Regulation of apoptosis during neuronal differentiation by ceramide and b-series complex gangliosides

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of c eramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramid e was coincident with the first appearance of b-series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F-11) cells with sialyltransferase-II (ST2) resulted in a 70% reduction of ceramide-induced apoptosis. This was most likely due to an 80% reduced exp ression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apop tosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynt hesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In s ections of day E14.5 mouse brain, the intermediate zone showed intensive st aining for complex gangliosides, but only low staining for apoptosis (TUNEL ) and PAR-4. Apoptosis and PAR-4 expression, however, were elevated in the ventricular zone which only weakly stained for complex gangliosides. Whole cell patch clamping revealed a 2-fold increased calcium influx in ST2-trans fected cells, the blocking of which with nifedipine restored apoptosis to t he level of untransfected cells. In serum-free culture, supplementation of the medium with IGF-1 was required to maintain MAPK phosphorylation and the anti-apoptotic effect of BCG expression. BCG-enhanced calcium influx and t he presence of insulin-like growth factor-1 may thus activate a cell surviv al mechanism that selectively protects developing neurons against ceramide- induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expressio n.