N. F. Avrova

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.

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.

Ganglioside-dependent factor, inhibiting lipid peroxidation in rat brain synaptosomes

Preincubation of rat brain synaptosomes with GM1, GD1a or GT1b (10(-10)-10(-6) M), as well as with phorbol 12-myristate, 13-acetate (10(-10)-10(-6) M) was found to have dose dependent inhibitory effect on Fe(2+)-ascorbate induced lipid peroxidation, while penetrating analogue of c-AMP markedly decreased the inhibitory effect of these compounds. In liposomes made of lipids isolated from synaptosomal membranes the degree of inhibition of induced LPO by gangliosides was practically absent. The inhibitory effect of GM1 on lipid peroxidation could not be revealed after thermal denaturation of synaptosomes or after treatment with polymyxin B (inhibitor of lipid-dependent protein kinases). These results and some other data provide evidence for the existence of ganglioside-dependent factor inhibiting lipid peroxidation in brain tissue. It may be suggested to be a protein kinase modulated by gangliosides.

Ganglioside GM1 protects cAMP 3′ 5′: Phosphodiesterase from inactivation caused by lipid peroxidation in brain synaptosomes of rats

The preincubation of synaptosomes with nanomolar concentrations of ganglioside GM1 was shown to protect Ca(2+)-dependent and Ca(2+)-independent cyclic nucleotide phosphodiesterase from inactivation caused by lipid peroxidation (LPO) induction. Thus, Ca(2+)-dependent phosphodiesterase activity decreased to approximately 34% of the initial value following 30 min of LPO induction, but it constituted more than 60% of the control activity if synaptosomes were preincubated with 10(-8)M GM1, the difference being statistically significant. 10(-6)M alpha-tocopherol had a similar effect. As far as the lipid matrix is concerned, gangliosides were found to prevent to a great extent malonic dialdehyde (MDA) accumulation and to protect polyenoic fatty acids from oxidative destruction. The ability of gangliosides to protect phosphodiesterase from inactivation caused by LPO induction appears to be owing not only to the inhibition of the accumulation of LPO products, but to the direct activation of the enzyme as well, 10(-7) M of ganglioside GM1 having the maximal activating effect. In contrast to alpha-tocopherol and other antioxidants reacting directly with free radicals, the inhibitory effect of gangliosides appears to be mediated by signal transduction systems.

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.

α-Tocopherol at Nanomolar Concentration Protects Cortical Neurons against Oxidative Stress

The aim of the present work is to study the mechanism of the α-tocopherol (α-T) protective action at nanomolar and micromolar concentrations against H2O2-induced brain cortical neuron death. The mechanism of α-T action on neurons at its nanomolar concentrations characteristic for brain extracellular space has not been practically studied yet. Preincubation with nanomolar and micromolar α-T for 18 h was found to increase the viability of cortical neurons exposed to H2O2; α-T effect was concentration-dependent in the nanomolar range. However, preincubation with nanomolar α-T for 30 min was not effective. Nanomolar and micromolar α-T decreased the reactive oxygen species accumulation induced in cortical neurons by the prooxidant. Using immunoblotting it was shown that preincubation with α-T at nanomolar and micromolar concentrations for 18 h prevented Akt inactivation and decreased PKCδ activation induced in cortical neurons by H2O2. α-T prevented the ERK1/2 sustained activation during 24 h caused by H2O2. α-T at nanomolar and micromolar concentrations prevented a great increase of the proapoptotic to antiapoptotic proteins (Bax/Bcl-2) ratio, elicited by neuron exposure to H2O2. The similar neuron protection mechanism by nanomolar and micromolar α-T suggests that a “more is better” approach to patients’ supplementation with vitamin E or α-T is not reasonable.

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.