A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption.
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.
Molecular Neuropathology Group, University of Copenhagen, Denmark.
Georg Johannes
GJ
Müller
Marie Aavang
MA
Geist
Lone Merete
LM
Veng
Mette Georgi
MG
Willesen
Flemming Fryd
FF
Johansen
Marcel
M
Leist
Elisabetta
E
Vaudano
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western / Q000379:methods; D002227:Carbazoles / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D003078:Colchicine / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013778:Tetrazolium Salts; D013844:Thiazoles
D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D016755:Proto-Oncogene Proteins c-jun; D013778:Tetrazolium Salts; D013844:Thiazoles; C106592:3,9-bis((ethylthio)methyl)-K-252a; D048031:JNK Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C022616:thiazolyl blue; D003078:Colchicine