Abstracts

Rationale: Temporal lobe epilepsy (TLE), the most prevalent form of epilepsy, is devastating and often refractory to treatment. Evidence from clinical and preclinical studies supports the idea that an isolated episode of prolonged seizures (status epilepticus [SE]) promotes development of TLE. Understanding the molecular mechanisms by which SE promotes TLE may provide novel targets for therapeutics for TLE. Recent studies from our laboratory demonstrate that SE-induced activation of the receptor tyrosine kinase TrkB is necessary for subsequent development of TLE. Specifically, transient inhibition of TrkB kinase initiated after SE prevents development of spontaneous recurrent seizures in mice. The objective of this study is to explore the molecular mechanisms by which TrkB promotes epileptogenesis. Electrophysiological data implicate plasticity of hippocampal excitatory synapses as a cellular mechanism of epileptogenesis. We therefore sought to elucidate the SE-induced modifications of the proteome of excitatory synapses that are dependent on TrkB activation. Methods: Using a chemical-genetic approach to pharmacologic TrkB inhibition, TrkB^F616A genetic mutant mice were given intra-amygdala kainate (KA) infusions to induce SE, and were then treated with TrkB inhibitor or vehicle. Twenty-four hours following SE, we biochemically isolated the PSD fractions from mouse hippocampi ipsilateral to KA infusion using subcellular fractionation. Purified PSD fractions were then analyzed by label-free quantitative differential proteomic analysis using multi-fraction LC/LC-MS/MS. Results: Proteomic analysis achieved high depth of protein coverage while maintaining rigorous quantitative performance; approximately 9400 unique peptides corresponding to 1450 proteins from PSD fractions were identified. Of these 1450 proteins, 192 proteins were found to have SE-induced changes in PSD protein level. SE-induced changes in the PSD proteome involved numerous pathways, including protein ubiquitination, calcium signaling, and actin cytoskeletal remodeling. Of these changes, a smaller subset of TrkB-dependent protein changes was identified. Conclusions: Characterization of synaptic proteomes provides insight into the molecular underpinnings of both global SE-induced PSD protein changes, as well as those changes that are TrkB-dependent. To our knowledge, this study represents the first attempt at using proteomic methods to elucidate the molecular mechanisms of epileptogenesis by globally analyzing the proteome of excitatory synapses. Those proteins with TrkB-dependent changes may represent potential components of a TrkB-epileptogenesis pathway, meriting further study. These proteins also represent possible targets for inhibition to prevent SE-induced epileptogenesis in the clinical setting.