Ceramide prevents motoneuronal cell death through inhibition of oxidative signal

We previously reported that cell death of rat spinal motoneurons, induced b y trophic factor-deprivation, was attenuated by the application of exogenou s cell-permeable ceramide (C-6-Cer), or bacterial sphingomyelinase (SMase). Recently, motoneuronal cell death was demonstrated to be mediated by the g eneration of reactive oxygen species (ROS), including superoxide and peroxi nitrite. 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. Stain ing with C-DCDHF-DA, a fluorescent probe for detection of ROS, demonstrated that application of C-6-Cer (2.5 mM) or bacterial SMase inhibited the incr ease of ROS generation. C-6-dihydro-Cer, a biologically inactive analogue o f C-6-Cer, sphingosine, and sphingosine-1-phosphate did not affect ROS gene ration. This specificity corresponded to the results of cell survival assay s. In addition, C-6-Cer was shown to specifically inhibit ROS-induced react ions, such as tyrosine nitration and lipid peroxidation, in studies using a ntibodies against peroxinitrite and 4-hydroxinonenal, respectively. A poten t neurotrophin for motoneurons, GDNF, had inhibitory effects against ROS ge neration and ROS-induced reactions. C-6-Cer was also effective in the preve ntion of cytotoxicity induced by 1-buthionine-sulfoximine, an inhibitor of glutathione synthesis. These observations suggest that Cer plays a protecti ve role in spinal motoneurons through inhibition of oxidative signals. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.