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

Abstract—Understanding the regulation of the apoptotic pro-gram in neurons by intracellular pathways is currently a sub-ject 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 cer-amide levels increase upon survival-factor withdrawal in pri-mary 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-in-duced 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 transacti-vation region. Our results show that DNc-Jun partially pro-tects 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 neurona