Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system.
c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.
Morgan
Louise
L
Eisai London Research Laboratories Ltd., Bernard Katz Building, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Neame
Stephen J
SJ
Child
Hannah
H
Chung
Raymond
R
Shah
Bina
B
Barden
Laura
L
Staddon
James M
JM
Patel
Toshal R
TR
D000818:Animals; D003292:Convulsants / Q000633:toxicity*; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006624:Hippocampus / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D048670:MAP Kinase Kinase 4 / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010433:Pentylenetetrazole / Q000633:toxicity*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000187:drug effects / Q000378:metabolism; D012640:Seizures / Q000139:chemically induced*
D003292:Convulsants; D047428:Protein Kinase Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D048670:MAP Kinase Kinase 4; D010433:Pentylenetetrazole