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Characterization of conditioned medium of cultured bone marrow stromal cells
Abstracts / Neuroscience Research 68S (2010) e109–e222
P1-f01 Characterization of conditioned medium of cultured bone marrow stromal cells Norihiko Nakano , Yoshiyasu Nakai, Tae-Boem Seo, Chizuka Ide Institute of Regeneration and Rehabilitation, Aino University, Ibaraki It has been recognized that bone marrow stromal cell (BMSC) transplantation has beneficial effects on spinal cord injury in animal models and therapeutic trials. It is hypothesized that BMSCs provide microenvironments suitable for axonal regeneration and secrete some trophic factors to rescue affected cells from degeneration. However, the molecular and cellular mechanisms of trophic factors involved remain still unclear. In the present study, we examined the effects of trophic factors secreted by rat BMSCs using bioassay by cultured hippocampal neurons. The conditioned medium (CM) as well as non-contact co-culture of BMSCs promoted neurite outgrowth, and suppressed TUNEL-positive cells compared to serum-free D-MEM. Protein analyses of the CM by antibody-based protein array analysis and ELISA revealed that the CM contained insulin-like growth factor (IGF)-1, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-1. DNA microarray analysis revealed that neurons highly expressed receptors of IGF-1 and TGF-1. However, their expression indices remained unchanged even after CM treatment. Individual trophic factors mentioned above or their combinations were less effective on neuronal survival and neurite outgrowth than the CM. The present study showed that BMSCs secreted into the culture medium various kinds of molecules including trophic factors to promote neuronal survival and neurite outgrowth. doi:10.1016/j.neures.2010.07.2206
P1-f02 Neuroprotective effect of pleiotrophin on dopaminergic neurons in vitro and in vivo Yoko Watanabe 1,2 , Sachiyo Misumi 1 , Tadashi Masuda 1 , Tamihide Matsunaga 2 , Tadashi Suzuki 2 , Hideki Hida 1 1
Dept Neurophysiol. & Brain Sci., Nagoya City Univ, Nagoya 2 Educ. Res. Cntr. of Clin. Phar., Nagoya City Univ, Nagoya
Pleiotrophin (PTN) is a secreted 18-kDa heparin binding protein which expression is enhanced in dopamine (DA) depleted striatum in Parkinson disease model rats. We have previously reported that PTN has effect on the survival and the differentiation of DAergic neurons. However, it is still unknown whether PTN has a protective effect on DAergic neurons. To investigate PTN effect on neuroprotection in vitro, tyrosine hydroxylase (TH)positive neurons from E15 rat mesencephalon were cultured in DMEM+10% FCS at a density of 1.0 × 105 cells/cm2 for 5 days. Dose-dependent specific toxicity of 6OHDA (0–40 M) was seen in TH-positive neurons. Pretreatment with PTN (100 ng/ml) prevented the decrease in TH-positive cell numbers by 20 M 6OHDA, although the neuroprotective effect was weaker than that of GDNF. To investigate the effect of PTN in vivo, PTN (2.5 l of 500 g/ml) was treated into the substantia nigra (SN) every 3-4 days in the rats that received 6-OHDA (20 g) in the striatum. Methamphetamine (Met)-induced rotation test was carried out at 14 days after 6-OHDA lesions, and the striatum and SN was investigated for TH immunostaining. Half of PTN-received rats showed the decrease of Met-induced rotation. Increases of remaining TH-positive neurons in SN and reduction of the damage by 6-OHDA in the striatum were observed in these rats, suggesting that PTN has a neuroprotective effect in vivo. To confirm PTN effect in vivo, data of continuous PTN treatment into the SN using osmotic mini-pump will be also presented Data suggest that PTN exhibits neuroprotective effects on DAergic neurons in vitro and in vivo. doi:10.1016/j.neures.2010.07.2207
P1-f03 Imaging analysis of the secretory vesicle-associated protein CAPS2 regulated BDNF secretion Yo Shinoda 1,2 , Tetsushi Sadakata 1,2 , Emi Kinameri 1 , Asako Katoh-Semba 1 , Kazuhito Nakao 3 , Hajime Furuya 1 , Ritsuko 3 1,2 Hirase , Teiichi Furuichi 1
Laboratory for Molecular Neurogenesis, Riken Bsi 2 CREST/JST 3 Hirase Unit, Riken Bsi Calcium-dependent protein for secretion 2 (CAPS2) is associated with densecore vesicles. Through the use of CAPS2 knockout (KO) mice, the present study analyzed the role of CAPS2 in BDNF secretion. CAPS2 KO mice had reduced hippocampal BDNF levels, and overexpression of exogenous CAPS2 significantly increased frequency, amplitude, and kinetics of depolarization-
e143
induced BDNF vesicle exocytosis in CAPS2 KO hippocampal neurons. The CAPS2 KO hippocampus displayed impaired GABAergic interneuron systems, including decreased GABAergic neuronal numbers in the juvenile stage, decreased number of synaptic vesicles in inhibitory synapses, and reduced frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Moreover, the CAPS2 KO mice exhibited reduced late-phase long-term potentiation (L-LTP) in CA3-CA1 synapses, decreased hippocampal theta oscillation frequencies, and increased anxiety-like behavior. These results suggest that CAPS2 promotes activity-dependent BDNF secretion, which is critical for the formation of a hippocampal GABAergic interneuronal network. doi:10.1016/j.neures.2010.07.2208
P1-f04 Expression of Neuronal leucine-rich repeat 4 (NLRR4) in various regions of the adult and developing brain Takayoshi Bando 1 , Yoshihiro Morikawa 1 , Atsushi MIyajima 2 , Emiko Senba 1 1
Dept Anato and Neurobiol, Wakayama Medical Univ, Wakayama Univ of Tokyo, Tokyo
2
IMCB,
NLRR4 is a type I transmembrane protein with leucine-rich repeat in its extracellular region. Previously, we have reported that NLRR4 is expressed in the hippocampus and functions in the hippocampus-dependent memory (Bando et al., Mol. Cell. Biol. 25: 4166-4175, 2005). However, the detailed expression pattern of NLRR4 has not been examined. In this study, we investigated the expression of NLRR4 in various regions of the brain using NLRR4 heterozygoous (NLRR-4+/-) mice in which the exon of NLRR-4 gene was replaced by the -galactosidase gene. Thus, the expression of NLRR4 can be monitored by -galactosidase staining in NLRR-4+/- mice. Using -galactosidase staining, the expression of NLRR4 was strongly detected in the piriform cortex, hippocampus, and cerebellum, and moderately in the neocortex, striatum, hypothalamus, thalamus, midbrain, and pons of the adult brain. In contrast, the expression of NLRR4 was observed in restricted brain regions at postnatal day (P) 0. The expression of NLRR4 was detected predominantly in the olfactory bulb, and piriform cortex, and at low levels in the hippocampus and neocortex at P0. Between P0 and P14, the expression of NLRR4 was gradually induced in most of regions of the brain as the development proceeds. These stages are considered as a major phase of synaptic maturation. In the developing olfactory bulb, NLRR4 expression was observed mainly between embryonic day (E) 15.5 and P7 peaking at P0, and down-regulated after P7. As the olfactory sensory neurons give the well-ordered axonal projection into the precise glomerulus of the olfctory bulb in this period, NLRR4 might be involved in this projection. In conclusion, the spatiotemporal expression pattern of NLRR4 suggests important roles of NLRR4 in the later developmental processes, in which axonal projection and synapse formation. doi:10.1016/j.neures.2010.07.2209
P1-f05 Analysis of mouse Cadherin-6 gene regulation for the mouse neocortical auditory area Saki Egusa 1,2 , Yukiko Inoue 2 , Junko Asami 2 , Mikio Hoshino 2 , Takayuki Sota 1 , Takayoshi Inoue 2 1
Faculty of Sci and Eng, Waseda Univ, Tokyo, Japan 2 National Institute of Neuroscience, NCNP, Kodaira, Japan The cerebral cortex (neocortex) controls higher brain function and only mammals elaborate it in vertebrate evolutionary history. It has six-layered structure with area specific variance and this area specificity well corresponds to brain functional mode, constituting basis of the cerebral cortex. Curiously, it is known that some case of disease like epilepsy and polymicrogyria show hypoplastic phenotypes with cortical area or layer specificity. Yet developmental process of each neocortical area remains to be elucidated. Here we focused on Cadherin-6, a mouse type II classic cadherin, expression of which demarcates the auditory area and somatosensory area in the postnatal mouse cerebral cortex, to reveal the mechanism of neocortical arealization. By using efficient transgenesis of modified bacterial artificial chromosomes, we narrowed down the range of putative transcriptional regulatory segment of the gene into 10–25 kb upstream of the transcription start site. Our result would exploit ways to clarify the parcellating machinery of the cerebral cortex. doi:10.1016/j.neures.2010.07.2210
P1-f01 Characterization of conditioned medium of cultured bone marrow stromal cells Norihiko Nakano , Yoshiyasu Nakai, Tae-Boem Seo, Chizuka Ide Institute of Regeneration and Rehabilitation, Aino University, Ibaraki It has been recognized that bone marrow stromal cell (BMSC) transplantation has beneficial effects on spinal cord injury in animal models and therapeutic trials. It is hypothesized that BMSCs provide microenvironments suitable for axonal regeneration and secrete some trophic factors to rescue affected cells from degeneration. However, the molecular and cellular mechanisms of trophic factors involved remain still unclear. In the present study, we examined the effects of trophic factors secreted by rat BMSCs using bioassay by cultured hippocampal neurons. The conditioned medium (CM) as well as non-contact co-culture of BMSCs promoted neurite outgrowth, and suppressed TUNEL-positive cells compared to serum-free D-MEM. Protein analyses of the CM by antibody-based protein array analysis and ELISA revealed that the CM contained insulin-like growth factor (IGF)-1, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-1. DNA microarray analysis revealed that neurons highly expressed receptors of IGF-1 and TGF-1. However, their expression indices remained unchanged even after CM treatment. Individual trophic factors mentioned above or their combinations were less effective on neuronal survival and neurite outgrowth than the CM. The present study showed that BMSCs secreted into the culture medium various kinds of molecules including trophic factors to promote neuronal survival and neurite outgrowth. doi:10.1016/j.neures.2010.07.2206
P1-f02 Neuroprotective effect of pleiotrophin on dopaminergic neurons in vitro and in vivo Yoko Watanabe 1,2 , Sachiyo Misumi 1 , Tadashi Masuda 1 , Tamihide Matsunaga 2 , Tadashi Suzuki 2 , Hideki Hida 1 1
Dept Neurophysiol. & Brain Sci., Nagoya City Univ, Nagoya 2 Educ. Res. Cntr. of Clin. Phar., Nagoya City Univ, Nagoya
Pleiotrophin (PTN) is a secreted 18-kDa heparin binding protein which expression is enhanced in dopamine (DA) depleted striatum in Parkinson disease model rats. We have previously reported that PTN has effect on the survival and the differentiation of DAergic neurons. However, it is still unknown whether PTN has a protective effect on DAergic neurons. To investigate PTN effect on neuroprotection in vitro, tyrosine hydroxylase (TH)positive neurons from E15 rat mesencephalon were cultured in DMEM+10% FCS at a density of 1.0 × 105 cells/cm2 for 5 days. Dose-dependent specific toxicity of 6OHDA (0–40 M) was seen in TH-positive neurons. Pretreatment with PTN (100 ng/ml) prevented the decrease in TH-positive cell numbers by 20 M 6OHDA, although the neuroprotective effect was weaker than that of GDNF. To investigate the effect of PTN in vivo, PTN (2.5 l of 500 g/ml) was treated into the substantia nigra (SN) every 3-4 days in the rats that received 6-OHDA (20 g) in the striatum. Methamphetamine (Met)-induced rotation test was carried out at 14 days after 6-OHDA lesions, and the striatum and SN was investigated for TH immunostaining. Half of PTN-received rats showed the decrease of Met-induced rotation. Increases of remaining TH-positive neurons in SN and reduction of the damage by 6-OHDA in the striatum were observed in these rats, suggesting that PTN has a neuroprotective effect in vivo. To confirm PTN effect in vivo, data of continuous PTN treatment into the SN using osmotic mini-pump will be also presented Data suggest that PTN exhibits neuroprotective effects on DAergic neurons in vitro and in vivo. doi:10.1016/j.neures.2010.07.2207
P1-f03 Imaging analysis of the secretory vesicle-associated protein CAPS2 regulated BDNF secretion Yo Shinoda 1,2 , Tetsushi Sadakata 1,2 , Emi Kinameri 1 , Asako Katoh-Semba 1 , Kazuhito Nakao 3 , Hajime Furuya 1 , Ritsuko 3 1,2 Hirase , Teiichi Furuichi 1
Laboratory for Molecular Neurogenesis, Riken Bsi 2 CREST/JST 3 Hirase Unit, Riken Bsi Calcium-dependent protein for secretion 2 (CAPS2) is associated with densecore vesicles. Through the use of CAPS2 knockout (KO) mice, the present study analyzed the role of CAPS2 in BDNF secretion. CAPS2 KO mice had reduced hippocampal BDNF levels, and overexpression of exogenous CAPS2 significantly increased frequency, amplitude, and kinetics of depolarization-
e143
induced BDNF vesicle exocytosis in CAPS2 KO hippocampal neurons. The CAPS2 KO hippocampus displayed impaired GABAergic interneuron systems, including decreased GABAergic neuronal numbers in the juvenile stage, decreased number of synaptic vesicles in inhibitory synapses, and reduced frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Moreover, the CAPS2 KO mice exhibited reduced late-phase long-term potentiation (L-LTP) in CA3-CA1 synapses, decreased hippocampal theta oscillation frequencies, and increased anxiety-like behavior. These results suggest that CAPS2 promotes activity-dependent BDNF secretion, which is critical for the formation of a hippocampal GABAergic interneuronal network. doi:10.1016/j.neures.2010.07.2208
P1-f04 Expression of Neuronal leucine-rich repeat 4 (NLRR4) in various regions of the adult and developing brain Takayoshi Bando 1 , Yoshihiro Morikawa 1 , Atsushi MIyajima 2 , Emiko Senba 1 1
Dept Anato and Neurobiol, Wakayama Medical Univ, Wakayama Univ of Tokyo, Tokyo
2
IMCB,
NLRR4 is a type I transmembrane protein with leucine-rich repeat in its extracellular region. Previously, we have reported that NLRR4 is expressed in the hippocampus and functions in the hippocampus-dependent memory (Bando et al., Mol. Cell. Biol. 25: 4166-4175, 2005). However, the detailed expression pattern of NLRR4 has not been examined. In this study, we investigated the expression of NLRR4 in various regions of the brain using NLRR4 heterozygoous (NLRR-4+/-) mice in which the exon of NLRR-4 gene was replaced by the -galactosidase gene. Thus, the expression of NLRR4 can be monitored by -galactosidase staining in NLRR-4+/- mice. Using -galactosidase staining, the expression of NLRR4 was strongly detected in the piriform cortex, hippocampus, and cerebellum, and moderately in the neocortex, striatum, hypothalamus, thalamus, midbrain, and pons of the adult brain. In contrast, the expression of NLRR4 was observed in restricted brain regions at postnatal day (P) 0. The expression of NLRR4 was detected predominantly in the olfactory bulb, and piriform cortex, and at low levels in the hippocampus and neocortex at P0. Between P0 and P14, the expression of NLRR4 was gradually induced in most of regions of the brain as the development proceeds. These stages are considered as a major phase of synaptic maturation. In the developing olfactory bulb, NLRR4 expression was observed mainly between embryonic day (E) 15.5 and P7 peaking at P0, and down-regulated after P7. As the olfactory sensory neurons give the well-ordered axonal projection into the precise glomerulus of the olfctory bulb in this period, NLRR4 might be involved in this projection. In conclusion, the spatiotemporal expression pattern of NLRR4 suggests important roles of NLRR4 in the later developmental processes, in which axonal projection and synapse formation. doi:10.1016/j.neures.2010.07.2209
P1-f05 Analysis of mouse Cadherin-6 gene regulation for the mouse neocortical auditory area Saki Egusa 1,2 , Yukiko Inoue 2 , Junko Asami 2 , Mikio Hoshino 2 , Takayuki Sota 1 , Takayoshi Inoue 2 1
Faculty of Sci and Eng, Waseda Univ, Tokyo, Japan 2 National Institute of Neuroscience, NCNP, Kodaira, Japan The cerebral cortex (neocortex) controls higher brain function and only mammals elaborate it in vertebrate evolutionary history. It has six-layered structure with area specific variance and this area specificity well corresponds to brain functional mode, constituting basis of the cerebral cortex. Curiously, it is known that some case of disease like epilepsy and polymicrogyria show hypoplastic phenotypes with cortical area or layer specificity. Yet developmental process of each neocortical area remains to be elucidated. Here we focused on Cadherin-6, a mouse type II classic cadherin, expression of which demarcates the auditory area and somatosensory area in the postnatal mouse cerebral cortex, to reveal the mechanism of neocortical arealization. By using efficient transgenesis of modified bacterial artificial chromosomes, we narrowed down the range of putative transcriptional regulatory segment of the gene into 10–25 kb upstream of the transcription start site. Our result would exploit ways to clarify the parcellating machinery of the cerebral cortex. doi:10.1016/j.neures.2010.07.2210