Ceramide prevents motoneuronal cell death through inhibition of oxidative signal

We previously reported that cell death of rat spinal motoneurons, induced by trophic factor-deprivation, was attenuated by the application of exogenous cell-permeable ceramide (C6-Cer), or bacterial sphingomyelinase (SMase). Recently, motoneuronal cell death was demonstrated to be mediated by the generation of reactive oxygen species (ROS), including superoxide and peroxinitrite. In this study, to investigate the protective mechanism of ceramide (Cer), we examined the effects of Cer and sphingolipid metabolites against ROS generation and oxidative injury in enriched motoneuron cultures. Staining with C-DCDHF-DA, a fluorescent probe for detection of ROS, demonstrated that application of C6-Cer (2.5 mM) or bacterial SMase inhibited the increase of ROS generation. C6-dihydro-Cer, a biologically inactive analogue of C6-Cer, sphingosine, and sphingosine-1-phosphate did not affect ROS generation. This specificity corresponded to the results of cell survival assays. In addition, C6-Cer was shown to specifically inhibit ROS-induced reactions, such as tyrosine nitration and lipid peroxidation, in studies using antibodies against peroxinitrite and 4-hydroxinonenal, respectively. A potent neurotrophin for motoneurons, GDNF, had inhibitory effects against ROS generation and ROS-induced reactions. C6-Cer was also effective in the prevention of cytotoxicity induced by 1-buthionine-sulfoximine, an inhibitor of glutathione synthesis. These observations suggest that Cer plays a protective role in spinal motoneurons through inhibition of oxidative signals.