Abstracts

Rationale: Activation of mTOR signaling occurs during epileptogenesis and contributes to mossy fiber sprouting. mTOR activation in epilepsy has been shown to occur through PI3K-Akt pathway; however, there is a lack of data concerning upstream signaling. Neurons express several receptor tyrosine kinases (RTKs) as well as Insulin-like Growth Factor 1 (IGF-1) and insulin receptors. We tested the involvement of these receptors in epileptogenesis and activation of PI3K-Akt-mTOR pathway. Methods: We applied receptor inhibitors or ligands to chronic organotypic hippocampal culture model of epilepsy, starting from 3 days in vitro (DIV). We used assays of lactate and lactate dehydrogenase (LDH) concentrations in culture supernatant as markers of epileptiform activity and neural death, respectively. We verified results with electrical field potential recordings and Nissl- or immunostained neuron counts. Protein phosphorylation levels were analyzed by Western Blotting.Results: We found that addition of IGF-1 and insulin significantly contributed to duration of electrographic seizures and seizure-dependent cell death. Further, we found that inhibition of IGF-1 and insulin receptors significantly decreased electrographic seizures and cell death. We also found that, during latent period of epileptogenesis, activation of Akt-mTOR signaling, assessed by phosphorylation level of Akt and S6 proteins, was significantly increased by both IGF-1 and insulin.Conclusions: IGF-1 and insulin receptors play a significant role in activation of Akt-mTOR signaling and contribute to epileptogenesis in organotypic hippocampal culture model of epilepsy.