In vivo role of caspases in excitotoxic neuronal death: generation and analysis of transgenic mice expressing baculoviral caspase inhibitor, p35, in postnatal neurons.
Caspases, a family of cysteine proteases, are thought to be critical mediators of apoptosis. To examine the role of neuronal caspases in excitotoxic neurodegeneration in vivo, we have generated transgenic mice expressing the baculovirus protein p35, a potent viral caspase inhibitor, using the neuron-specific calmodulin dependent kinase-II alpha (CaMKII-alpha) promoter. The expression of p35 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. We analyzed caspase activation and cell death by employing an experimental paradigm, in which the excitotoxin kainate (KA) was injected into CA1 of hippocampus and the distribution of the caspase-generated actin fragment was detected immunohistochemically. While kainate treatment led to selective neuronal death in the CA1, CA3 and CA4 of non-transgenic control mice, we observed restricted caspase activation only in the CA3 sector. The transgenic expression of p35 consistently inhibited the kainate-induced caspase activation, but failed to influence the death of neurons to any extent. In addition, we observed concomitant early calpain activation in the specific areas where neurons underwent degeneration in both the transgenic and non-transgenic mice. These results indicate that p35-inhibitable caspases play rather minor roles in the kainate-induced excitotoxicity and that the relative contribution of calpain is likely to be greater than that of caspases.
Tomioka
Masanori
M
Laboratory for Proteolytic Neuroscience, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
Shirotani
Keiro
K
Iwata
Nobuhisa
N
Lee
Hahn-Jun
HJ
Yang
Fusheng
F
Cole
Greg M
GM
Seyama
Yousuke
Y
Saido
Takaomi C
TC
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology*; D004789:Enzyme Activation; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology / Q000633:toxicity; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D007608:Kainic Acid / Q000494:pharmacology / Q000633:toxicity; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009498:Neurotoxins / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D061945:Caspase Inhibitors; D018690:Excitatory Amino Acid Agonists; D009498:Neurotoxins; D014764:Viral Proteins; C081618:p35 protein, Nucleopolyhedrovirus; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517671:Camk2a protein, mouse; D002154:Calpain; D020169:Caspases; D007608:Kainic Acid