Abstracts

Rationale: Many brain insults, including TBI, stroke and status epilepticus (SE), are followed by the development of epilepsy. These epilepsies frequently progress over time and no treatments are available to prevent progression. In animal models of epilepsy, signaling pathways, such as JAK/STAT and MTOR, and proteins, such as BDNF, are activated. It is less known, however, if these same pathways are activated in brains of human patients with epilepsy. Understanding the molecular basis of epilepsies may facilitate identification of new therapeutic targets. To this end, we are profiling pathway perturbations in a mouse model of epilepsy and comparing them to those in brain tissue from pediatric patients with medically intractable epilepsy.Methods: Male FVB mice were injected with scopolamine followed by pilocarpine to induce SE or with saline to serve as controls. Mice (n=5 at each time point) were sacrificed at onset of SE, at 1, 6, and 24 hours after SE, and during the epileptogenic and chronic periods (when animals were exhibiting spontaneous seizures). Protein lysates were prepared by standard methods from hippocampus, cortex and cerebellum of mouse brains, and from 152 samples surgically resected from 84 patients. Protein levels were quantified using Reverse Phase Protein Arrays, where lysates are robotically printed onto nitrocellulose–coated microscope slides and screened with antibodies of interest in a protocol similar to that for Western blots.Results: Levels were quantified for ~50 protein/protein modifications, including components of the MTOR, JAK/STAT, JNK, MAPK and apoptotic pathways, and subunits of ionotropic glutamate receptors. In hippocampus and cortex from mice, strongest perturbations of the MTOR pathway, including levels of pMTOR, pP70S6, pS6 and RAPTOR, occurred at onset, at 24 hours and during the latent phase; levels of STAT3, pJNK, pCAMKII and pERK were also strongly elevated. In cerebellum, perturbations were extensive but less severe and largely occurred at onset and in the chronic phase. In human samples, levels of MTOR pathway components were highly correlated in hippocampus, and were correlated with MAPK components, BRAF, pELK and RSK in both hippocampus and temporal lobe. No gender differences in perturbations were detected, but age differences were noted in particular for levels of pMTOR which were significantly higher in patients >10 years vs. <5 years old.Conclusions: Many signaling pathways activated in the pilocarpine model of epilepsy overlap with those activated in human patients. This is observed in spite of the variability in age, type of epilepsy and clinical history associated with patient samples. Correlations seen between components of MTOR and MAPK suggest the involvement MAPK in epilepsy. These data suggest a novel line of research to investigate potentially druggable targets in the treatment of epilepsy.