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 ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide 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 expression of prostate apoptosis response-4 (PAR-4). PAR-4 expression and apoptosis were restored by preincubation of ST2-transfected cells with N-butyl deoxinojirimycin (NB-DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 mitogen-activated protein (MAPK) kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining 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-transfected cells, the blocking of which with nifedipine restored apoptosis to the 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 the presence of insulin-like growth factor-1 may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide-induced apoptosis by up-regulation of MAPK and reduction of PAR-4 expression.
Bieberich
E
E
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA. ebieberich@mail.mcg.edu
MacKinnon
S
S
Silva
J
J
Yu
R K
RK
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001921:Brain / Q000196:embryology* / Q000473:pathology; D002352:Carrier Proteins / Q000096:biosynthesis; D002454:Cell Differentiation; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005732:Gangliosides / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008564:Membrane Potentials; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D002518:Ceramides; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005732:Gangliosides; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C101296:prostate apoptosis response-4 protein; D049452:Green Fluorescent Proteins; D017485:1-Deoxynojirimycin; D019950:Mitogen-Activated Protein Kinase 1; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one