Region-specific alterations of AMPA receptor phosphorylation and signaling pathways in the pilocarpine model of epilepsy Mark William Lopes a , Samantha Cristiane Lopes b , Ana Paula Costa c , Filipe Marques Gonçalves a , Débora Kurrle Rieger a , Tanara Vieira Peres c , Helena Eyng a , Rui Daniel Prediger b,c , Alexandre Paim Diaz d , Jean Costa Nunes d , Roger Walz c,d,e , Rodrigo Bainy Leal a,c, * a Programa de Pós-graduação em Bioquímica, Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil b Programa de Pós-graduação em Farmacologia, Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil c Programa de Pós-graduação em Neurociências, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil d Centro de Neurociências Aplicadas (CeNAp), Hospital Universitário (HU), Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil e Departamento de Clínica Médica, Hospital Universitário (HU), Centro de Ciências da Saúde, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil ARTICLE INFO Article history: Received 26 January 2015 Received in revised form 6 May 2015 Accepted 7 May 2015 Available online Keywords: Epilepsy Pilocarpine Hippocampus AMPA receptor phosphorylation Signaling pathways A B ST R AC T Disturbances in glutamatergic transmission and signaling pathways have been associated with tempo- ral lobe epilepsy (TLE) in humans. However, the profile of these alterations within specific regions of the hippocampus and cerebral cortex has not yet been examined. The pilocarpine model in rodents repro- duces the main features of TLE in humans. The present study aims to characterize specific alterations of the glutamatergic transmission and signaling pathways in the dorsal (DH) and ventral hippocampus (VH) and temporal cortex (Ctx) of male adult Wistar rats 60 days after pilocarpine treatment (chronic period). The western blotting analyzes show a decrease of AMPA glutamate receptor subunit (GluA1)-Ser 845 phos- phorylation; reduction of ERK1 and PKA activity; up-regulation of GFAP and down-regulation of the glutamate transporter EAAT2 expression in the DH. In contrast, in the VH it was observed a decrease of GluA1-Ser 831 phosphorylation and JNKp54 and PKC activity. In the Ctx, only ERK1 phosphorylation/ activity decreased. The level of GluA1-Ser 845 phosphorylation and PKA activity (DH) and the level of GluA1- Ser 831 phosphorylation and PKC activity (VH) appear to be correlated, respectively. These findings suggest a differential imbalance of the signaling pathways involved in the site-specific phosphorylation of AMPA receptor in the hippocampus. Furthermore, we suggest that dorsal hippocampus is probably more sus- ceptible to the impairment of glutamate uptake and gliose, since only this area displayed a significant decrease of EAAT2 and increment of GFAP. Taken together, our study suggests that specific neurochemi- cal alterations take place in hippocampal sub regions. This approach may be valuable for understanding the onset of seizures and the alterations of neuronal excitability in specific regions and may help to es- tablish therapeutic targets for treatment of this neuropathology. © 2015 Elsevier Ltd. All rights reserved. Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; APS, ammonium persulfate; BSA, bovine serum albumin; CaMKII α and β, Ca 2+ -calmodulin-dependent protein kinases II α and β; CaN, calcineurin; CNS, central nervous system; Ctx, temporal cortex; DH, dorsal hippocampus; EAAT1 and 2, excitatory amino acid transporters 1 and 2; ERK 1/2, extracellular signal-regulated kinases 1 and 2; GABA, γ-aminobutyric acid; GFAP, glial fibrillary acidic protein; GluA1-Ser 831 , glutamate receptor subunit serine 831; GluA1-Ser 845 , glutamate receptor subunit serine 845; HRP, horseradish peroxidase; JNK1/2/3, c-Jun amino-terminal kinases 1–3; LumiGLO, luminol chemiluminescent substrate; MAPKs, mitogen-activated protein kinases; NMDA, N-methyl-D-aspartate; NMDAR, N-methyl-D-aspartate receptor; OD, optic density; p38 MAPK α, β, γ and δ, p38 mitogen-activated protein kinase α, β, γ and δ; PKA, cAMP- dependent protein kinase; PKC, protein kinase C; PP1, protein phosphatase 1; PP1ca, protein phosphatase 1 (catalytic subunit); PP2A, protein phosphatase 2A; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SE, Status epilepticus; SRS, spontaneous recurrent seizures; TBS-T, Tris-buffered saline with Tween; TBS, Tris- buffered saline; TLE, temporal lobe epilepsy; VH, ventral hippocampus. * Corresponding author. Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil - 88040-900. Tel.: +55 48 37215045; fax: +55 48 3721 9672. E-mail address: rbleal@gmail.com (R.B. Leal). http://dx.doi.org/10.1016/j.neuint.2015.05.003 0197-0186/© 2015 Elsevier Ltd. All rights reserved. Neurochemistry International ■■ (2015) ■■–■■ ARTICLE IN PRESS Please cite this article in press as: Mark William Lopes, Samantha Cristiane Lopes,Ana Paula Costa, Filipe Marques Gonçalves, Débora Kurrle Rieger, Tanara Vieira Peres, Helena Eyng, Rui Daniel Prediger, Alexandre Paim Diaz, Jean Costa Nunes, Roger Walz, Rodrigo Bainy Leal, Region-specific alterations of AMPA receptor phosphorylation and signaling pathways in the pilocarpine model of epilepsy, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.05.003 Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci