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 c(2)-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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis*
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Blotting, Western
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Cell Survival / drug effects
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Ceramides / pharmacology*
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Cerebral Cortex / cytology
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Cerebral Cortex / drug effects
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Cerebral Cortex / metabolism
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Culture Media, Serum-Free / pharmacology
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Culture Techniques
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DNA-Binding Proteins / metabolism
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Early Growth Response Protein 1
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Embryo, Mammalian
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Enzyme Inhibitors / pharmacology
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Female
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Gene Expression Regulation / drug effects
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Green Fluorescent Proteins
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Imidazoles / pharmacology
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Immediate-Early Proteins*
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Immunohistochemistry
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JNK Mitogen-Activated Protein Kinases*
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Luminescent Proteins / metabolism
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MAP Kinase Kinase 4
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Mice
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Mitogen-Activated Protein Kinase Kinases / metabolism
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Mitogen-Activated Protein Kinases / metabolism*
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Neurons / drug effects*
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Neurons / metabolism
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Neurons / pathology
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Phosphorylation / drug effects
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Pregnancy
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Proteins / metabolism
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Proto-Oncogene Proteins c-jun / metabolism*
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Pyridines / pharmacology
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Sphingosine / analogs & derivatives*
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Sphingosine / pharmacology
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Subcellular Fractions / drug effects
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Subcellular Fractions / metabolism
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Time Factors
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Transcription Factors / metabolism
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Transfection / methods
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p38 Mitogen-Activated Protein Kinases
Substances
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Ceramides
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Culture Media, Serum-Free
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DNA-Binding Proteins
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Early Growth Response Protein 1
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Egr1 protein, mouse
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Enzyme Inhibitors
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Imidazoles
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Immediate-Early Proteins
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Luminescent Proteins
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N-acetylsphingosine
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Proteins
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Proto-Oncogene Proteins c-jun
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Pyridines
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Transcription Factors
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Green Fluorescent Proteins
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JNK Mitogen-Activated Protein Kinases
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 4
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Mitogen-Activated Protein Kinase Kinases
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Sphingosine
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SB 203580