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Protection against cell death by docosahexaenoic acid in human neuroblastoma SH-SY5Y cells
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Abstracts / Neuroscience Research 68S (2010) e223–e334
(rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether) on the cell death were further examined by the same procedure. Neuroprotective effects were found in hirsutine, hirsuteine, and geissoschizine methyl ether, which also ameliorated the glutamate-induced decrease in GSH levels. These results suggest that yokukansan protects against PC12 cell death induced by glutamatemediated oxidative stress via the cystine/glutamate antiporter system of glutamate, which may be mainly attributed to a synergistic effect of the hirsutine, hirsuteine, and geissoschizine methyl ether in Uncaria thorn. doi:10.1016/j.neures.2010.07.1133
P2-e22 Protection against cell death by docosahexaenoic acid in human neuroblastoma SH-SY5Y cells Kotaro Inoue , Hiroko Inoue Graduate School of Advanced Science and Engineering, Waseda Univ, Tokyo, Japan Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phospholipids and can exhibit increases in cellular membrane fluidity, promotion of neurite extension. Furthermore, antiapoptotic effect of DHA has been indicated in neuronal cells. DHA is broadly neuroprotective via multiple mechanisms that include neuroprotective DHA metabolites, reduced arachidonic acid metabolites, and increased trophic factors or downstream trophic signal transduction. In mouse neuroblastoma cells, DHA has been shown to protect cells from apoptotic death induced by staurosporine (STS) and serum deprivation-induced apoptosis. The protective effect of DHA was suggested to be mediated in part through phosphatidylserine (PS) accumulation in neuronal membranes. In this study, we examined effects of DHA on STS-induced apoptosis in human neuroblastoma cell line SH SY5Y. The cell viability was significantly decreased by exposure to 200 nM STS for 5 h, and the loss of viability was suppressed by pretreatment with 25 M DHA for 48 h. DHA reduced the percentage of the cells that induced chromatin condensation, but it did not suppress an increase in cleaved caspase-3 level induced by STS. We also investigated effects of DHA on serum deprivation in SH SY5Y cells. The cell viability was decreased by incubation with serum free medium for 48 h, and the loss of viability was reduced by pretreatment with 25 M DHA for 48 h. DHA reduced the percentage of the cells that induced chromatin condensation, but it did not suppress an increase in cleaved caspase-3 level induced serum deprivation. doi:10.1016/j.neures.2010.07.1134
P2-e23 Characterization of the signaling pathway of apoptosis induced by -amyloid Aiko Tanaka , Hiroko Inoue
Graduate School of Advanced Science and Engineering, Waseda Univ, Tokyo, Japan Alzheimer’s disease (AD) is one of the most common forms of dementia, and one of the neuropathological hallmarks of AD is the neuronal degeneration associated to senile plaques. Such plaques are composed of compacted amyloid -peptide (A), which is a 40–43 amino-acid peptide. A is originated by the proteolytic processing of a transmembrane glycoprotein called -amyloid precursor protein (-APP), which can be cleaved by the action of - and ␥-secretases. The deposition of soluble A produces the aggregation of the peptide-forming amyloid fibrils, which have been shown to generate intracellular hydrogen peroxide and other reactive oxygen species and increase intracellular Ca2+ concentration. Previously, we found that incubation of human neuroblastoma SH-SY5Y cells with 10–20 M A(1–42) for 48 h induced chromatin condensation, although cleaved caspase-3 level was not increased. To investigate whether the signaling pathway activated by A(25–35) is different from that by A(1–42), we examined effects of A(25–35) on SH SY5Y cells in this study. A(25–35) is a shorter hydrophobic fragment of A(1–42), though not present in biological systems, is widely used together with, or instead of, the endogenous fragment A, and is found to be at least as toxic as the full-length fragment. The percentage of the cells that induced chromatin condensation was increased by incubation of cells with 20 M A(25–35) for 72 h, and was suppressed by treatment with a caspase-3 inhibitor, z-VAD-fmk. However, cleaved caspase-3 level was not elevated significantly. We also examined effects of A on intracellular Ca2+ concentration using fura-2, and found that it elevated Ca2+ concentration. doi:10.1016/j.neures.2010.07.1135
P2-e24 Effects of hydroxylated fullerene on light-induced cell death in human retinal pigment epithelial cells Iwai Ryo , Hiroko Inoue Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Component of the visible light spectrum can be absorbed by biologic chromophores in the retinal pigment epithelial (RPE) cells, causing cellular dysfunction and even death of cells. Cell culture studies revealed that blue light directly induces the production of reactive oxygen spices (ROS) in RPE mitochondria and leads to apoptosis, potentially triggered by ROS damage to mitochondrial DNA. This can lead to permanent or transient loss of vision. Fullerene (C60 ), the large carbon cage molecule has the ability to quench various free radicals, behaving as a “free radical sponge”. Water-soluble fullerene, hydroxylated fullerene (fullerol) has several clinical applications including use as effective scavengers for ROS. Conversely, photosensitization of fullerene has been shown to yield highly reactive singlet oxygen or superoxide anion. Fullerol absorbs light in both the UVA and visible light regions and therefore have the potential to be phototoxic to the skin and the eye. In this study we examined whether fullerol had radical scavenging effects in the human RPE cells (ARPE-19), which was exposed to light. Analysis of cell viability showed that approximately 50% of cells were died by 6 h exposure to blue light, and fullerol, C60 (OH)12 had no effect on cell viability in the dark at concentrations of up to 25 M. When cells were exposed to blue light with 25 M fullerol for 6 h, the cell viability was about 10% of the control value (25 M fullerol in the dark). We examined involvement of ROS in cell death increased by light in the presence of fullerol. Cell exposed to light with fullerol showed a significant increase ROS relative to the level seen in cells with fullerol in the dark. Furthermore, we found that the antioxidant N-acetyl cysteine did not reduce the ROS levels generated by light and fullerol. doi:10.1016/j.neures.2010.07.1136
P2-e25 The development of new glaucoma mice model with the obstruction of axonal flow Toru Nakazawa , Morin Ryu, Shanab Ahmed Department of Ophthalmology, Tohoku University Graduate School of Medicine Purpose: Retinal ganglion cell (RGC) death is involved in the various retinal disorders, including glaucoma, diabetic retinopathy and vessel occlusion. Establishment of the neuroprotective treatment is an important strategy for the prevention of blindness. Evidences suggest that axonal damage is involved in the pathogenesis of glaucoma due to the deformity of lamina cribrosa and the ischemic insult in the optic nerve head. The purpose of this study is to develop the mice model of glaucoma with the disturbed axonal flow without the elevation of intraocular pressure (IOP). Methods: Adult male C57/BL6 mice were used in this study. To investigate the role of axonal damage in glaucoma-induced RGC death, we performed the optic nerve crush (NC) with forceps for 10 s or leaving the Spongel around the optic nerve soaked with vinblastine (0.1 to 10 mM), a microtubule disassembly drug. Obstruction of axonal flow was demonstrated by sequential retrograde labeling with fluorogold (FG) and DiI. To investigate the causative roles of the axonal flow obstruction and RGC death, RGC was labeled by FG and counted the density of survival RGCs 3, 5, 7, 10, and 14 days after injury. Results: Both of NC and vinblastine induced axonal damage demonstrated by the discontinuous immunoreactivity of neurofilament on the longitudinal section of optic nerve and the suppression of retrograde transport of FG. The density of surviving RGCs was significantly decreased more than 1 mM of vinblastine 7 days after injury in a dose-dependent manner. By day 3 after injury, the density of surviving RGCs began to decrease significantly and, by day 7 and 28, almost 80% of RGCs were lost. BDNF and Tat-BH4, an inhibitor of mitochondrial activation, partially prevented the NC-induced RGC death. Conclusions: The obstruction of axonal flow in optic nerve had a causative role on the RGC death. This mice model is valuable for the research on the glaucoma especially normal tension glaucoma. doi:10.1016/j.neures.2010.07.1137
P2-e26 Ischemic preconditioned neurons protect astrocytes against oxidative stress during ischemia reperfusion Xiaomei Wu , Fang Du, Chun Hu, Ya Ke School of Biomedical Science, The Chinese University of Hong Kong Ischemia-preconditioning refers to the phenomenon that a sub-lethal ischemia enables the brain to tolerate subsequent lethal ischemia and astro-
Abstracts / Neuroscience Research 68S (2010) e223–e334
(rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsutine, hirsuteine, and geissoschizine methyl ether) on the cell death were further examined by the same procedure. Neuroprotective effects were found in hirsutine, hirsuteine, and geissoschizine methyl ether, which also ameliorated the glutamate-induced decrease in GSH levels. These results suggest that yokukansan protects against PC12 cell death induced by glutamatemediated oxidative stress via the cystine/glutamate antiporter system of glutamate, which may be mainly attributed to a synergistic effect of the hirsutine, hirsuteine, and geissoschizine methyl ether in Uncaria thorn. doi:10.1016/j.neures.2010.07.1133
P2-e22 Protection against cell death by docosahexaenoic acid in human neuroblastoma SH-SY5Y cells Kotaro Inoue , Hiroko Inoue Graduate School of Advanced Science and Engineering, Waseda Univ, Tokyo, Japan Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phospholipids and can exhibit increases in cellular membrane fluidity, promotion of neurite extension. Furthermore, antiapoptotic effect of DHA has been indicated in neuronal cells. DHA is broadly neuroprotective via multiple mechanisms that include neuroprotective DHA metabolites, reduced arachidonic acid metabolites, and increased trophic factors or downstream trophic signal transduction. In mouse neuroblastoma cells, DHA has been shown to protect cells from apoptotic death induced by staurosporine (STS) and serum deprivation-induced apoptosis. The protective effect of DHA was suggested to be mediated in part through phosphatidylserine (PS) accumulation in neuronal membranes. In this study, we examined effects of DHA on STS-induced apoptosis in human neuroblastoma cell line SH SY5Y. The cell viability was significantly decreased by exposure to 200 nM STS for 5 h, and the loss of viability was suppressed by pretreatment with 25 M DHA for 48 h. DHA reduced the percentage of the cells that induced chromatin condensation, but it did not suppress an increase in cleaved caspase-3 level induced by STS. We also investigated effects of DHA on serum deprivation in SH SY5Y cells. The cell viability was decreased by incubation with serum free medium for 48 h, and the loss of viability was reduced by pretreatment with 25 M DHA for 48 h. DHA reduced the percentage of the cells that induced chromatin condensation, but it did not suppress an increase in cleaved caspase-3 level induced serum deprivation. doi:10.1016/j.neures.2010.07.1134
P2-e23 Characterization of the signaling pathway of apoptosis induced by -amyloid Aiko Tanaka , Hiroko Inoue
Graduate School of Advanced Science and Engineering, Waseda Univ, Tokyo, Japan Alzheimer’s disease (AD) is one of the most common forms of dementia, and one of the neuropathological hallmarks of AD is the neuronal degeneration associated to senile plaques. Such plaques are composed of compacted amyloid -peptide (A), which is a 40–43 amino-acid peptide. A is originated by the proteolytic processing of a transmembrane glycoprotein called -amyloid precursor protein (-APP), which can be cleaved by the action of - and ␥-secretases. The deposition of soluble A produces the aggregation of the peptide-forming amyloid fibrils, which have been shown to generate intracellular hydrogen peroxide and other reactive oxygen species and increase intracellular Ca2+ concentration. Previously, we found that incubation of human neuroblastoma SH-SY5Y cells with 10–20 M A(1–42) for 48 h induced chromatin condensation, although cleaved caspase-3 level was not increased. To investigate whether the signaling pathway activated by A(25–35) is different from that by A(1–42), we examined effects of A(25–35) on SH SY5Y cells in this study. A(25–35) is a shorter hydrophobic fragment of A(1–42), though not present in biological systems, is widely used together with, or instead of, the endogenous fragment A, and is found to be at least as toxic as the full-length fragment. The percentage of the cells that induced chromatin condensation was increased by incubation of cells with 20 M A(25–35) for 72 h, and was suppressed by treatment with a caspase-3 inhibitor, z-VAD-fmk. However, cleaved caspase-3 level was not elevated significantly. We also examined effects of A on intracellular Ca2+ concentration using fura-2, and found that it elevated Ca2+ concentration. doi:10.1016/j.neures.2010.07.1135
P2-e24 Effects of hydroxylated fullerene on light-induced cell death in human retinal pigment epithelial cells Iwai Ryo , Hiroko Inoue Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan Component of the visible light spectrum can be absorbed by biologic chromophores in the retinal pigment epithelial (RPE) cells, causing cellular dysfunction and even death of cells. Cell culture studies revealed that blue light directly induces the production of reactive oxygen spices (ROS) in RPE mitochondria and leads to apoptosis, potentially triggered by ROS damage to mitochondrial DNA. This can lead to permanent or transient loss of vision. Fullerene (C60 ), the large carbon cage molecule has the ability to quench various free radicals, behaving as a “free radical sponge”. Water-soluble fullerene, hydroxylated fullerene (fullerol) has several clinical applications including use as effective scavengers for ROS. Conversely, photosensitization of fullerene has been shown to yield highly reactive singlet oxygen or superoxide anion. Fullerol absorbs light in both the UVA and visible light regions and therefore have the potential to be phototoxic to the skin and the eye. In this study we examined whether fullerol had radical scavenging effects in the human RPE cells (ARPE-19), which was exposed to light. Analysis of cell viability showed that approximately 50% of cells were died by 6 h exposure to blue light, and fullerol, C60 (OH)12 had no effect on cell viability in the dark at concentrations of up to 25 M. When cells were exposed to blue light with 25 M fullerol for 6 h, the cell viability was about 10% of the control value (25 M fullerol in the dark). We examined involvement of ROS in cell death increased by light in the presence of fullerol. Cell exposed to light with fullerol showed a significant increase ROS relative to the level seen in cells with fullerol in the dark. Furthermore, we found that the antioxidant N-acetyl cysteine did not reduce the ROS levels generated by light and fullerol. doi:10.1016/j.neures.2010.07.1136
P2-e25 The development of new glaucoma mice model with the obstruction of axonal flow Toru Nakazawa , Morin Ryu, Shanab Ahmed Department of Ophthalmology, Tohoku University Graduate School of Medicine Purpose: Retinal ganglion cell (RGC) death is involved in the various retinal disorders, including glaucoma, diabetic retinopathy and vessel occlusion. Establishment of the neuroprotective treatment is an important strategy for the prevention of blindness. Evidences suggest that axonal damage is involved in the pathogenesis of glaucoma due to the deformity of lamina cribrosa and the ischemic insult in the optic nerve head. The purpose of this study is to develop the mice model of glaucoma with the disturbed axonal flow without the elevation of intraocular pressure (IOP). Methods: Adult male C57/BL6 mice were used in this study. To investigate the role of axonal damage in glaucoma-induced RGC death, we performed the optic nerve crush (NC) with forceps for 10 s or leaving the Spongel around the optic nerve soaked with vinblastine (0.1 to 10 mM), a microtubule disassembly drug. Obstruction of axonal flow was demonstrated by sequential retrograde labeling with fluorogold (FG) and DiI. To investigate the causative roles of the axonal flow obstruction and RGC death, RGC was labeled by FG and counted the density of survival RGCs 3, 5, 7, 10, and 14 days after injury. Results: Both of NC and vinblastine induced axonal damage demonstrated by the discontinuous immunoreactivity of neurofilament on the longitudinal section of optic nerve and the suppression of retrograde transport of FG. The density of surviving RGCs was significantly decreased more than 1 mM of vinblastine 7 days after injury in a dose-dependent manner. By day 3 after injury, the density of surviving RGCs began to decrease significantly and, by day 7 and 28, almost 80% of RGCs were lost. BDNF and Tat-BH4, an inhibitor of mitochondrial activation, partially prevented the NC-induced RGC death. Conclusions: The obstruction of axonal flow in optic nerve had a causative role on the RGC death. This mice model is valuable for the research on the glaucoma especially normal tension glaucoma. doi:10.1016/j.neures.2010.07.1137
P2-e26 Ischemic preconditioned neurons protect astrocytes against oxidative stress during ischemia reperfusion Xiaomei Wu , Fang Du, Chun Hu, Ya Ke School of Biomedical Science, The Chinese University of Hong Kong Ischemia-preconditioning refers to the phenomenon that a sub-lethal ischemia enables the brain to tolerate subsequent lethal ischemia and astro-