ABSTRACT
Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase (JNK) pathway. Cultured murine cerebellar granule cells were exposed to 1 microm colchicine for 24 h. Activation of the JNK pathway was detected by western blotting as well as immunocytochemistry using antibodies against phospho-c-Jun (p-c-Jun). Next, adult male rats were injected intracerebroventricularly with colchicine (10 microg), and JNK pathway activation in dentate granule cells (DGCs) was detected by antibodies against p-c-Jun. The second part of the study tested the involvement of mixed lineage kinases (MLK) as upstream activators of the JNK pathway in colchicine toxicity, using CEP-1347, a potent MLK inhibitor. In vitro, significant inhibition of the JNK pathway, activated by colchicine, was achieved by 100-300 nm CEP-1347, which blocked both activation of cell death proteases and apoptosis. Moreover, CEP-1347 markedly delayed neurite fragmentation and cell degeneration. In vivo, CEP-1347 (1 mg/kg) significantly prevented p-c-jun increase following injection of colchicine, and enhanced survival of DGCs. We conclude that colchicine-induced neuronal apoptosis involves the JNK/MLK pathway, and that protection of granule cells can be achieved by MLK inhibition.
Subject(s)
Apoptosis/physiology , Cerebellum/cytology , Cytoskeleton/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Kinase Kinases/metabolism , Neurons/cytology , Animals , Animals, Newborn , Apoptosis/drug effects , Blotting, Western/methods , Carbazoles/pharmacology , Caspase 3 , Caspases/metabolism , Cell Count/methods , Cells, Cultured , Colchicine/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Immunohistochemistry/methods , Indoles/pharmacology , Male , Mice , Mice, Inbred BALB C , Neurons/drug effects , Proto-Oncogene Proteins c-jun/metabolism , Rats , Rats, Wistar , Signal Transduction/drug effects , Signal Transduction/physiology , Tetrazolium Salts , ThiazolesABSTRACT
In neuronal stress and degeneration, mitogen-activated protein (MAP) kinase signaling pathways play an important role. We studied the pattern of activation of the c-Jun N terminal kinase (JNK) signal transduction pathway during the course of a subacute MPTP mouse model of Parkinson's disease. In this model, there was no significant neuronal loss, but the function of the dopaminergic neurons was significantly decreased. During MPTP administration, phosphorylation of p-Jun was increased in the substantia nigra, and MKK4 was increased both in the striatum and substantia nigra. We conclude that after MPTP intoxication in the mouse, activation of the JNK pathway occurs both in the striatum and in the substantia nigra. This activation does not seem to corrrelate with loss of neuronal cell bodies but might represent a response to damage/loss of axonal terminals.
