Abstracts

Rationale: Cortical dysplasia (CD) is commonly associated with drug resistant epilepsy and microarray studies of cytomegalic neurons found in CD have shown activation of mammalian target of rapamycin (mTOR) pathway. The cause of mTOR pathway hyperactivity is not known but one possible explanation for hyperactivation of mTOR pathway is phosphorylation of its upstream regulator, TSC2, at the S664 site by extracellular regulated kinase (ERK). Though the interaction between ERK and mTOR is well characterized in cancer biology, it has not yet been evaluated in neurons. In the studies presented, we sought to characterize ERK and mTOR pathway activation and interaction in epilepsy. Methods: Human brain tissue sections obtained after resection from epilepsy surgery were stained with antibodies against phosphorylated ERK (pERK), TSC2 (ERK phosphorylation site S664; pTSC2), eIF4E (ERK phosphorylation site S209; peIF4E) and S6 (phosphorylation site S235/236; pS6). Staining data was correlated with the neuropathological diagnosis and clinical history. Furthermore, we evaluated mTOR pathway activation subsequent to ERK phosphorylation in HEK293 cells and mice cortical neurons along with its effect on neuronal morphology by transfecting them with constitutively active MEK1 (caMEK1) copGFP. Activation of ERK and mTOR signaling in these cells was determined using immunofluorescence (IF) and western blotting (WB) against above mentioned phosphoantibodies. Dendritic morphological analysis of transfected mouse cortical neurons also was performed using Neurolucida software. Results: IHC analysis of brain sections obtained from individuals undergoing epilepsy surgery showed aberrant pERK, pTSC2, peIF4E and pS6 labeling of cytomegalic neurons. IF and WB showed that activation of ERK correlates with increased levels pTSC2 and pS6 in HEK293 cells (WB: p<0.001, n=5) and mouse cortical neurons. A pilot experiment of dendritic morphological analyses of cultured neurons with high levels of pERK showed a decrease in the number of dendrites (p<0.001, n=5) and dendritic branching (p<0.001, n=5) compared to controls expressing copGFP. This effect was reversed by 200nM rapamycin treatment of cultures for 4 days. Conclusions: Our findings suggest that ERK and mTOR pathways are aberrantly activated in cortical dysplasia. Additionally, there is ERK and mTOR pathway cross-talk at the level of TSC2 in neurons under physiologic conditions and in epilepsy. Preliminary studies suggesting interplay between these two pathways in the central nervous system may provide support for targeting both pathways in epilepsy.