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Adenosine facilitates the neuronal differentiation of neural stem cells derived from mouse hippocampus
Abstracts / Neuroscience Research 68S (2010) e335–e446
P3-c28 Inhibition by bisphenol A of mesencephalic neural stem-cell migration in a neurosphere assay in vitro Junko Suzuki , Masami Ishido Natl Inst for Environmental Studies, Tsukuba, Japan Developmental neurotoxicity of environmental chemicals has been recognized worldwide as a serious threat to human health. The large numbers of environmental chemicals that now exist have made assessing their neurodevelopmental risks challenging. Therefore, in the previous study we have established an in vitro neurosphere assay system to evaluate for environmental chemical toxicity. In this study, we extended to quantify neurodevelopmental toxicity of endocrine disruptors, such as bisphenol A. Neural stem cells isolated from rat E16 mesencephalone were plated in serum-free DMEM/ F12 (1:1) medium in the presence of bFGF and EGF to form neurospheres. Cells emerged from the neurosphere in adhesive dishes and migrated along the radial axis. The migrating populations were positive for nestin, a marker of neural stem cells. Exposure to bisphenol A resulted in inhibition of cell migration and proliferation in a dose-dependent manner. Quantitative analysis revealed a linear function between the inhibition of migration and the logarithm of bisphnol A concentration (0–100 M); the percentage of inhibition by 1 M bisphnol A of migration was 35% (p < 0.05). Thus, we showed for the first time that bisphenol A inhibited migration as well as proliferation of neural stem cells in vitro and that a neurosphere assay system in vitro is very useful for rapid quantification of neurodevelopmental toxicity of environmental chemicals. doi:10.1016/j.neures.2010.07.1581
P3-d01 Adenosine facilitates the neuronal differentiation of neural stem cells derived from mouse hippocampus Aya Hariu , Takahiro Moriya, Yuko Suzuki, Takashi Katura, Norimichi Nakahata Department of Cell Signal, Grad. School Pharm. Sci., Tohoku University, Sendai Neural stem cells (NSCs) possess the ability of the proliferation and the neuronal and glial differentiation and play crucial roles in the neurogenesis not only in developing, but also in adult brain. Adenosine is known to have widespread physiological roles in the CNS, including the modulation of synaptic activity. We have previously shown that adenosine suppresses the cell growth of NSCs derived from embryonic mouse hippocampus not via adenosine receptors, but through equilibrative nucleoside transporter 1 (ENT1), known as a plasma membrane adenosine transporter. The effect of adenosine on neuronal differentiation of NSCs, however, remains to be clarified. In this study, we investigated effects of adenosine on the differentiation of NSCs derived from embryonic mouse hippocampus in vitro and examined ENT1 mRNA levels in the course of neural differentiation from NSCs. We found that adenosine increased the proportion of TuJ1-positive neurons even when treated with EGF (20 ng/ml). The concentration-dependency for adenosine-induced neuronal differentiation (30–300 M) was well similar to that for cell growth inhibition. Furthermore, mRNA levels of ENT1 were decreased when NSCs were differentiated by EGF withdrawal and 1% FCS. These results suggest that adenosine has the ability to facilitate the neuronal differentiation of hippocampal NSCs. doi:10.1016/j.neures.2010.07.1582
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obtained cells were fixed for immunostaining of nestin and GFAP. TMT led to a significant increase in the nestin-positive cells, without affecting that of GFAP-positive cells. Cells cultured floatingly in DMEM/F-12 medium containing EGF/bFGF resulted in the formation of round spheres immunoreactive to nestin in 27 days in vitro (DIV). The number of neurospheres was significantly increased compared with those derived from naive animals. Cells were seeded on dishes that had been previously coated with poly-l-ornithine and then cultured in DMEM/F12 medium containing bFGF/EGF. Under these conditions, cells grew at monolayer without forming neurospheres. Most cells were immunoreactive with both nestin and GFAP. ELISA for 5 -bromo-2 deoxyuridine revealed that a marked increase in the proliferative activity was seen by treatment with TMT at 28 DIV. Our data suggest that hippocampal neural stem/progenitor cells can be abundantly prepared from the dentate gyrus of mice treated with TMT. Thus, TMT-treated mice are an attractive model for achieving the neural stem/progenitor cell from hippocampal dentate gyrus. doi:10.1016/j.neures.2010.07.1583
P3-d03 Modulation by Ifrd1 as a novel differentiation regulator in neural progenitors Takeshi Takarada , Shiho Konishi, Yukio Yoneda Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology We have previously identified interferon-related developmental regulator-1 (Ifrd1) as the gene responsible for the predominant suppression of neuronal differentiation in neural progenitors isolated from adult mouse hippocampus. In the mouse embryonic carcinoma P19 cells endowed to differentiate into neuronal and astroglial cells, marked but transient expression was at first seen in nestin mRNA within 4 days in culture under floating conditions, followed by MAP2 mRNA expression during culture under adherent conditions after dispersion and subsequent Ifrd1 mRNA expression in line with GFAP mRNA. In P19 cells with transient overexpression of Ifrd1, a significant decrease was found in mRNA expression of MAP2, but not of GFAP, within 72 h after transfection. Prior to the decrease in MAP mRNA expression, a significant decrease was seen in mRNA expression of the proneural gene NeuroD1 in P19 cells 48 h after the transfection of Ifrd1 expression vector. By contrast, no significant changes were found in mRNA expression of different proneural genes up to 72 h after transfection. These included Hes1, Hes5, Mash1, Math1, Math3, Neurogenin2 and Neurogenin3. In P19 cells transfected with a luciferase reporter plasmid linked to NeuroD1 promoter, a drastic decrease was seen in luciferase activity in cells with a NeuroD1 promoter, but not in those with an empty vector, within 4 days in culture with retinoic acid. On analysis using mutated deletion constructs, however, a significant decrease was still seen in luciferase activity in cells transfected with a reporter construct containing −112bp upstream of NeuroD1 promoter. These results suggest that Ifrd1 may suppress neuronal differentiation through a mechanism relevant to predominant inhibition of transactivation of NeuroD1 in undifferentiated neural progenitors. doi:10.1016/j.neures.2010.07.1584
P3-d04 Neurogliogenesis and angiogenesis in adult circumventricular organs Shoko Morita , Shinri Ukai, Seiji Miyata Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan
P3-d02 Neural stem/progenitor cells derived from adult hippocampal dentate gyrus can be abundantly prepared from mice treated with trimethyltin Masanori Yoneyama , Tatsuo Shiba, Kiyokazu Ogita Department of Pharmacology, Faculty of Pharmaceutical Sciences Various neurological injuries are widely recognized as promoting endogenous neurogenesis in hippocampal dentate gyrus of adulthood. Our previous studies demonstrated that the granule cells in the hippocampal dentate gyrus are injured and disappeared by treatment with trimethyltin chloride (TMT), with being regenerated in the dentate granule cell layer after neuronal loss. Using TMT-treated mouse as an in vivo model, we established the efficient culture system of adult mouse dentate gyrus-derived neural stem/progenitor cell after injury. Hippocampal dentate gyrus was isolated from 5-week-old Std-ddY male mice day 3 after injection of TMT, and then cell suspensions were maintained in DMEM/F-12 supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). The
Neurogenesis in adult mammalian nervous system is well known in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. The circumventricular organs (CVOs) are brain areas that lack the BBB or tight junctions between endothelial cells, and therefore are able to access to circulating substances in blood and cerebrospinal fluids. Recently, it is shown that adult CVOs cells are able to proliferate and undergo constitutive neurogenesis and gliogenesis. Nerve/glial antigen 2 (NG2) is highly expressed in immature and angiogenic microvascular pericytes at early developmental period of the central nervous system (CNS), but its expression becomes lower in microvascular pericytes of the adult CNS. In the present study, we found the prominent NG2 expression and determined the fate of bromodeoxyuridine positive cells in adult CVOs such as the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median eminence (ME), area postrema (AP), neurohypophysis (NH), and pineal gland (PG), using confocal microscopic immunohistochemistry. Furthermore, we analyzed the cell proliferation changes by salt-loading, known as the chronic stimulation to cause the structural remodeling. The present study thus demonstrates that NG2 is
P3-c28 Inhibition by bisphenol A of mesencephalic neural stem-cell migration in a neurosphere assay in vitro Junko Suzuki , Masami Ishido Natl Inst for Environmental Studies, Tsukuba, Japan Developmental neurotoxicity of environmental chemicals has been recognized worldwide as a serious threat to human health. The large numbers of environmental chemicals that now exist have made assessing their neurodevelopmental risks challenging. Therefore, in the previous study we have established an in vitro neurosphere assay system to evaluate for environmental chemical toxicity. In this study, we extended to quantify neurodevelopmental toxicity of endocrine disruptors, such as bisphenol A. Neural stem cells isolated from rat E16 mesencephalone were plated in serum-free DMEM/ F12 (1:1) medium in the presence of bFGF and EGF to form neurospheres. Cells emerged from the neurosphere in adhesive dishes and migrated along the radial axis. The migrating populations were positive for nestin, a marker of neural stem cells. Exposure to bisphenol A resulted in inhibition of cell migration and proliferation in a dose-dependent manner. Quantitative analysis revealed a linear function between the inhibition of migration and the logarithm of bisphnol A concentration (0–100 M); the percentage of inhibition by 1 M bisphnol A of migration was 35% (p < 0.05). Thus, we showed for the first time that bisphenol A inhibited migration as well as proliferation of neural stem cells in vitro and that a neurosphere assay system in vitro is very useful for rapid quantification of neurodevelopmental toxicity of environmental chemicals. doi:10.1016/j.neures.2010.07.1581
P3-d01 Adenosine facilitates the neuronal differentiation of neural stem cells derived from mouse hippocampus Aya Hariu , Takahiro Moriya, Yuko Suzuki, Takashi Katura, Norimichi Nakahata Department of Cell Signal, Grad. School Pharm. Sci., Tohoku University, Sendai Neural stem cells (NSCs) possess the ability of the proliferation and the neuronal and glial differentiation and play crucial roles in the neurogenesis not only in developing, but also in adult brain. Adenosine is known to have widespread physiological roles in the CNS, including the modulation of synaptic activity. We have previously shown that adenosine suppresses the cell growth of NSCs derived from embryonic mouse hippocampus not via adenosine receptors, but through equilibrative nucleoside transporter 1 (ENT1), known as a plasma membrane adenosine transporter. The effect of adenosine on neuronal differentiation of NSCs, however, remains to be clarified. In this study, we investigated effects of adenosine on the differentiation of NSCs derived from embryonic mouse hippocampus in vitro and examined ENT1 mRNA levels in the course of neural differentiation from NSCs. We found that adenosine increased the proportion of TuJ1-positive neurons even when treated with EGF (20 ng/ml). The concentration-dependency for adenosine-induced neuronal differentiation (30–300 M) was well similar to that for cell growth inhibition. Furthermore, mRNA levels of ENT1 were decreased when NSCs were differentiated by EGF withdrawal and 1% FCS. These results suggest that adenosine has the ability to facilitate the neuronal differentiation of hippocampal NSCs. doi:10.1016/j.neures.2010.07.1582
e357
obtained cells were fixed for immunostaining of nestin and GFAP. TMT led to a significant increase in the nestin-positive cells, without affecting that of GFAP-positive cells. Cells cultured floatingly in DMEM/F-12 medium containing EGF/bFGF resulted in the formation of round spheres immunoreactive to nestin in 27 days in vitro (DIV). The number of neurospheres was significantly increased compared with those derived from naive animals. Cells were seeded on dishes that had been previously coated with poly-l-ornithine and then cultured in DMEM/F12 medium containing bFGF/EGF. Under these conditions, cells grew at monolayer without forming neurospheres. Most cells were immunoreactive with both nestin and GFAP. ELISA for 5 -bromo-2 deoxyuridine revealed that a marked increase in the proliferative activity was seen by treatment with TMT at 28 DIV. Our data suggest that hippocampal neural stem/progenitor cells can be abundantly prepared from the dentate gyrus of mice treated with TMT. Thus, TMT-treated mice are an attractive model for achieving the neural stem/progenitor cell from hippocampal dentate gyrus. doi:10.1016/j.neures.2010.07.1583
P3-d03 Modulation by Ifrd1 as a novel differentiation regulator in neural progenitors Takeshi Takarada , Shiho Konishi, Yukio Yoneda Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology We have previously identified interferon-related developmental regulator-1 (Ifrd1) as the gene responsible for the predominant suppression of neuronal differentiation in neural progenitors isolated from adult mouse hippocampus. In the mouse embryonic carcinoma P19 cells endowed to differentiate into neuronal and astroglial cells, marked but transient expression was at first seen in nestin mRNA within 4 days in culture under floating conditions, followed by MAP2 mRNA expression during culture under adherent conditions after dispersion and subsequent Ifrd1 mRNA expression in line with GFAP mRNA. In P19 cells with transient overexpression of Ifrd1, a significant decrease was found in mRNA expression of MAP2, but not of GFAP, within 72 h after transfection. Prior to the decrease in MAP mRNA expression, a significant decrease was seen in mRNA expression of the proneural gene NeuroD1 in P19 cells 48 h after the transfection of Ifrd1 expression vector. By contrast, no significant changes were found in mRNA expression of different proneural genes up to 72 h after transfection. These included Hes1, Hes5, Mash1, Math1, Math3, Neurogenin2 and Neurogenin3. In P19 cells transfected with a luciferase reporter plasmid linked to NeuroD1 promoter, a drastic decrease was seen in luciferase activity in cells with a NeuroD1 promoter, but not in those with an empty vector, within 4 days in culture with retinoic acid. On analysis using mutated deletion constructs, however, a significant decrease was still seen in luciferase activity in cells transfected with a reporter construct containing −112bp upstream of NeuroD1 promoter. These results suggest that Ifrd1 may suppress neuronal differentiation through a mechanism relevant to predominant inhibition of transactivation of NeuroD1 in undifferentiated neural progenitors. doi:10.1016/j.neures.2010.07.1584
P3-d04 Neurogliogenesis and angiogenesis in adult circumventricular organs Shoko Morita , Shinri Ukai, Seiji Miyata Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan
P3-d02 Neural stem/progenitor cells derived from adult hippocampal dentate gyrus can be abundantly prepared from mice treated with trimethyltin Masanori Yoneyama , Tatsuo Shiba, Kiyokazu Ogita Department of Pharmacology, Faculty of Pharmaceutical Sciences Various neurological injuries are widely recognized as promoting endogenous neurogenesis in hippocampal dentate gyrus of adulthood. Our previous studies demonstrated that the granule cells in the hippocampal dentate gyrus are injured and disappeared by treatment with trimethyltin chloride (TMT), with being regenerated in the dentate granule cell layer after neuronal loss. Using TMT-treated mouse as an in vivo model, we established the efficient culture system of adult mouse dentate gyrus-derived neural stem/progenitor cell after injury. Hippocampal dentate gyrus was isolated from 5-week-old Std-ddY male mice day 3 after injection of TMT, and then cell suspensions were maintained in DMEM/F-12 supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). The
Neurogenesis in adult mammalian nervous system is well known in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. The circumventricular organs (CVOs) are brain areas that lack the BBB or tight junctions between endothelial cells, and therefore are able to access to circulating substances in blood and cerebrospinal fluids. Recently, it is shown that adult CVOs cells are able to proliferate and undergo constitutive neurogenesis and gliogenesis. Nerve/glial antigen 2 (NG2) is highly expressed in immature and angiogenic microvascular pericytes at early developmental period of the central nervous system (CNS), but its expression becomes lower in microvascular pericytes of the adult CNS. In the present study, we found the prominent NG2 expression and determined the fate of bromodeoxyuridine positive cells in adult CVOs such as the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median eminence (ME), area postrema (AP), neurohypophysis (NH), and pineal gland (PG), using confocal microscopic immunohistochemistry. Furthermore, we analyzed the cell proliferation changes by salt-loading, known as the chronic stimulation to cause the structural remodeling. The present study thus demonstrates that NG2 is