[Metabolic effects of ganglioside GM1 on PC12 cells at oxidative stress depend on modulation of activity of tyrosine kinase of trk receptor].

Evidence has been obtained that only GM1, but also other main brain gangliosides (GD1a, GD1b, and GT1b) increase viability of ells of the PC12 neuronal line submitted to action of H2O2. By the example of GM1 and GD1a, gangliosides have been shown to induce a protective effect when acting on PC12 cells under conditions of oxidative stress both at micro- and nanomolar concentrations that are physiological concentrations of gangliosides in cerebrospinal fluid. It has been shown for the first time that GM1 at nanomolar concentrations decrease the H2O2-induced formation of reactive oxygen species (ROS). It was found that in the presence of K-252a, an inhibitor of tyrosine kinase of Trk receptors, GM1 at concentrations of 10 microM and 10 nM lost the ability to increase viability of these cells under conditions of oxidative stress. The dependence of protective and metabolic effects of ganglioside GM 1 in PC 12 cells at action on them of H2O2 on modulation of activity of tyrosine kinase of Trk receptors (i. e., on the same signal system) agrees with concept of the essential role of the GM1 antioxidant effect in its increase of cell viability.

[Protective effect of gangliosides against the toxic action of bacterial lipopolysaccharide of various serotypes on PC12 cells].

The effect of bacterial lipopolysaccharide (LPS) from E. coli on the viability of PC12 neuronal cell line was studied. LPS of 0111:B4 serotype and LPS of 055:B5 serotype were shown to have toxic effect on PC12 cells. But the toxic effect of LPS of 0127:B8 serotype was not revealed. Preincubation of PC12 cells with GM1 or GD1a gangliosides was found to diminish significantly the death of the cells and the formation of reactive oxygen species induced by LPS of 0111:B4 and 055:B5 serotypes in them. The protective effect of GM1 and GD1a was found not to depend on the presence of the inhibitor of Trk receptor tyrosine kinase in the medium, though activation of this protein kinase was previously shown to mediate the protective effect of gangliosides against the action of excitatory amino acids, pro-oxidants and other toxins on neurons and cells of neuronal cell lines. The protective effect of gangliosides against the LPS action on PC12 cells was similar to the effect of methyl-beta-cyclodextrin, which was shown to disturb cell membrane raft structure. The suggestion is put forward that the pronounced diminution of toxic effect of LPS on PC12 cells by gangliosides may be explained by the alteration of structural organization of lipid rafts caused by the incorporation of exogenous gangliosides in cell plasma membranes, which diminishes the translocation of TLR4 receptor into the rafts and their activation by LPS.

[Role of tyrosine kinase of Trk-receptors in realization of antioxidant effect of ganglioside GM1 in PC12 cells].

Ganglioside GM1 has been shown to increase viability of PC12 cells at their induction of oxidative stress by hydrogen peroxide. However, in the presence of inhibitor of tyroxine kinase Trk-receptors K-252a this GM1 effect decreases or virtually disappears. To understand mechanism of the protective effect, there was studied action of H2O2, GM1, and inhibitor K-252a on formation of reactive oxygen species (ROS). It has been shown that ganglioside GM1 decreases significantly the H2O2-induced ROS accumulation in PC12 cells; however, in the presence of inhibitory of tyrosine kinase of Trk-receptors, this GM1 effect is not revealed. It has been found that inhibitors of each of protein kinases present at the signal realization stages following the stages of activation of tyrosine kinase Trk-receptors--Erk 1/2, PI3-kinases, and PKC, decreased the GM1 ability to reduce the H2O2-induced ROS accumulation, while in the combined use of inhibitors of these three protein kinases, the GM1 effect was completely absent. Thus, the ganglioside GM1 antioxidant effect on PC12 is mediated by activation of tyrosine kinase Trk-receptors and protein kinases perceiving signal from this enzyme.

[A decrease of neuroprotector effect of ganglioside GM1 on PC12 cells under conditions of oxidative stress in the presence of inhibitor of tyrosine kinase of Trk-receptors].

Effects of inhibitors of tyrosine kinases (K-252a, genistein) and of phospholipase A2 (bromophenacetyl bromide) on viability of PC12 cells are studied in the presence of hydrogen peroxide and ganglioside GM1. The degree of inhibition of hydrogen peroxide cytotoxic effect by ganglioside GM1 amounted to 52.8 +/- 4.3 %. However, in the presence in the medium of 0.1 and 1 microM inhibitors of tyrosine kinase of Trk-receptors (K-252a) it was as low as 32.7 +/- 6.5 % and 11.7 +/- 9.8 %, respectively. GM1 prevented Na+, K+-ATPase produced by H2O2, but in the presence of 1 microM K-252a this effect was practically not pronounced. In the presence of another inhibitor of tyrosine kinases--genistein, a tendency for a decrease of the GM1 protective effect was observed at its concentrations 0.1 and 1 microM, whereas at a higher concentration 10 microM genistein depressed the GM1 neuroprotective effect statistically significantly. It was found that inhibitor of phospholipase A2 bromophenacetyl bromide did not affect the action of GM1 aimed at increasing the viability of cells under action of hydrogen peroxide on them. It seems that this enzyme is not involved in the cascade of reactions participating in realization of the ganglioside protective effect. Thus, inhibitor of tyrosine kinase of Trk-receptors K-252 decreases or practically prevents the ganglioside GM1 neuroprotective effect of PC12 cells under stress conditions; the same ability is characteristic of genistein--an inhibitor of tyrosine kinases of the wider spectrum of action.

[PC12 cells transfected with human mutant gene causing one of Alzheimer's disease forms have a high sensitivity to oxidative stress].

Used in this work are PC12 cells transfected with human gene expressing amyloid precursor protein of beta-peptide and carrying the so-called "Swedish mutation" leading to the appearance of one Alzheimer's disease family forms. It has been shown that the PC12 cells transfected with this mutant gene, at action of various hydrogen peroxide concentrations, die to the significant greater degree than the used for comparison PC12 cells transfected with analogous human gene of the wild type or than vector-transfected cells. It has been found that ganglioside GM1 at micro- or nanomolar concentrations is able to increase viability of the PC12 cells transfected with the mutant gene causing a significant accumulation of endogenous amyloid beta-peptide. The obtained data confirm an important role of oxidative stress in injury and death of brain nerve cells in Alzheimer's disease.