が修復されるかを検討した。その結果、Wntシグナルが神経新生の元になるラジアルグリア(RG)の増殖と神経細胞への分化に関与することを明らかにした(Shimizu et al., Glia 2018)。Wntシグナル以外にも、ShhシグナルやNotchシグナルの関与も示唆されるため、これらのシグナル伝達の関与も検討を進めている。研究成果が、ヒトの神経系の再生医療への応用につながることが期待される。

  • ゼブラフィッシュにおけるグリア細胞内シグナルと機能連携の可視化

    2016  

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    成体脳において神経新生が行われる現象は、ヒトを含む哺乳類では限られた脳領域で行なわれているが、ゼブラフィッシュなどの硬骨魚類では、比較的広範囲の脳領域で行なわれている。本研究では、神経幹細胞であるラジアルグリア細胞の増殖・分化のプロセスと、Ca動態を可視化することにより、グリア内細胞シグナルと幹細胞増殖・分化の関連を明らかにすることを目的とした。バイオリソースセンターよりGFAP:GCaMP6a導入して、解析を開始した。また、Gal4-UAS系を用いて、GFAP:GalFF Tg fish(Shimizuら2015)とUAS:GCaMP7を交配する予定である。

  • Cdk5活性化サブユニットp35の統合失調症への関与の解明

    2015  

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    統合失調症患者脳の解析からCdk5活性化サブユニットp35タンパク質量の減少が報告された。また、統合失調症患者の神経細胞のスパイン密度が減少していることが報告されている(Gareyら1993, 1995,1998; Glantz&Lewis 1995, 1996)。本研究では、条件付けp35遺伝子改変マウス(p35cKOマウス)におけるスパイン密度を解析した。その結果、発達期と成熟期のいずれにおいてもスパイン密度が減少していること、さらにその減少が大脳皮質と海馬の神経細胞で観察し、学術誌に報告した(MitaらCerebral cortex2016)。

  • Cdk5の神経機能における役割の解明

    2013  

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    <研究目的>サイクリン依存性キナーゼ(Cdk)は増殖細胞で細胞周期を制御する最も重要な因子である。Cdk5はCdkのメンバーでありながら、最終分化した神経細胞で機能するユニークなCdkである。Cdk5の活性化サブユニットであるp35欠損マウスでの空間学習記憶の障害を報告した(Ohshima et al., JNC 2005)が、p35欠損マウスには脳形成の異常があり、空間学習記憶の障害が脳形成の異常に起因する可能性がある。H21-23文科省基盤研究C「活性化サブユニットp35コンディショナルKOを用いたCdk5の機能解析」で確立したp35コンディショナルKO(p35cKO)の行動解析を行なうとともに、Cdk5のもう一つのサブユニットであるp39欠損も併せ持つp35KO;p39KOマウスを作製し、脳高次機能を解析することで、Cdk5のキナーゼとしての機能を明らかにすることを目的とした。 <研究結果>p35コンディショナルKOマウスは、タモキシフェン投与により誘導されるタイプのCreERマウスとの交配により確立した。このマウスラインを用いて研究を行なった。タモキシフェンを3日間経口投与し、1週後に脳各部位のp35タンパク質量をコントロールと比較し、p35タンパク質量が顕著に減少していることを確認した。3ヶ月齢以降でタモキシフェンを投与し、p35遺伝子欠損を誘導した。これにより脳構造に異常を有しない状態でのp35遺伝子欠損による機能的変化を調べることが可能となった。これまでにモリス水迷路試験を行ない、空間記憶・学習機能の障害が明らかになっている。現在、その障害の原因となる海馬のシナプス可塑性についての電気生理学的な検討と、生化学的な解析を行なっている。さらに、p39KOマウスと交配することで、誘導型p35cKO;p39KOマウスラインを確立した。今後このマウスラインを用いた研究を行なって行く予定である。

  • CRMPの神経回路形成および神経再生における役割の解明と脊髄損傷治療への応用

    2013  

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    【研究目的】中枢神経系損傷に対する決定的な治療法は未だない。そのため、中枢神経系の限られた再生能力の分子機構解明が急がれている。Collapsin-response mediator protein (CRMP)は軸索ガイダンス分子であるセマファリンの細胞内シグナル伝達物質として同定され、その後の研究で、軸索伸長などへの関与が示された。CRMPはファミリータンパク質でCRMP1-5が別々の遺伝子にコードされている。これらはヘテロ4量体を形成して機能していると考えられているが、生体内での役割は不明な点が多い。本研究は、CRMP遺伝子の改変マウスを用いて、中枢神経損傷後のCRMPの機能解明を目的とする。我々はこれまでに、CRMP4欠損マウス由来の後根神経節細胞の培養実験では中枢神経系神経再生を阻害する物質に対する反応が減弱していることを見出して報告している(Nagaiら2012)。従って本研究では特にCRMP4欠損(Crmp4-/-)マウスを用いた研究を行なった。【研究成果】野生型マウスの損傷した脊髄において再生阻害活性あるいは細胞毒性のあるCRMP4 のいくつかのformが上昇していることが確認された。こうした結果を踏まえて、本研究ではCrmp4-/-マウスを用いることにより、脊髄損傷後の再生過程におけるCRMP4の機能解明を試みた。脊髄損傷4週間後、Crmp4+/+マウスにおいては損傷部位以降の脊髄神経軸索消失、半身不随が観察されたが、Crmp4-/-マウスの脊髄では神経軸索が長距離にわたり再生し、後肢に加重ができるほど運動機能が回復していることが確認された。組織学的にも、再生する軸索のマーカーであるGAP43の増加が、生化学的および組織学的に示された。従って、CRMP4の機能を阻害することで、脊髄損傷の回復が改善される可能性がin vivoで示された。これらの結果から、CRMP4を標的とした中枢神経系損傷に対する新規治療開発の可能性が示唆された。

  • 成体神経新生促進因子スクリーニングのためのin vivoモニタリング系の開発

    2012  

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    成体脳での神経新生は、失われた神経機能を回復させる神経再生医療の面で注目されている。主に神経前駆細胞を補う細胞療法と、神経新生に係わる因子を用いて、内在性の神経新生機能を促進することで神経再生を成し遂げようとする、神経再生因子療法の2つに大別される。後者の場合、どの様な因子が有用であるかの研究が盛んに行われている。本研究では、生物モデルであるゼブラフィッシュ成魚で神経新生をモニタリング出来るラインを確立し、成体での神経新生促進因子を個体が生きたままスクリーニング出来るシステムを構築することを目的とする。具体的には、神経幹細胞特異的にルシフェラーゼと蛍光タンパク質を発現させ、ルシフェリンを化学発光させ、検出器IVISを用いて生きたままで成体脳での神経新生を経時的にモニタリングする系を確立することを目指した。手法としてはGal4-UASシステムを応用し、神経幹細胞特異的Gal4を発現しているTgゼブラフュッシュとUSAによりルシフェラーゼと蛍光タンパク質の発現が調整されているTgゼブラフィッシュの両系統を交配して得る。神経幹細胞のマーカーであるGFAPの遺伝子発現制御領域下でGal4の改良型であるGFFを発現させるTgライン(GFAP:GFF)を作成した。 このGFAP:GFF Tg fishとUAS-GFP fishと交配したF1を生後3日、1ヵ月、3か月齢でGFPの発現がGFAPの発現と同様であるかを検討した。方法として、1) GFAP-GFP fishでのGFP発現と比較を行う。2) GFAP陽性細胞の別のマーカーであるBLBPの免疫染色で確認した。1),2)の方法により、内在性のGFAP陽性の神経幹細胞と同様の発現を示すTg fishラインが得られたことが確認できた。さらにUAS下にルシフェラーゼと蛍光タンパク質を発現させるコンストラクトを作成した。今後このコンストラクトをGFAP:GFF Tg fishの受精卵に導入して一過性に発現させ、Gal4-UAS systemが機能するかを確認した上で、UAS Tg fishの作成を行なう計画である。

  • ゼブラフィッシュ成魚脳を用いた神経幹細胞の増殖・分化の分子機構の解析

    2012  

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    成体脳での神経新生は、失われた神経機能を回復させる神経再生医療の面で注目されている。しかし、神経幹細胞・神経前駆細胞の増殖・分化を制御する分子機構は不明な点が多い。本研究では、哺乳類に比較して広範囲で神経新生が行われている生物モデルであるゼブラフィッシュ成魚を用いて、神経幹細胞の増殖・分化に係わる分子機構の解明を目的とした。我々は眼の視神経の投射先である視蓋における神経新生について研究を行なってきた。マウスなど哺乳類の研究で神経新生への関与が示唆されているシグナル伝達経路が、ゼブラフィッシュ成魚視蓋においてどのように神経幹細胞の増殖・分化に関与するかを、シグナル伝達の阻害剤や遺伝子発現制御により明らかにすることを目指した。 今回の解析では、特にNotchシグナルとWntシグナルに着目して研究を進めた。Notchシグナルの活性化にはリガンドであるNotchのgamma-セクレターゼによる切断が必要であることから、Notchシグナルの阻害剤として用いられる。実験では、gamma-セクレターゼ阻害剤LY450139を飼育水中に加え、BrdU取り込みで細胞増殖を評価することにより、ゼブラフィッシュ成魚視蓋での神経幹細胞増殖を増加させることが明らかとなった。また、Wntシグナルの阻害剤であるIWR1を投与することにより、Wntシグナルの関与を検討したところ、視蓋における細胞増殖は抑制された。逆に、Wntシグナル活性化状態を模倣するGSK3抑制剤の投与により、細胞増殖は増加傾向であった。従って、阻害剤などの薬剤を用いた実験結果より、神経幹細胞増殖・神経前駆細胞に対して、Notchシグナルは抑制的に、Wntシグナルは促進的に働いていることが示唆された。 Wntシグナルに関しては、薬剤投与による実験から得られた結果をさらに検討するため、ヒートショックによりWntシグナルが抑制されるトランスジェニックゼブラフィッシュを用いてさらに検討を行なっている。

  • 軸索ガイダンス関連因子CRMPのリン酸化による機能制御の個体レベルでの解析と応用

    2011  

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    脳機能の発現には正しく神経回路が形成される必要があり、その過程で軸索ガイダンスという機構が重要である。細胞外に分泌される軸索ガイダンス分子とそれを感受するシステムの組み合わせがあり、Sema3Aというガイダンス分子を感受し、それに反応するために必要な分子として同定されたものがCRMPである。我々はCdk5の基質としてCRMP2を同定し、そのSer522をCdk5がリン酸化することを示した。本研究では、CRMPの機能をそのリン酸化による調節を含めて、マウスとゼブラフィッシュをモデル生物として用いて個体レベルで明らかにすることを目的として研究を進めてきた。CRMPはCRMP1-5の5つの遺伝子でコードされたホモロジーの高いファミリータンパク質であり、それがヘテロテトラマーを形成して機能している。遺伝子改変マウスはCRMP1,CRMP4のKOマウスとSer522をAlaに1アミノ酸置換したCRMP2のみを有するCRMP2KIマウスを用いて研究を進めた。CRMP1KO;CRMP2KIの大脳皮質の神経細胞は特にbasal dendriteの投射領域が変化する異常が明らかとなり(Yamashitaら2012)、CRMP4KOマウスでは先に報告されたSema3AKOマウスと同様に、海馬CA1の錐体神経細胞のapical dendriteの分岐の異常がそれぞれ観察された(Niisatoら2012)。また、ゼブラフィッシュの胚に遺伝子のノックダウンが可能なCRMPのAMOを導入してその効果を調べたところ、脊髄内感覚神経細胞(RB細胞)の位置の異常がCRMP2, CRMP4AMOで観察され、同様の異常がCdk5AMOで起こることが判った。さらに、Cdk5AMOの異常がCRMP2,4のリン酸化により改善することから、CRMP2,4のリン酸化がRB細胞の位置の決定に重要であることが示唆された(Tanakaら投稿中)。以上より、神経系の発達、特に樹状突起の形成や細胞の位置決定にCRMPのリン酸化が必要であることが、個体レベルで明らかになった。

  • 細胞内サイクリン依存性キナーゼ5活性のモニタリング法の開発

    2010  

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    サイクリン依存性キナーゼ5(Cyclin-dependent kinase 5, Cdk5)は、脳形成過程での役割に加え、様々な脳機能への関与が知れらているが、その活性上昇が神経変性過程で報告されている。しかし、活性上昇が神経細胞死の原因であるとする報告があるものの、結果にすぎないとする反論があり、結論が出ていない。神経細胞中のCdk5活性を生きたままモニタリング出来れば、細胞死に先立って活性上昇があるのか、細胞死後に活性が上がるのかを検討できる。本研究では、Fluorescence Resonant Energy Transfer (FRET)を用いた生細胞でのCdk5活性のモニタリング法を開発して、神経細胞内での活性測定を試みることを目的とした。 FRETによるCdk5活性のモニタリング法は、1. Cdk5活性と活性型Cdk5の局在-Cdk5及びp35キメラタンパク質のヘテロ二量体形成による解析、2. Cdk5の基質タンパク質にCdk5のリン酸化依存的に起きるタンパク質相互作用を利用したものを用いた。1のCdk5/p35ヘテロ二量体形成に伴うFRETを培養神経細胞で観察した。グルタミン酸添加により一過性の活性上昇が知られているが、FRETの上昇が観察され、Cdk5/p35のヘテロ二量体形成の増加が推定された。2の基質リン酸化依存的なタンパク室結合を、FRETシグナルとして培養COS細胞を用いた系で観察することに成功した。これらの結果を踏まえて今後神経細胞にCdk5活性を上昇させる刺激を加えた時、細胞死を誘導した時などのFRETの変化を検討する予定である。本研究によるCdk5活性のFRET法を用いたモニタリング系をさらに発展させることで、神経細胞死におけるCdk5活性の意義、即ち原因であるか結果であるかを明らかにすることが可能であると考えた。

  • 膵β細胞におけるCdk5の役割の解明と糖尿治療への応用

    2009  

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    我々は、Cdk5がL-typeカルシウムチャネルのリン酸化を介してインシュリン分泌を負に制御している事を報告し、Cdk5阻害剤の糖尿病治療薬への応用を示唆した(WeiらNat. Med 11, 1104, 2005)。2008年度の特定課題研究Aではbeta細胞特異的なCdk5抑制モデルマウスを作成し解析した。予想に反し、beta細胞特異的なCdk5抑制マウスは生後にbeta細胞の減少を示し、6週齢で糖尿病状態を示した。この事は、Cdk5がbeta細胞の生存に不可欠である事を示唆しているが、Cdk5活性の低下が、生存そのものに必須なのか、増殖・分化に必須なのか、不明である。Cdk5阻害剤の糖尿病治療薬としての応用を考えた場合には、Cdk5のbeta細胞への作用は、明確にする必要があり、以下の実験を行なった。1) beta細胞特異的なCdk5抑制モデルマウスのbata細胞の細胞死について、TUNEL染色で検討。2) beta細胞特異的なCdk5抑制モデルマウスのbeta細胞の増殖について、BrdUのラベルやKi67などの増殖細胞のマーカーで検討。3) beta細胞特異的なCdk5抑制モデルマウスとアポトーシスシグナル構成因子のBax欠損マウスを交配し、beta細胞の数を週齢ごとに、Tg;Bax+/+、 Tg;Bax-/-、 nonTg;Bax-/-の3群で比較。<結果と考察>1) beta細胞特異的なCdk5抑制モデルマウスにおいて、beta細胞が減少する原因を検討するため、TUNEL染色を行なったが、Tgマウスにおいて、明確なTUNEL陽性細胞数の上昇はなかった。2) 同様に、増殖細胞数に明らかな変化はなかった3) Tg;Bax-/- vs Tg;Bax+/+間で明確なbeta細胞が減少に差はなかった。すなわち、明確なrescueは得られなかった。Tg作成より、5-7世代目の解析となり、Tgの発現レベルが低下していた。このため、Tg単独でのbeta細胞の減少程度が軽度となり、細胞死、細胞増殖低下などの評価が困難となった。このため、1)-3)で行なった検討で、明確な結果が得られなかった。Tgは世代が進むにつれてTg由来の遺伝子産物の発現レベルが低下するという問題点があり、今回の解析でも、明確な結論が得られなかった大きな要因となった。今後検討を進めるために、再度Tgを作成する方向も検討することとした。

  • 神経特異的サイクリン依存性キナーゼ5の生理的機能と神経変性疾患における役割の解明

    2008  

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    目的神経特異的サイクリン依存性キナーゼ5(Cdk5)は、中枢神経系の形成過程や、機能発現に寄与するセリン・スレオニンキナーゼであり、我々は、Cdk5欠損マウスや活性化サブユニットp35欠損マウスの解析を通じて、研究を行ってきた。、Cdk5欠損マウスは胎生致死であり、p35欠損マウスは脳の形成に軽度の異常がある。我々はこれまでにp35欠損マウスにおける記憶・学習の異常を報告したが、異常が脳組織構築の異常によるものか、機能喪失によるものかは結論を得ていない。胎生致死などの解決するため、近年の技術進歩により、時間・空間的に遺伝子の機能喪失を起こさせるコンディショナルKOマウスの技術が、Cre-loxP系を応用して確立されている。我々は、本研究において、p35遺伝子のコンディショナルマウスを作製する事を目的として、p35遺伝子座に2ヶ所loxPを有するマウス(p35-floxマウス)の作成を行った。結果まず、p35遺伝子を含むBACクローンを入手し、ターゲティングコンストラクトを作成し、ES細胞に導入して、相同組換えを起こしたクローンを得た。これをブラストシストへ導入し、キメラマウスを得る事が出来た。このキメラ♂を野生型B6♀マウスと交配して、F1ヘテロマウスを作製する事に成功している。今後は、frtにフランクされたネオマイシン耐性遺伝子を除くために、FLP Tgマウスと交配した後に、ヘテロのp35-loxPマウス同志を交配し、p35-loxPのホモマウスを得たのち、creマウスと交配する事により、コンディショナルKOマウスを得る予定である。結語今回の研究により、p35遺伝子座loxPサイトが導入されたマウスを作製する事に成功し、今後のp35コンディショナルKOマウス作成の基盤が確立できた。

  • インシュリン分泌機構の遺伝子改変マウスを用いた検討と糖尿治療への応用

    2008  

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    我々は、Cdk5がL-typeカルシウムチャネルのリン酸化を介してインシュリン分泌を負に制御している事を報告し、Cdk5阻害剤の糖尿病治療薬への応用を示唆した(WeiらNat. Med 11, 1104, 2005)。Cdk5阻害によるインシュリン分泌の亢進作用は、高血糖時に顕著となるため、糖尿病薬の副作用である低血糖が起こさず、従来の治療薬とは作用点の異なる薬剤となる可能性がある。2005年の報告の際には、Cdk5活性が低下したマウスモデルとしてp35欠損マウスを用いた。p35はCdk5の活性化サブユニットであり、Cdk5はp35とのヘテロダイマーで活性型となる。p35欠損はCdk5の活性低下を来すが、全身性に遺伝子欠損の影響が出るため、Cdk5活性低下によるインシュリン分泌亢進がベータ細胞での直接的効果である事を示すに至らなかった。この問題をクリアするため、より適切なモデルマウスを作製する必要がある。 本研究では、insulin-promoter下にCdk5DNを発現するトランスジェニック(Tg)マウスラインを確立し、その解析を行った。ラインを確立した4系統のうち2系統でTg由来のCdk5DNタンパク質の膵臓ベータ細胞特異的な発現が確認された。Cdk5DNの発現により、インシュリンの分泌が亢進する事が期待されたが、Tgマウスにおいては、Cdk5DNの発現レベル依存的に、ベータ細胞が減少し、6週齢で糖尿病状態を示した。ベータ細胞の減少が観察される時期を検討した結果、生後10日目には既にラ氏島の縮小とベータ細胞数の減少が認められる事が明らかとなった。今後は、このベータ細胞の減少が、増殖の低下によるものか、アポトーシスなどの細胞死の増加によるものか、検討を行う予定である。今回の研究により、Cdk5活性の低下はベータ細胞の生存にも関与する事が初めて明らかとなった

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