T. V. Sokolova

Inhibition of glutamate-induced intensification of free radical reactions by gangliosides: possible role in their protective effect in rat cerebellar granule cells and brain synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 μM) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na+, K+-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.

Mitochondrial Electron Transport Chain in Heavy Metal-Induced Neurotoxicity: Effects of Cadmium, Mercury, and Copper

To clarify the role of mitochondrial electron transport chain (mtETC) in heavy-metal-induced neurotoxicity, we studied action of Cd2+, Hg2+, and Cu2+ on cell viability, intracellular reactive oxygen species formation, respiratory function, and mitochondrial membrane potential of rat cell line PC12. As found, the metals produced, although in a different way, dose- and time-dependent changes of all these parameters. Importantly, Cd2+ beginning from 10 [mu]M and already at short incubation time (3 h) significantly inhibited the FCCP-uncoupled cell respiration; besides, practically the complete inhibition of the respiration was reached after 3 h incubation with 50 [mu]M Hg2+ or 500 [mu]M Cd2+, whereas even after 48 h exposure with 500 [mu]M Cu2+, only a 50% inhibition of the respiration occurred. Against the Cd2+-induced cell injury, not only different antioxidants and mitochondrial permeability transition pore inhibitors were protective but also such mtETC effectors as FCCP and stigmatellin (complex III inhibitor). However, all mtETC effectors used did not protect against the Hg2+- or Cu2+-induced cell damage. Notably, stigmatellin was shown to be one of the strongest protectors against the Cd2+-induced cell damage, producing a 15–20% increase in the cell viability. The mechanisms of the mtETC involvement in the heavy-metal-induced mitochondrial membrane permeabilization and cell death are discussed.

Neuroprotective Effect of Ganglioside GM1 on the Cytotoxic Action of Hydrogen Peroxide and Amyloid β-peptide in PC12 cells

Ganglioside GM1 was shown to increase the viability of PC12 cells exposed to hydrogen peroxide or amyloid β-peptide (Aβ25–35). The PC12 cells transfected with mutant gene (expressing APPSW) were found to be more sensitive to oxidative stress than the cells transfected with wild type gene (expressing APPWT) or vector-transfected cells, GM1 being effective in enhancing the viability of the cells transfected with mutant gene. The exposure to hydrogen peroxide or Aβ25–35 results in a partial inactivation of Na+,K+-ATPase in PC12 cells, H2O2 increases MDA accumulation in these cells. But these effects could be partially prevented or practically abolished by GM1 ganglioside. In the presence of the inhibitor of tyrosine kinase of Trk receptors (K-252a) the protective and metabolic effects of GM1 on PC12 cells in conditions of oxidative stress caused by hydrogen peroxide are not observed or are markedly diminished.

α-Tocopherol at Nanomolar Concentration Protects PC12 Cells from Hydrogen Peroxide-Induced Death and Modulates Protein Kinase Activities

The aim of this work was to compare protective and anti-apoptotic effects of α-tocopherol at nanomolar and micromolar concentrations against 0.2 mM H2O2-induced toxicity in the PC12 neuronal cell line and to reveal protein kinases that contribute to α-tocopherol protective action. The protection by 100 nM α-tocopherol against H2O2-induced PC12 cell death was pronounced if the time of pre-incubation with α-tocopherol was 3–18 h. For the first time, the protective effect of α-tocopherol was shown to depend on its concentration in the nanomolar range (1 nM < 10 nM < 100 nM), if the pre-incubation time was 18 h. Nanomolar and micromolar α-tocopherol decreased the number of PC12 cells in late apoptosis induced by H2O2 to the same extent if pre-incubation time was 18 h. Immunoblotting data showed that α-tocopherol markedly diminished the time of maximal activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt)-induced in PC12 cells by H2O2. Inhibitors of MEK 1/2, PI 3-kinase and protein kinase C (PKC) diminished the protective effect of α-tocopherol against H2O2-initiated toxicity if the pre-incubation time was long. The modulation of ERK 1/2, Akt and PKC activities appears to participate in the protection by α-tocopherol against H2O2-induced death of PC12 cells. The data obtained suggest that inhibition by α-tocopherol in late stage ERK 1/2 and Akt activation induced by H2O2 in PC12 cells makes contribution to its protective effect, while total inhibition of these enzymes is not protective.

α-Tocopherol Prevents ERK1/2 Activation in PC12 Cells under Conditions of Oxidative Stress and Its Contribution to the Protective Effect

Preincubation with 100 nM and 100 μM α-tocopherol for 18 h prevented long-term activation of extracellular signal-activated kinase (ERK1/2), induced by H2O2 in PC12 cells. α-Tocopherol significantly reduced H2O2-induced death of PC12 cell, but its protective effect was signifi cantly lower in the presence of ERK1/2 inhibitor. These data show that prevention of long-term activation of ERK1/2 by α-tocopherol contributes to the increase in viability of PC12 cells exposed to H2O2. This fact suggests that inhibition of ERK1/2 activity by α-tocopherol reduces neuronal cell death in the brain under conditions of oxidative stress in vivo.

Effect of ganglioside GM1 on mitochondrial respiration and viability of PC12 cells under oxidative stress

50 μg/ml of penicillin and 50 U/ml of streptomy-cin (Biolot, Russia). Cell viability was evaluated by the method based on determination of activity of lactate dehydrogenase (LDH), released from destroyed cells to incubation medium, with the total LDH activity in the samples taken as 100%. PC12 cells were incubated with 10 μM GM1 (or without it) for 1 h, then subjected to action of 1 mM H 2 O 2 for 2 h, the experiments were performed in serum-free DMEM medium. The LDH activity in the samples was determined from the changes in NADH level measured at 340 nm on a Specord M40 spectrophotometer (Carl Zeiss, Germany), as described earlier [1]. Respiration rates of PC12 cells (in nmol O 2 • min –1 • 10 –6 cells –1) were measured at 37oC in serum-free DMEM medium, in the chamber of 1.5 ml volume with closed Clarks type platinum electrode on an Expert-001 analyzer (NPO Econic-expert, Russia). The experiments were performed on PC12 cells—the control ones, those treated with 0.5 mM H 2 O 2 for 1 h, and the cells incubated for 1 h with 10 μM GM1, and then with 0.5 mM H 2 O 2 for 1 h. Hydrogen peroxide induces development of Gangliosides, typical components of nerve cell membranes, are glycosphingolipids containing sialic acids. The major brain gangliosides (GM1, GD1a, GD1b, and GT1b) upon their administration to animals with ischemic, traumatic, and toxic brain lesions are known to prevent its nerve cells from death. They increase viability of neurons and of cells of neuronal lines under action on them of glutamate and other toxins. Most often used in experiments of such type is the most stable of these gangliosides—GM1. Understanding the mechanism of protective gangliosides action, elucidation of their capacity to prevent or decrease the disturbances in functional activity of brain cells mitochondria, induced by oxidative stress, are of great interest. The goal of present work was to study effect of GM1 ganglio-side on viability and mitochondrial respiration of cells of neuronal line PC12 under action on them of toxic concentrations of hydrogen peroxide. The experiments were performed on the PC12 cells (ATCC). They were grown in CO 2-incubator at 37°C in the DMEM culture medium supplemented with L-glutamine and containing 10% of bovine fetal serum and 5% of horse blood serum,

Dependence of Protective Effect of α-Tocopherol on Its Concentration and Time of Action on PC12 Cells under Conditions of Oxidative Stress

At the short-term incubation (0.5 and 1.5 h) of cells of the PC12 neuronal line with α-tocopherol, its protective effect against the cytotoxic hydrogen peroxide action was increased with rise of its concentration in samples; the protection was practically absent at action of nanomolar antioxidant concentrations, but was well expressed at its micromolar concentrations. These data agree with the concept that α-tocopherol increases the cell viability by reacting directly with free radicals, which leads to formation of the less reactive compounds deprived of non-paired electron. The evidence is obtained that at the long-term action on PC12 cells, α-tocopherol not only in micro-, but also in nanomolar concentrations increases significantly the cell viability under conditions of oxidative stress. As follows from the obtained data, an important role in realization of the α-tocopherol protective effect at the long-term incubation seems to be played by modulation by this antioxidant of activity of protein kinase activated by extracellular signaling, phosphatidylinosite 3-kinase, and protein kinase C.

Stimulation by Gangliosides of Viability of Rat Brain Neurons and of Neuronal PC12 Cell Line under Conditions of Oxidative Stress

We studied effect of gangliosides on viability of brain neurons and neuronal PC12 cell line exposed to toxic concentrations of compounds activating free radical reactions. It is found that preincubation of cerebellar granule cells and PC12 cells with micromolar concentrations of ganglioside GM1 increases statistically significantly viability of these cells submitted to inductors of oxidative stress, such as hydrogen peroxide and the Fe2+-ascorbate system However, the effect of ganglioside GM1 in the PC12 cells failed to be revealed 1–2 days after treatment of the cells with trypsin, which indicates an importance of interaction of gangliosides with surface proteins for realization of their protective action. GM1, GD1a, and other gangliosides were shown to produce the neuroprotective effect on cerebellar granule cells in the presence of toxic glutamate concentrations. Not only micro-, but also nanomolar concentrations of these gangliosides increased statistically significantly the neuronal viability, although at micromolar concentrations this effect as a rule was more pronounced. The obtained data allow suggesting that the neuroprotective action of gangliosides is determined to a considerable degree by their ability to inhibit free-radical reactions in nerve cells.

Metabolic effects of ganglioside GM1 on PC12 cells in oxidative stress depend on modulation of activity of tyrosine kinase Trk of receptors

There have been obtained evidences that not only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of cells of the neuronal line PC12 under action of H2O2. By the example of GM1 and GD1a, gangliosides have been shown to produce a protective effect on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. For the first time, GM1 at nanomolar concentrations was shown to decrease the H2O2-induced formation of reactive oxygen species (ROS). It was found that in the presence of inhibitor of tyrosine kinase Trk of receptors K-252a, GM1 at concentrations of 10 μM and 10 nM lost its ability to produce such metabolic effects as a decrease of ROS accumulation and of the degree of oxidative inactivation of Na+,K+-ATPase in PC12 cells, as well as ceased to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of gangliosides GM1 in PC12 cells treated with H2O2 on modulation of activity of activity of tyrosine kinase Trk receptors (i.e., from the same signal system) agrees with concept about the essential role of oxidant effect of GM1 in its increase of cell viability.

Alpha-tocopherol at nanomolar concentrations increases the viability of PC12 cells under oxidative stress conditions. The effects of modulation of signaling systems

We found that long-term preincubation of neuronal-like PC12 cells with α-tocopherol at micro- as well as nanomolar concentrations significantly increased cell viability under oxidative stress conditions. We discovered that the protective effect of α-tocopherol increases with an increase in its concentration in the 1–100 nM range, while its effects at concentrations of 100 nM, 1, 10, and 100 μM were similar when α-tocopherol was applied to cells 12–18 h prior to H2O2. An important role in the protective effect of long-term pre-incubation of cells with α-tocopherol at various concentrations is probably related to its modulatory influence on the activities of protein kinase C, extracellular signal-activated protein kinase, and phosphatidylinositol 3-kinase. Short-term preincubation of PC12 cells with this antioxidant at nanomolar concentrations for 0.5 or 1.5 h practically did not influence cell viability, while the protective effect of α-tocopherol at micromolar concentrations was probably related to its ability to terminate free-radical reactions due to direct interactions with free radicals.