RESUMEN
Hippocampal sclerosis is a common acquired disease that is a major cause of drug-resistant epilepsy. A mechanism that has been proposed to lead from brain insult to hippocampal sclerosis is the excessive generation of reactive oxygen species, and consequent mitochondrial failure. Here we use a novel strategy to increase endogenous antioxidant defences using RTA 408, which we show activates nuclear factor erythroid 2-related factor 2 (Nrf2, encoded by NFE2L2) through inhibition of kelch like ECH associated protein 1 (KEAP1) through its primary sensor C151. Activation of Nrf2 with RTA 408 inhibited reactive oxygen species production, mitochondrial depolarization and cell death in an in vitro model of seizure-like activity. RTA 408 given after status epilepticus in vivo increased ATP, prevented neuronal death, and dramatically reduced (by 94%) the frequency of late spontaneous seizures for at least 4 months following status epilepticus. Thus, acute KEAP1 inhibition following status epilepticus exerts a neuroprotective and disease-modifying effect, supporting the hypothesis that reactive oxygen species generation is a key event in the development of epilepsy.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Epilepsia/metabolismo , Epilepsia/terapia , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Animales , Animales Recién Nacidos , Anticonvulsivantes/química , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Epilepsia/inducido químicamente , Agonistas de Aminoácidos Excitadores/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glutatión/metabolismo , Ácido Kaínico/toxicidad , Proteína 1 Asociada A ECH Tipo Kelch/genética , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/genética , Ratones Transgénicos , Mutación/genética , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Ratas , Ratas Sprague-Dawley , Triterpenos/química , Triterpenos/uso terapéuticoRESUMEN
Fast inhibitory signalling in the mammalian brain is mediated by gamma-aminobutyric acid type A receptors (GABAARs), which are targets for anti-epileptic therapy such as benzodiazepines. GABAARs undergo tightly regulated trafficking processes that are essential for maintenance and physiological modulation of inhibitory strength. The trafficking of GABAARs to and from the membrane is altered during prolonged seizures such as in Status Epilepticus (SE) and has been suggested to contribute to benzodiazepine pharmacoresistance in patients with SE. However, the intracellular signalling mechanisms that cause this modification in GABAAR trafficking remain poorly understood. In this study, we investigate the surface stability of GABAARs during SE utilising the low Mg(2+) model in hippocampal rat neurons. Live-cell imaging of super ecliptic pHluorin (SEP)-tagged α2 subunit containing GABAARs during low Mg(2+) conditions reveals that the somatic surface receptor pool undergoes down-regulation dependent on N-methyl-d-aspartate receptor (NMDAR) activity. Analysis of the intracellular Ca(2+) signal during low Mg(2+) using the Ca(2+)-indicator Fluo4 shows that this reduction of surface GABAARs correlates well with the timeline of intracellular Ca(2+) changes. Furthermore, we show that the activation of the phosphatase calcineurin was required for the decrease in surface GABAARs in neurons undergoing epileptiform activity. These results indicate that somatic modulation of GABAAR trafficking during epileptiform activity in vitro is mediated by calcineurin activation which is linked to changes in intracellular Ca(2+) concentrations. These mechanisms could account for benzodiazepine pharmacoresistance and the maintenance of recurrent seizure activity, and reveal potential novel targets for the treatment of SE.