CellularC-jun N-terminal kinases/c-Jun and p38 pathways cooperate in ceramide-induced neuronal apoptosis
Section snippets
Materials
C2-ceramide (N-acetylsphingosine, Biomol Research Laboratory, Plymouth Meeting, PA, USA) was prepared as a 25 mM stock in ethanol and incubated in the culture media as described. The specificity of c2-ceramide was evaluated by comparing its effects with those of c2-dihydro-ceramide (Biomol Research Laboratory, Plymouth Meeting, PA, USA), an analog lacking the 4–5 trans double bond in the sphingosine moiety of c2-ceramide, which has been shown to be incapable of activating the sphingomyelin
Serum withdrawal-induced neuronal apoptosis increases ceramide levels in cortical neurons
Cortical neurons were deprived of serum after a 5-day maturation period in vitro and assayed for cell viability at various time points after treatment using the MTT metabolism assay (Fig. 1A). No significant loss of viability was detected during the first 6 h of treatment; however, after this lag phase the neurons degenerated progressively. After 24 and 42 h, 52% and 37% of the cells survived in the culture respectively (Fig. 1A).
We measured ceramide levels, a stress and apoptotic lipid second
Discussion
Ceramide generation has been reported as a mechanism for the induction of apoptosis in response to different stress stimuli in a variety of non-neuronal cells types. The present study demonstrates that the second-messenger ceramide increases upon survival-factor withdrawal in primary cortical neurons. Serum deprivation increased endogenous ceramide levels in a two-peak manner: the first peak occurred very rapidly within the first hour and the second peak at 16 h after the apoptotic process had
Acknowledgements
We are grateful to C. Jarvis for critical review of the manuscript. We thank M. Yaniv for the DNc-Jun plasmid, T. Levade and S. Carpentier for the ceramide dosage. This work was partially supported by grants from Association France-Alzheimer (to S.W.M.), Association pour la Recherche sur le Cancer (to S.W.M.), Fondation Lejeune (to K.B.C.) and European Community (TMR Neuril no. FMRX CT97-0149 and Biomed Cybrainet no. BMH4 CT97-2492 to J.M.).
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