Elsevier

Neuroscience

Volume 119, Issue 2, 27 June 2003, Pages 387-397
Neuroscience

Cellular
C-jun N-terminal kinases/c-Jun and p38 pathways cooperate in ceramide-induced neuronal apoptosis

https://doi.org/10.1016/S0306-4522(02)00996-XGet rights and content

Abstract

Understanding the regulation of the apoptotic program in neurons by intracellular pathways is currently a subject of great interest. Recent results suggest that c-Jun N-terminal kinases (JNK), mitogen-activated protein kinases and the transcription factor c-Jun are important regulators of this cell death program in post-mitotic neurons following survival-factor withdrawal. Our study demonstrates that ceramide levels increase upon survival-factor withdrawal in primary cultured cortical neurons. Furthermore, survival-factor withdrawal or addition of exogenous c2-ceramide induces JNK pathway activation in these cells. Western blot analyses of JNK and c-Jun using phospho-specific antibodies reveal that JNK and subsequent c-Jun phosphorylation occur hours before the initiation of apoptosis, reflected morphologically by neurite retraction and fragmentation, cell-body shrinkage and chromatin fragmentation. Immunocytochemistry using the same antibodies shows that phospho-JNK are localized in the neurites of control neurons and translocate to the nucleus where phospho-c-Jun concurrently appears upon ceramide-induced apoptosis. To determine if ceramide-induced c-Jun activation is responsible for the induction of the apoptotic program, we performed transient transfections of a dominant negative form of c-Jun, truncated in its transactivation region. Our results show that DNc-Jun partially protects cortical neurons from ceramide-induced apoptosis. Treatment of dominant negative c-Jun-expressing neurons with the pharmacological inhibitor of p38 kinase, SB203580, completely blocked neuronal death. Thus our data show that p38 and JNK/c-Jun pathways cooperate to induce 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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