RESUMEN
The consequences of activation of tumour necrosis factor receptor 1 (TNFR1) during neuronal injury remain controversial. The apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, can mediate cell death downstream of TNFR1. Presently, we examined the formation of the TNFR1 signalling cascade and response of ASK1 during seizure-induced neuronal death. Brief (40 min) seizures were induced in rats by intra-amygdala microinjection of kainic acid, which elicited unilateral hippocampal CA3 neuronal death. Seizures caused a rapid decline in the expression of the silencer of death domains protein within injured CA3. Co-immunoprecipitation analysis revealed a commensurate assembly of a TNFR1 scaffold complex containing TNFR-associated death domain protein, receptor interacting protein and TNFR-activating factor 2. In addition, recruitment of TNFR-activating factor 2 was likely promoted by Bcl10-mediated sequestering of cellular inhibitor of apoptosis protein 2. Apoptosis signal-regulating kinase 1 was sequestered in a complex that contained the molecular chaperone 14-3-3beta and protein phosphatase 5. Seizures triggered its dissociation, and the phosphorylation of the ASK1 substrates, mitogen-activated protein kinase kinase 3/6 and 4. Subsequently, protein phosphatase 5 translocated into the nuclei of degenerating CA3 neurons, while ASK1 colocalized with the adaptor proteins Daxx and TNFR-activating factor 2 at the outer membrane of injured CA3 neurons. Neutralizing antibodies to TNFalpha reduced the numbers of DNA damaged cells within the injured hippocampus. These data suggest ASK1 may be involved in the mechanism of seizure-induced neuronal death downstream of a TNFR1 death-signalling complex.
Asunto(s)
Antígenos CD/metabolismo , Apoptosis/fisiología , Epilepsia/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Células Piramidales/enzimología , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas 14-3-3 , Animales , Anticuerpos/farmacología , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Epilepsia/inducido químicamente , Epilepsia/patología , Proteína Ligando Fas , Inmunohistoquímica , MAP Quinasa Quinasa Quinasa 5 , Masculino , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas/metabolismo , Células Piramidales/patología , Ratas , Ratas Sprague-Dawley , Receptores Tipo I de Factores de Necrosis Tumoral , Transducción de Señal , Factor 2 Asociado a Receptor de TNF , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Regulación hacia ArribaRESUMEN
The caspase family of cell death proteases has been implicated in the mechanism of neuronal death following seizures. We investigated the expression and processing of caspases 6 and 7, putative executioner caspases. Brief limbic seizures were evoked by intraamygdala kainic acid to elicit unilateral death of target hippocampal CA3 neurons in the rat. Seizures rapidly induced cleavage of constitutively expressed caspase-6, followed by elevated VEIDase activity and the proteolysis of lamin A. Neuronal caspase-6 immunoreactivity was markedly upregulated within cortex and hippocampus in relation to bursts of polyspike paroxysmal discharges. In contrast, while caspase-7 expression also increased within cortical and hippocampal neuronal populations in response to the same seizure patterns, caspase-7 was not proteolytically activated. These data highlight differences in expression and activation of caspases 6 and 7 in response to identifiable seizure patterns, focusing potential therapeutic targets for neuroprotection in epilepsy.
Asunto(s)
Caspasas/metabolismo , Electroencefalografía/efectos de los fármacos , Hipocampo/patología , Proteínas del Tejido Nervioso/metabolismo , Convulsiones/enzimología , Amígdala del Cerebelo , Animales , Apoptosis/efectos de los fármacos , Caspasa 6 , Caspasa 7 , Convulsivantes/toxicidad , Diazepam/farmacología , Activación Enzimática , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Agonistas del GABA/farmacología , Glioma/patología , Células HeLa/efectos de los fármacos , Células HeLa/patología , Hipocampo/enzimología , Humanos , Infarto de la Arteria Cerebral Media/patología , Células Jurkat/efectos de los fármacos , Células Jurkat/patología , Ácido Kaínico/toxicidad , Lamina Tipo A , Laminas , Masculino , Microinyecciones , Proteínas Nucleares/metabolismo , Ratas , Ratas Sprague-Dawley , Convulsiones/inducido químicamente , Convulsiones/patología , Estaurosporina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patologíaRESUMEN
Activation of cysteinyl aspartate-specific proteases (caspases) may underlie apoptotic cell death in brain. Terminal, executioner caspases 3, 6 and 7 likely contribute to such cell death in a stimulus- and cell type-specific manner. Here we investigate the processing and activation of caspases 3, 6 and 7 in rat C6 glioma cells induced to undergo apoptosis by staurosporine (STS) treatment as a model of apoptosis in glia. Proteolysis and activation of caspases 3 and 7 as determined by immunoblotting and substrate-specific cleavage assay (DEVDase) preceded caspase-6 proteolysis and increased VEIDase activity following STS treatment. Activation of caspase-6 was paralleled by cleavage of the nuclear envelope protein lamin-A. These results highlight temporal differences in the activation of the triad of executioner caspases 3, 6 and 7 during glial cell apoptosis.