Abstracts

Rationale: Mammalian target of Rapamycin (mTOR) is a downstream effector of the PI3K/ AKT pathway that is involved in the regulation of mRNA translation and protein synthesis. In hippocampal and cortical neurons, mTOR regulates cellular and dendritic plasticity, including ion channel expression. Constitutive activation of this pathway due to mutations in the upstream modulators PTEN or TSC, has been implicated in the development of neuronal and dendritic hypertrophy, abnormal behavior and seizures in developing mice. However, little is known of the effects of seizures on mTOR pathway signaling. Therefore, we investigated the effects of prolonged seizure activity (status epilepticus; SE) on mTOR signaling in hippocampus of juvenile rats using phospho S6 as a marker of mTOR pathway activation. Methods: Sprague Dawley rats at postnatal day 30 were treated with pilocarpine to induce 1 hr of prolonged seizure activity (SE). Sham-treated animals (control) were assessed in parallel. We used western blotting (WB) and immunohistochemistry (IHC) to evaluate levels and distribution of phospho S6 (S 240/244), Ribosomal S6 protein, AKT and phospho AKT (S 473) in the hippocampus. For WB, hippocampi was harvested following 1 hr, 1-, 3-, 7- and 14 days after SE (n = 3-8 / group). For IHC, tissue was perfused with 4% PFA 1 day following SE. Results: We found that SE induced a significant and long-lasting increase in the activation of phospho S6 in the hippocampus. We found that the levels of phospho S6 are significantly increased following 1 hr of SE (~2 fold, P < 0.01) compared to controls. Phospho-S6 levels peaked 1 day after SE (~4 fold, P < 0.0001) and remained significantly increased for up to 2 weeks (~ 1 fold, P < 0.05). In contrast, we found that phospho AKT levels are significantly reduced 1 and 3 days after SE (P<0.05) compared to controls. Phospho AKT levels were not different from controls 1 week after SE. IHC confirmed the WB results and revealed that the hippocampal regions CA3, DG and slm have the strongest phospho S6 immunoreactivity compared to controls. Conclusions: Our findings reveal a significant activation of mTOR pathway signaling immediately following SE that lasts for at least 2 weeks in selective regions of the hippocampus. Future studies will be aimed at determining the role of mTOR pathway activation following SE.