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Development of a pentylenetetrazole-induced seizure model to evaluate kinase inhibitor efficacy in the central nervous system
- Source :
- Neuroscience Letters. 395:143-148
- Publication Year :
- 2006
- Publisher :
- Elsevier BV, 2006.
-
Abstract
- 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.
- Subjects :
- Male
inorganic chemicals
Cell signaling
MAP Kinase Kinase 4
Proto-Oncogene Proteins c-jun
Immunoblotting
Phosphatase
Convulsants
macromolecular substances
Biology
Mitogen-activated protein kinase kinase
Hippocampus
environment and public health
Neuroprotection
Mice
Seizures
Animals
Protein phosphorylation
Phosphorylation
Protein Kinase Inhibitors
Kinase
Activator (genetics)
General Neuroscience
Immunohistochemistry
Cell biology
Enzyme Activation
Mice, Inbred C57BL
Disease Models, Animal
enzymes and coenzymes (carbohydrates)
Biochemistry
Pentylenetetrazole
bacteria
Electrophoresis, Polyacrylamide Gel
Subjects
Details
- ISSN :
- 03043940
- Volume :
- 395
- Database :
- OpenAIRE
- Journal :
- Neuroscience Letters
- Accession number :
- edsair.doi.dedup.....3e8f08aafb8cb69e4d92d9a2aa6cd6f0