Takumi Satoh

Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells.

We examined the effects of various types of oxidative stress on cell survival and on mitochondrial membrane potential (ΔΨm) in PC12 cells transfected with BCL‐2. Several types of oxidative stress such as exposure to hydrogen peroxide, 13‐L‐hydroperoxylinoleic acid, and xanthine + xanthine oxidase triggered apoptotic nuclear condensation and DNA fragmentation in normal PC12 cells. These types of oxidative stress induced significant increases in level of reactive oxygen species (ROS) before cell death. By contrast, BCL‐2 prevented the apoptosis induced by these oxidative stresses. However, BCL‐2 did not reduce ROS levels, indicating that it functions downstream of ROS generation. We measured ΔΨm as a potential target of ROS during oxidative stress–induced cell death. Hydrogen peroxide, 13‐L‐hydroperoxylinoleic acid, and xanthine + xanthine oxidase induced a significant loss of ΔΨm simultaneously with cell death. BCL‐2 prevented the decrease in ΔΨm as well as apoptosis induced by oxidative stress. These observations suggest that the oxidative stress triggers apoptosis associated with both increased generation of ROS and decreases in level of ΔΨm and that BCL‐2 prevents cell death as well as Δ Ψm but not ROS production. J. Neurosci. Res. 50:413–420, 1997.

Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells.

To investigate the involvement of reactive oxygen species (ROS) in neuronal apoptosis, we performed confocal and flow cytometric analysis with a ROS-specific fluorogen, 6-carboxy-2, 7-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Serum deprivation significantly increased the level of ROS in PC12 cells and rat cortical neurons. N,N-diphenyl-p-phenylenediamine (DPPD), an antioxidant, reduced ROS production induced by serum deprivation and recovered cell survival. However, some survival factors such as nerve growth factor and Bcl-2, which prevented the apoptosis of PC12 cells, did not affect the up-regulation of ROS induced by serum deprivation. Epidermal growth factor which prevented the apoptosis of cortical neurons, did not affect the increase of ROS. These data suggest that survival factors rescue the serum deprivation induced apoptosis independently of ROS production.

Generation of free radicals during lipid hydroperoxide-triggered apoptosis in PC12h cells.

Abstract The compound 13- l -hydroperoxylinoleic acid (LOOH) triggered the death of clonal rat pheochromocytoma PC12h cells (LD 50 =about 8 μ M). LOOH induced nuclear condensation and DNA fragmentation, which was prevented by cycloheximide (a protein synthesis inhibitor) and NGF, indicating that LOOH triggered apoptosis in PC12h cells. LOOH produced reactive oxygen species (ROS) in PC12h cells in a time- and dose-dependent manner, as measured by flow cytometry using the ROS-specific fluorescent indicator, 6-carboxy-2,7-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Antioxidants such as N,N′ -diphenyl- p -phenylenediamine (DPPD), vitamin E and N -acetylcysteine, and a ferric iron chelator, deferoxamine, inhibited the LOOH-triggered apoptosis and simultaneously decreased the generation of ROS, whereas an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO), enhanced the apoptosis and increased the generation of ROS. These results indicate that LOOH triggers the apoptosis of PC12h cells by increasing the production of ROS. A confocal analysis with the Ca 2+ -specific fluorescent indicator, fluo-3, demonstrated that LOOH at concentrations up to 200 μ M, did not increase the intracellular Ca 2+ concentration. These data indicate that LOOH induces apoptosis of PC12h cells through the enhanced production of ROS, not through increasing the permeability of Ca 2+ .

Generation of reactive oxygen species, release of L-glutamate and activation of caspases are required for oxygen-induced apoptosis of embryonic hippocampal neurons in culture.

Oxygen-induced cell death in embryonic neurons is a useful in vitro model of neuronal apoptosis to study the molecular mechanisms underlying the cell death induced by oxidative stress. In the present study, we examined the involvement of reactive oxygen species and glutamate in the high (50%) oxygen-induced death of cultured hippocampal neurons. During the course of cell death, increases in O2- and hydrogen peroxide (H2O2) levels were observed. On the other hand, superoxide dismutase (SOD), catalase and deferoxamine (DFX), which have inhibitory effects on the generation of O2-, H2O2 and hydroxyl radicals, respectively, protected the neurons. These results suggested that both O2- and H2O2 play important roles in this apoptosis. Antagonists of NMDA and AMPA/kinate (AMPA/KA) receptors and an inhibitor of glutamate release partially prevented the apoptosis, suggesting that exposure to high oxygen enhances glutamate release, which results in activation of NMDA receptor and AMPA/KA receptor. In addition, specific nitric oxide (NO) scavenger and NO synthetase inhibitors blocked the apoptosis, indicating that NO and/or peroxynitrite are involved in this mechanism of cell death. Caspase inhibitors also blocked the neuronal apoptosis. These results suggested that multiple effectors including generation of reactive oxygen species, release of L-glutamate and activation of caspases are activated during the death induced by high oxygen.

Production of reactive oxygen species and release of L-glutamate during superoxide anion-induced cell death of cerebellar granule neurons

Abstract: Enhanced production of superoxide anion (O2−) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O2− generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C‐DCDHF‐DA) for O2− and other ROS and hydroethidine (HEt) specifically for O2− by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO‐induced cell death. Catalase effectively reduced C‐DCDHF‐DA but not HEt fluorescence, whereas SOD reduced HEt but not C‐DCDHF‐DA fluorescence, indicating that HEt and C‐DCDHF‐DA fluorescence correlated with O2− and hydrogen peroxide, respectively. The NMDA antagonist MK‐801 prevented the death. XA + XO induced an increase in l‐glutamate release from cerebellar granule neurons. These results indicate that elevation of O2− induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l‐glutamate.

Oxygen Toxicity Induces Apoptosis in Neuronal Cells

Abstract1. A high oxygen atmosphere induced apoptosis in cultured neuronal cells including PC12 cells and rat embryonic cortical, hippocampal, and basal forebrain neurons associated with DNA fragmentation and nuclear condensation.2. The sensitivity of CNS neurons to a high-oxygen atmosphere was the following order; cortex > basal forebrain > hippocampus.3. Cycloheximide and actinomycin-D inhibited the apoptosis, indicating that it depends on new macromolecular synthesis. In contrast, cultured postnatal CNS neurons were resistant to oxidative stress.4. Neurotrophic factors such as nerve growth factor (NGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) blocked the apoptosis induced by a high-oxygen atmosphere.

Brain-derived neurotrophic factor prevents superoxide anion-induced death of PC12h cells stably expressing TrkB receptor via modulation of reactive oxygen species

Abstract In our previous report (Satoh et al., 1999. Regulation of reactive oxygen species by nerve growth factor but not by Bcl-2 as a novel mecahnism of protection of PC12 cells from superoxide anion-induced death. J. Biochem. 125, 952–959), we reported that nerve growth factor (NGF) protected PC12 cells from superoxide anion ( O 2 − )-induced cell death through a novel regulation of reactive oxygen species (ROS) which increased O 2 − and decreased hydrogen peroxide (H 2 O 2 ), indicating that decreasing conversion from O 2 − to H 2 O 2 is a critical process for the protection by NGF. In the present study, we performed a comparative study on protective mechanisms between NGF and brain-derived neurotrophic factor (BDNF) using TrkB-expressing PC12h cells. When compared with NGF, BDNF induced a weaker but significant protective effect on the cells from O 2 − induced death. BDNF did not seem to change the total amount of ROS in the cells treated with xanthine and xanthine oxidase. On the other hand, BDNF increased O 2 − and decreased H 2 O 2 levels in the same cells, although not so strongly as NGF. These results suggest that decreasing conversion from O 2 − to H 2 O 2 is also critical for the protection by BDNF, which is considered to play a central role in survival and differentiation of CNS neurons.

Flow cytometric analysis of serum deprivation-induced apoptosis of PC12 cells, with special reference to role of bcl-2.

To investigate the mechanism of serum deprivation-induced apoptosis in PC12 cells, we performed flow cytometry with the viable cell-specific fluorogen, fluorescein diacetate (FDA). Intact PC12 cells were positive when stained with FDA, and those cells were identical with the major population which possess larger forward and side scattered light. Apoptotic PC12 cells decreased the forward and side scatter light associated with the loss of FDA-positive cells. Nerve growth factor (NGF) and the bcl-2 protein protected the cells from serum deprivation-induced apoptosis. These data suggested that the process of apoptosis in PC12 cells can be analyzed with flow cytometry.

A novel estimation method of road condition for pedestrian navigation

In this paper, we design and develop a ground surface condition recognition algorithm using shoe-mounted inertial sensors. We use a pair of small sensor boxes mounted on feet with accelerometers and gyro sensors inside and detect walking steps using them. Firstly, we detect stationary stance phase using accelerometers and gyro sensors. Then, based on this information, we estimate the “Angle of Inclination” (AoI) and stability of the ground. Moreover, we estimate whether the road surface is flat or not(unstable or unpaved (covered by gravel rubble or dirt) otherwise) based on variance of AoI. In addition, as for small undulation of surface due to dips, humps and bumps, which is hard to recognize only by a few samplings, we rely on continuous sensing data aggregated spatially from multiple users based on dead-reckoning techniques. We have developed a prototype of the proposed method. In the experiments, we show that our method cannot estimate not only walking steps correctly and but also AoIs of roads in rough trends.