Phosphorylation of translation initiation factor eIF2a in the brain during pilocarpine-induced status epilepticus in mice Larissa S. Carnevalli a,1 , Ca ´tia M. Pereira a,1 , Beatriz M. Longo b,1 , Carolina B. Jaqueta b , Marcelo Avedissian b , Luiz Euge ˆnio A.M. Mello b , Beatriz A. Castilho a, * a Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de Sa ˜o Paulo, Rua Botucatu 862, 58 andar, Sa ˜o Paulo, SP 04023-062, Brazil b Departamento de Fisiologia, Escola Paulista de Medicina, Universidade Federal de Sa ˜ o Paulo, Rua Botucatu 862, 38 andar, Sa ˜o Paulo, SP 04023-062, Brazil Received 26 August 2003; received in revised form 15 December 2003; accepted 15 December 2003 Abstract In this work, we show extensive phosphorylation of the alpha subunit of translation initiation factor 2 (eIF2a) occurring in the brain of mice subjected to 30 min of status epilepticus induced by pilocarpine. eIF2a(P) immunoreactivity was detected in the hippocampal pyramidal layer CA1 and CA3, cortex layer V, thalamus and amygdala. After 2 h of recovery, there was a marked decrease in total brain eIF2a(P), with the cortex layer V showing the most pronounced loss of anti-eIF2a(P) labeling, whereas the CA1 subregion had a significant increase in eIF2a(P). These results indicate that inhibition of protein synthesis in experimental models of epilepsy might be due to low levels of eIF2-GTP caused by the phosphorylation of eIF2a, and suggest that translational control may contribute to cell fate in the affected areas. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: eIF2a phosphorylation; Epilepsy; Pilocarpine; Translation initiation A drastic reduction in brain protein synthesis during status epilepticus (SE) has been previously shown in experimen- tal models of epilepsy, with polysomal profiles suggesting defects in translation initiation [4,6,18]. Protein synthesis initiation can be affected by the activity or availability of initiation factors, mainly eIF2 and eIF4F (reviewed in ref. [3]). Translation initiation factor 2 (eIF2) forms a complex with GTP and Met-tRNA Met , which then binds to the 40S ribosomal subunit along with other factors. This pre- initiation complex associates with the heterotrimeric factor eIF4F, bound to the 5 0 cap structure on the mRNA through its eIF4E subunit, and initiates scanning of the message until an AUG codon is encountered. At this point GTP is hydrolyzed to GDP, with the release of eIF2-GDP and other initiation factors. The eIF2-bound GDP is exchanged to GTP by eIF2B. eIF2 in mammals is the target of four known kinases, GCN2, HRI, PKR and PEK/PERK, which phosphorylate specifically the Ser 51 residue of its alpha subunit (eIF2a), and are activated by amino acid starvation, low levels of heme, double stranded RNA and accumulation of unfolded proteins in the endoplasmic reticulum, respectively. The phosphorylated form of eIF2 [eIF2a(P)] acts as a competitive inhibitor of eIF2B, thus lowering or blocking translation initiation. In the brain ischemia-reperfusion model, phosphoryl- ation of eIF2a is observed in selected vulnerable neurons, and has been correlated with the activation of PERK [7]. In this work, we investigated the phosphorylation status of brain eIF2a following the induction of temporal lobe epilepsy by pilocarpine in mice [2]. This study was conducted under protocols approved by the Animal Care and Use Ethic Committee of the Universidade Federal de Sa ˜o Paulo, in accordance with the Guide for Care and Use of Laboratory Animals adopted by the National Institutes of Health. Seizures were induced in male Swiss albino mice (20–30 g) by i.p. injections of pilocarpine hydrochloride (200 mg/kg, Merck, Quimitra, Brazil). For most animals, seizures progressed to SE within approximately 10 min after pilocarpine injection. SE occurrence in the pilocarpine model has been extensively characterized and previously defined in both rats and mice 0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.093 Neuroscience Letters 357 (2004) 191–194 www.elsevier.com/locate/neulet 1 L.S.C., C.M.P. and B.M.L. contributed equally to this work. * Corresponding author. Tel.: þ55-11-5576-4537; fax: þ 55-11-5571- 6504. E-mail address: bac@ecb.epm.br (B.A. Castilho).