Characterization of ceramide-induced apoptotic death in cerebellar granulecells in culture

Sphingomyelin signalling system has been involved in several examples of ce ll death through apoptosis. We have characterised the effect of exposure to the cell permeable ceramide analogue, C-2-ceramide, on cultures of differe ntiated cerebellar granule cells. C-2-ceramide was toxic to granule cells i n a dose- and time-dependent way at concentrations higher than 10 muM. Cera mide exposure was accompanied by characteristic alterations of cell morphol ogy, namely swollen cell bodies and punctuate appearance and arcuate direct ion of processes. The final outcome of ceramide exposure was a form of cell death largely apoptotic in nature. Hoechst stain, followed by counts of nu clei with normal appearance and size or with condensed chromatin and reduce d size, revealed a large increase of the proportion of shrunken nuclei in t reated cultures. In situ visualisation of fragmented DNA through the TUNEL technique, additionally marked cells undergoing apoptosis as a consequence of ceramide treatment. accordingly, the DNA extracted from cultures exposed to C-2-ceramide and subjected to agarose gel electrophoresis showed the pe culiar ladder of fragmented low molecular weight DNA. Treatments with inhib itors of two caspases or of nitric oxide synthase were unable to rescue neu rons exposed to ceramide, thus suggesting a neurotoxic action not primarily dependent on activation of death proteases or on nitric oxide production. (C) 2001 Elsevier Science Ltd. All rights reserved.