Namikawa kazuhiko | Technische Universität Braunschweig (original) (raw)
Papers by Namikawa kazuhiko
Developmental Cell, 2006
TC10, a Rho family GTPase, has been shown to play an important role in the exocytosis of GLUT4 an... more TC10, a Rho family GTPase, has been shown to play an important role in the exocytosis of GLUT4 and other proteins, primarily by tethering the vesicles at the plasma membrane. Using a newly developed probe based on fluorescence resonance energy transfer, we found that TC10 activity at tethered vesicles dropped immediately before vesicle fusion in HeLa cells stimulated with epidermal growth factor (EGF), suggesting that GTP hydrolysis by TC10 is a critical step in vesicle fusion. In support of this model, a GTPasedeficient TC10 mutant potently inhibited EGF-induced vesicular fusion in HeLa cells and depolarizationinduced neuronal secretion. Furthermore, we found that GTP hydrolysis by TC10 in the vicinity of the plasma membrane was dependent on Rac and the redox-regulated Rho GAP, p190RhoGAP-A. We propose that an EGF-stimulated GAP accelerates GTP hydrolysis of TC10, thereby promoting vesicle fusion.
Journal of Neurochemistry, 2002
Abstract: An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve i... more Abstract: An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve injury was demonstrated by differential display PCR using single arbitrary primer coupled with in situ hybridization screening called in situ display. Differential display PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. Several gene fragments were increased after nerve injury; one was identified as GS. Subsequent emulsion autoradiography of hybridization tissue sections revealed that the increase in GS mRNA was observed in injured motoneurons. As GS is a key enzyme participating in the metabolism of the major excitatory neurotransmitter glutamate, we examined the significance of increased GS expression on glutamate-uptake kinetics. GS-transfected human embryonic kidney cells showed an up-regulation in glutamate-uptake kinetics. Therefore, newly expressed GS together with an increased expression of the neuronal glutamate transporter EAAC1 in the injured motoneurons accelerates glutamate uptake. The present results may suggest that the glutamate-uptake system involving the neuronal glutamate transporter and GS in injured neurons is enhanced so as to provide resistance against neurotoxic glutamate accumulation during the early process of nerve regeneration.
Human Molecular Genetics, 2003
Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal... more Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.
Biological Psychiatry, 2004
Background: Methamphetamine (MA) induces degeneration of various regions of the brain, resulting ... more Background: Methamphetamine (MA) induces degeneration of various regions of the brain, resulting in neuropsychiatric damage. Although the underlying mechanisms of MA-induced neurotoxicity have been studied, there are few reports to date regarding the factor(s) that can effectively prevent MA-induced neurotoxicity. Because brain-derived neurotrophic factor (BDNF) has been known to prevent many kinds of neuronal cell death, we investigated whether BDNF inhibits MA-induced neuronal death. Methods: Using primary cortical neurons, we examined the effect of BDNF on MA-induced neuronal death. In addition, using pharmacologic and molecular biological tools, we elucidated which pathways are involved in this effect. Results: Brain-derived neurotrophic factor dose-dependently blocked MA-induced neuronal death, and this effect was inhibited by phosphatidylinositol-3-kinase inhibitors. In addition, overexpression of activated Akt protects neurons against MA. Furthermore, expression of kinase-defective Akt blocked the effect of BDNF on MA-induced neuronal death. Conclusions: Brain-derived neurotrophic factor effectively blocks MA-induced neuronal death, and Akt activation is necessary and sufficient for this effect.
Gastroenterology, 2005
Abbreviations used in this paper: GFP, green fluorescent protein; HSC, hepatic stellate cell; IFN... more Abbreviations used in this paper: GFP, green fluorescent protein; HSC, hepatic stellate cell; IFN, interferon; TbRE, transforming growth factor -responsive element; TGF, transforming growth factor; ␣-SMA, ␣-smooth muscle actin.
Journal of Neurochemistry, 2003
The rat collapsin response mediator protein-2 (CRMP-2) is a member of CRMP family (CRMP-1–5). The... more The rat collapsin response mediator protein-2 (CRMP-2) is a member of CRMP family (CRMP-1–5). The functional consequence of CRMP-2 during embryonic development, particularly in neurite elongation, is relatively understood; however, the role in nerve regeneration is unclear. Here we examined the role of CRMP-2 during nerve regeneration using rat hypoglossal nerve injury model. Among the members, CRMP-1, CRMP-2, CRMP-5 mRNA expressions increased after nerve injury, whereas CRMP-3 and CRMP-4 mRNA did not show any significant change. In the N1E-115 cells, CRMP-2 has the most potent neurite elongation activity among the CRMP family members. In dorsal root ganglion (DRG) organ culture, CRMP-2 overexpression by adenoviral vector demonstrated substantial neurite elongation. On the other hand, CRMP-2 (ΔC381), which acts as a dominant negative form of CRMP-2, inhibited neurite formation. Collectively, it would be plausible that CRMP-2 has potent nerve regeneration activity after nerve injury. We therefore examined whether CRMP-2 overexpression in the injured hypoglossal motor neurons accelerates nerve regeneration. A retrograde-tracer, Fluoro-Gold (FG), was used to evaluate the number of reprojecting motor neurons after nerve injury. CRMP-2-overexpressing motor neurons demonstrated the accelerated reprojection. The present study suggests that CRMP-2 has potent neurite elongation activity in nerve regeneration in vivo.
Laboratory Investigation, 2004
We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad i... more We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFb/ activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 ll of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n ¼ 56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and a-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFb1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.
We developed a new method, designated N-linked glycosylation signal (NGS) differential display (D... more We developed a new method, designated N-linked glycosylation signal (NGS) differential display (DD)-PCR, that enables the identification of genes encoding N-linked glycosylated molecules that exhibit varying patterns of expression. Using this innovative technique, we identified an N-linked glycosylated 11-transmembrane domain protein that is upregulated in response to axotomy. Expression levels increased 3 d after axotomy, reached maximal levels at approximately postoperative days 5-7, and then gradually decreased through day 20. The protein was termed axotomy-induced glycosylated/Golgicomplex protein 1 (AIGP1). AIGP1 immunoreactivity is specifically localized in neurons, with subcellular localization within the Golgi, indicating that AIGP1 is a resident Golgi protein.
Biochemical and Biophysical Research Communications, 2005
In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-assoc... more In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-associated protein (PAP) family members following hypoglossal nerve injury in rats. In addition to four rat family members (RegI, Reg-2/PAP I, PAP II, and PAP III) that had been identified, we newly cloned and sequenced a type-IV Reg gene in rats. Among these five family members, the expression of Reg-2/PAP I mRNA was predominantly enhanced in injured motor neurons after axotomy. Furthermore, a marked induction of PAP III mRNA was observed in the distal part of the injured nerve. A polyclonal antibody was raised against PAP III, and a Western blotting analysis using this antibody confirmed an increased level of PAP III protein in the injured nerve. These results suggest that Reg family members would be new mediators among injured neurons and glial cells, and may play pivotal roles during nerve regeneration.
Journal of Neurochemistry, 2002
Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demo... more Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRα1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRα1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRα1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.
Molecular Brain Research, 2004
Type Ia phosphatidylinositol 3-kinase (PI3K) generates lipid products that operate as one of majo... more Type Ia phosphatidylinositol 3-kinase (PI3K) generates lipid products that operate as one of major second messengers following activation of tyrosine kinase receptors. PI3K is a heterodimer composed of a 110-kDa catalytic subunit and a regulatory subunit. In this study, we determined the expression of mRNA for the regulatory subunits after injury of rat hypoglossal nerves. In situ hybridization histochemistry revealed that the expression of PI3K regulatory subunit alpha isoforms (p85alpha, p55alpha, and p50alpha) was significantly enhanced in injured motor neurons, whereas other regulatory subunits such as p85beta or p55gamma were not detected. Of the alpha isoforms, the greatest increase was observed in p55alpha mRNA levels, while there were smaller increases in p85alpha and p50alpha mRNA expression. These results were confirmed by RT-PCR analysis. Further immunohistochemical analysis also confirmed the increased level of p55alpha protein in injured motor neurons. Taken together with the previously reported induction of the p110alpha catalytic subunit in injured neurons, these results suggest that PI3K, consisting of p55alpha and p110alpha, plays a crucial role in the process of nerve regeneration.
Molecular Brain Research, 2002
Journal of Neurochemistry, 2001
Ciliary neurotrophic factor (CNTF) can prevent injury-induced motor neuron death. However, it is ... more Ciliary neurotrophic factor (CNTF) can prevent injury-induced motor neuron death. However, it is also evident that expression of CNTF in Schwann cells is suppressed during nerve regeneration. In this report, we have addressed the mechanism underlying the down-regulation of CNTF expression in injured nerves using a mouse Schwann cell line IMS32 and mouse sciatic nerve. In IMS32 cells, activation of the Ras extracellular-signal-regulated kinase (ERK) pathway by adenoviral vector-mediated expression of dominant active MEK1 did not alter a basal level of CNTF expression, whereas inhibition of the Ras-ERK pathway by using adenoviral vectors resulted in a marked increase in CNTF expression. This inverse relation between before and after axotomy was also observed in mouse sciatic nerve. In the axotomized sciatic nerve, the phosphorylated ERK was markedly increased; in contrast, the expression of CNTF was markedly decreased. These findings suggest that an inactive state of ERK is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF. This might well explain why CNTF is highly expressed in quiescent Schwann cells in the peripheral nervous system, and also why CNTF is not abundant in axotomized nerves or cultured Schwann cells in which the proliferation signal is obviously active.
Brain Research, 2006
The aim of this study was to establish a novel adenovirus-based gene therapy system targeting ast... more The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.
Motoneurons require neurotrophic factors for their survival and axonal projection during developm... more Motoneurons require neurotrophic factors for their survival and axonal projection during development, as well as nerve regeneration. By using the axotomy-induced neuronal death paradigm and adenovirus-mediated gene transfer, we attempted to gain insight into the functional significances of major growth factor receptor downstream cascades, Ras-extracellular signalregulated kinase (Ras-ERK) pathway and phosphatidylinositol-3 kinase-Akt (PI3K-Akt) pathway. After neonatal hypoglossal nerve transection, the constitutively active Akt-overexpressing neurons could survive as well as those overexpressing Bcl-2, whereas the constitutively active ERK kinase (MEK)-overexpressing ones failed to survive. A dominant negative Akt experiment demonstrated that inhibition of Akt pathway hastened axotomy-induced neuronal death in the neonate. In addition, the dominant active Akt-overexpressing adult hypoglossal neurons showed accelerated axonal regeneration after axotomy. These results suggest that Akt plays dual roles in motoneuronal survival and nerve regeneration in vivo and that PI3K-Akt pathway is probably more vital in neuronal survival after injury than Ras-ERK pathway.
Photochemistry and Photobiology, 2001
(1 ؋ 10 6 ) were transfected at 1 ؋ 10 8 plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cell... more (1 ؋ 10 6 ) were transfected at 1 ؋ 10 8 plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 ؋ 10 9 PFU in 200 L. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m 2 ) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results in- ¶Posted on the website on dicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.
Neuroscience Research, 2004
This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), whic... more This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), which packages acetylcholine into synaptic vesicles, can be used as a marker for regenerating motor axon terminal. We examined motor axon regeneration in the tongue after hypoglossal nerve axotomy, using an anterograde tracer biotin-dextran (BD), retrograde tracer Fluoro-Gold (FG), electron microscopic (EM) observation, and VAChT immunocytochemistry. BD study demonstrated that outgrowth of thin regenerating axons into the frontal area of the tongue was firstly observed at 14 post-operative days, and presynaptic formation of neuromuscular junction (NMJ) was observed from 21 post-operative days. Under electron microscopic observation, reconstruction of new NMJs was observed within the interval between 21 and 28 days. VAChT-immunoreactive nerve terminals disappeared by 3 days after axotomy, slightly appeared at 14 post-operative days, and thereafter gradually increased in number from 21 to 28 post-operative days. The re-expression of VAChT positive presynaptic terminal was almost the same as those obtained in BD, FG and EM studies. Regenerating axons tip in the crush model of the hypoglossal nerve exhibited prominent VAChT immunoreactivity in growing tip of regenerating axons. These indicate that VAChT is an excellent morphological indicator for regenerating nerve terminals of motor neurons.
Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term po... more Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1 m , or dominant negative Ras by means of adenovirus vectors. Gap1 m expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNFinduced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.
Developmental Cell, 2006
TC10, a Rho family GTPase, has been shown to play an important role in the exocytosis of GLUT4 an... more TC10, a Rho family GTPase, has been shown to play an important role in the exocytosis of GLUT4 and other proteins, primarily by tethering the vesicles at the plasma membrane. Using a newly developed probe based on fluorescence resonance energy transfer, we found that TC10 activity at tethered vesicles dropped immediately before vesicle fusion in HeLa cells stimulated with epidermal growth factor (EGF), suggesting that GTP hydrolysis by TC10 is a critical step in vesicle fusion. In support of this model, a GTPasedeficient TC10 mutant potently inhibited EGF-induced vesicular fusion in HeLa cells and depolarizationinduced neuronal secretion. Furthermore, we found that GTP hydrolysis by TC10 in the vicinity of the plasma membrane was dependent on Rac and the redox-regulated Rho GAP, p190RhoGAP-A. We propose that an EGF-stimulated GAP accelerates GTP hydrolysis of TC10, thereby promoting vesicle fusion.
Journal of Neurochemistry, 2002
Abstract: An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve i... more Abstract: An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve injury was demonstrated by differential display PCR using single arbitrary primer coupled with in situ hybridization screening called in situ display. Differential display PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. Several gene fragments were increased after nerve injury; one was identified as GS. Subsequent emulsion autoradiography of hybridization tissue sections revealed that the increase in GS mRNA was observed in injured motoneurons. As GS is a key enzyme participating in the metabolism of the major excitatory neurotransmitter glutamate, we examined the significance of increased GS expression on glutamate-uptake kinetics. GS-transfected human embryonic kidney cells showed an up-regulation in glutamate-uptake kinetics. Therefore, newly expressed GS together with an increased expression of the neuronal glutamate transporter EAAC1 in the injured motoneurons accelerates glutamate uptake. The present results may suggest that the glutamate-uptake system involving the neuronal glutamate transporter and GS in injured neurons is enhanced so as to provide resistance against neurotoxic glutamate accumulation during the early process of nerve regeneration.
Human Molecular Genetics, 2003
Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal... more Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.
Biological Psychiatry, 2004
Background: Methamphetamine (MA) induces degeneration of various regions of the brain, resulting ... more Background: Methamphetamine (MA) induces degeneration of various regions of the brain, resulting in neuropsychiatric damage. Although the underlying mechanisms of MA-induced neurotoxicity have been studied, there are few reports to date regarding the factor(s) that can effectively prevent MA-induced neurotoxicity. Because brain-derived neurotrophic factor (BDNF) has been known to prevent many kinds of neuronal cell death, we investigated whether BDNF inhibits MA-induced neuronal death. Methods: Using primary cortical neurons, we examined the effect of BDNF on MA-induced neuronal death. In addition, using pharmacologic and molecular biological tools, we elucidated which pathways are involved in this effect. Results: Brain-derived neurotrophic factor dose-dependently blocked MA-induced neuronal death, and this effect was inhibited by phosphatidylinositol-3-kinase inhibitors. In addition, overexpression of activated Akt protects neurons against MA. Furthermore, expression of kinase-defective Akt blocked the effect of BDNF on MA-induced neuronal death. Conclusions: Brain-derived neurotrophic factor effectively blocks MA-induced neuronal death, and Akt activation is necessary and sufficient for this effect.
Gastroenterology, 2005
Abbreviations used in this paper: GFP, green fluorescent protein; HSC, hepatic stellate cell; IFN... more Abbreviations used in this paper: GFP, green fluorescent protein; HSC, hepatic stellate cell; IFN, interferon; TbRE, transforming growth factor -responsive element; TGF, transforming growth factor; ␣-SMA, ␣-smooth muscle actin.
Journal of Neurochemistry, 2003
The rat collapsin response mediator protein-2 (CRMP-2) is a member of CRMP family (CRMP-1–5). The... more The rat collapsin response mediator protein-2 (CRMP-2) is a member of CRMP family (CRMP-1–5). The functional consequence of CRMP-2 during embryonic development, particularly in neurite elongation, is relatively understood; however, the role in nerve regeneration is unclear. Here we examined the role of CRMP-2 during nerve regeneration using rat hypoglossal nerve injury model. Among the members, CRMP-1, CRMP-2, CRMP-5 mRNA expressions increased after nerve injury, whereas CRMP-3 and CRMP-4 mRNA did not show any significant change. In the N1E-115 cells, CRMP-2 has the most potent neurite elongation activity among the CRMP family members. In dorsal root ganglion (DRG) organ culture, CRMP-2 overexpression by adenoviral vector demonstrated substantial neurite elongation. On the other hand, CRMP-2 (ΔC381), which acts as a dominant negative form of CRMP-2, inhibited neurite formation. Collectively, it would be plausible that CRMP-2 has potent nerve regeneration activity after nerve injury. We therefore examined whether CRMP-2 overexpression in the injured hypoglossal motor neurons accelerates nerve regeneration. A retrograde-tracer, Fluoro-Gold (FG), was used to evaluate the number of reprojecting motor neurons after nerve injury. CRMP-2-overexpressing motor neurons demonstrated the accelerated reprojection. The present study suggests that CRMP-2 has potent neurite elongation activity in nerve regeneration in vivo.
Laboratory Investigation, 2004
We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad i... more We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFb/ activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 ll of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n ¼ 56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and a-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFb1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.
We developed a new method, designated N-linked glycosylation signal (NGS) differential display (D... more We developed a new method, designated N-linked glycosylation signal (NGS) differential display (DD)-PCR, that enables the identification of genes encoding N-linked glycosylated molecules that exhibit varying patterns of expression. Using this innovative technique, we identified an N-linked glycosylated 11-transmembrane domain protein that is upregulated in response to axotomy. Expression levels increased 3 d after axotomy, reached maximal levels at approximately postoperative days 5-7, and then gradually decreased through day 20. The protein was termed axotomy-induced glycosylated/Golgicomplex protein 1 (AIGP1). AIGP1 immunoreactivity is specifically localized in neurons, with subcellular localization within the Golgi, indicating that AIGP1 is a resident Golgi protein.
Biochemical and Biophysical Research Communications, 2005
In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-assoc... more In this study, we examined the expression of mRNAs for Regenerating gene (Reg)/pancreatitis-associated protein (PAP) family members following hypoglossal nerve injury in rats. In addition to four rat family members (RegI, Reg-2/PAP I, PAP II, and PAP III) that had been identified, we newly cloned and sequenced a type-IV Reg gene in rats. Among these five family members, the expression of Reg-2/PAP I mRNA was predominantly enhanced in injured motor neurons after axotomy. Furthermore, a marked induction of PAP III mRNA was observed in the distal part of the injured nerve. A polyclonal antibody was raised against PAP III, and a Western blotting analysis using this antibody confirmed an increased level of PAP III protein in the injured nerve. These results suggest that Reg family members would be new mediators among injured neurons and glial cells, and may play pivotal roles during nerve regeneration.
Journal of Neurochemistry, 2002
Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demo... more Axotomy-induced neuronal death occurs in neonatal motoneurons, but not in adult rat. Here we demonstrated that during the course of postnatal development, nerve injury induced down-regulation of the glial cell line-derived neurotrophic factor (GDNF) receptor GFRα1 in axotomized hypoglossal motoneurons of rat are gradually converted to the adult up-regulation pattern of response. The compensatory expression of GFRα1 specifically in the injured motoneurons of neonates by adenovirus succeeded in rescuing the injured neurons without an application of growth factors. To the contrary, the nuclear antisense RNA for GFRα1 expression accelerates the axotomy-induced neuronal death in pups. These findings suggest that the receptor expression response after nerve injury is critical for the determination of injured motoneuron fate.
Molecular Brain Research, 2004
Type Ia phosphatidylinositol 3-kinase (PI3K) generates lipid products that operate as one of majo... more Type Ia phosphatidylinositol 3-kinase (PI3K) generates lipid products that operate as one of major second messengers following activation of tyrosine kinase receptors. PI3K is a heterodimer composed of a 110-kDa catalytic subunit and a regulatory subunit. In this study, we determined the expression of mRNA for the regulatory subunits after injury of rat hypoglossal nerves. In situ hybridization histochemistry revealed that the expression of PI3K regulatory subunit alpha isoforms (p85alpha, p55alpha, and p50alpha) was significantly enhanced in injured motor neurons, whereas other regulatory subunits such as p85beta or p55gamma were not detected. Of the alpha isoforms, the greatest increase was observed in p55alpha mRNA levels, while there were smaller increases in p85alpha and p50alpha mRNA expression. These results were confirmed by RT-PCR analysis. Further immunohistochemical analysis also confirmed the increased level of p55alpha protein in injured motor neurons. Taken together with the previously reported induction of the p110alpha catalytic subunit in injured neurons, these results suggest that PI3K, consisting of p55alpha and p110alpha, plays a crucial role in the process of nerve regeneration.
Molecular Brain Research, 2002
Journal of Neurochemistry, 2001
Ciliary neurotrophic factor (CNTF) can prevent injury-induced motor neuron death. However, it is ... more Ciliary neurotrophic factor (CNTF) can prevent injury-induced motor neuron death. However, it is also evident that expression of CNTF in Schwann cells is suppressed during nerve regeneration. In this report, we have addressed the mechanism underlying the down-regulation of CNTF expression in injured nerves using a mouse Schwann cell line IMS32 and mouse sciatic nerve. In IMS32 cells, activation of the Ras extracellular-signal-regulated kinase (ERK) pathway by adenoviral vector-mediated expression of dominant active MEK1 did not alter a basal level of CNTF expression, whereas inhibition of the Ras-ERK pathway by using adenoviral vectors resulted in a marked increase in CNTF expression. This inverse relation between before and after axotomy was also observed in mouse sciatic nerve. In the axotomized sciatic nerve, the phosphorylated ERK was markedly increased; in contrast, the expression of CNTF was markedly decreased. These findings suggest that an inactive state of ERK is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF. This might well explain why CNTF is highly expressed in quiescent Schwann cells in the peripheral nervous system, and also why CNTF is not abundant in axotomized nerves or cultured Schwann cells in which the proliferation signal is obviously active.
Brain Research, 2006
The aim of this study was to establish a novel adenovirus-based gene therapy system targeting ast... more The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.
Motoneurons require neurotrophic factors for their survival and axonal projection during developm... more Motoneurons require neurotrophic factors for their survival and axonal projection during development, as well as nerve regeneration. By using the axotomy-induced neuronal death paradigm and adenovirus-mediated gene transfer, we attempted to gain insight into the functional significances of major growth factor receptor downstream cascades, Ras-extracellular signalregulated kinase (Ras-ERK) pathway and phosphatidylinositol-3 kinase-Akt (PI3K-Akt) pathway. After neonatal hypoglossal nerve transection, the constitutively active Akt-overexpressing neurons could survive as well as those overexpressing Bcl-2, whereas the constitutively active ERK kinase (MEK)-overexpressing ones failed to survive. A dominant negative Akt experiment demonstrated that inhibition of Akt pathway hastened axotomy-induced neuronal death in the neonate. In addition, the dominant active Akt-overexpressing adult hypoglossal neurons showed accelerated axonal regeneration after axotomy. These results suggest that Akt plays dual roles in motoneuronal survival and nerve regeneration in vivo and that PI3K-Akt pathway is probably more vital in neuronal survival after injury than Ras-ERK pathway.
Photochemistry and Photobiology, 2001
(1 ؋ 10 6 ) were transfected at 1 ؋ 10 8 plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cell... more (1 ؋ 10 6 ) were transfected at 1 ؋ 10 8 plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 ؋ 10 9 PFU in 200 L. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m 2 ) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results in- ¶Posted on the website on dicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.
Neuroscience Research, 2004
This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), whic... more This study was designed to evaluate whether the vesicular acetylcholine transporter (VAChT), which packages acetylcholine into synaptic vesicles, can be used as a marker for regenerating motor axon terminal. We examined motor axon regeneration in the tongue after hypoglossal nerve axotomy, using an anterograde tracer biotin-dextran (BD), retrograde tracer Fluoro-Gold (FG), electron microscopic (EM) observation, and VAChT immunocytochemistry. BD study demonstrated that outgrowth of thin regenerating axons into the frontal area of the tongue was firstly observed at 14 post-operative days, and presynaptic formation of neuromuscular junction (NMJ) was observed from 21 post-operative days. Under electron microscopic observation, reconstruction of new NMJs was observed within the interval between 21 and 28 days. VAChT-immunoreactive nerve terminals disappeared by 3 days after axotomy, slightly appeared at 14 post-operative days, and thereafter gradually increased in number from 21 to 28 post-operative days. The re-expression of VAChT positive presynaptic terminal was almost the same as those obtained in BD, FG and EM studies. Regenerating axons tip in the crush model of the hypoglossal nerve exhibited prominent VAChT immunoreactivity in growing tip of regenerating axons. These indicate that VAChT is an excellent morphological indicator for regenerating nerve terminals of motor neurons.
Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term po... more Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long-term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli, including calcium influx through NMDA receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and cAMP response element-binding protein (CREB) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase-activating protein, Gap1 m , or dominant negative Ras by means of adenovirus vectors. Gap1 m expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed similar effects. On the other hand, Rap1GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. In contrast to MAP kinase activation, the inactivation of Ras activity did not inhibit significantly NMDA-induced CREB phosphorylation, whereas BDNFinduced CREB phosphorylation was inhibited almost completely. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons and further suggest that CREB phosphorylation depends on multiple pathways.