Abstracts

Rationale: The mammalian target of rapamycin (mTOR) and extracellular regulated kinase (ERK) pathways are aberrantly regulated in epilepsy. Interaction between the ERK and mTOR pathways occurs at various upstream and downstream levels and is well-described in tumor biology. For example, activated ERK can phosphorylate tuberous sclerosis complex 2 (TSC2) and S6 in the mTOR pathway. However, the physiological role of ERK and mTOR pathway cross-talk in neurons is not well-understood. Furthermore, while both pathways are activated in some forms of epilepsy, it is unclear if there is ERK and mTOR cross-talk in this context. In the studies presented here we sought to further characterize ERK pathway coupling to mTOR signaling in neurons and in human brain tissue resected for treatment of drug resistant epilepsy. Methods: HEK293 cells and cortical neurons were transfected with constitutively active MEK1 (caMEK1) copGFP to elicit ERK activation or a copGFP only (control). Activation of ERK and mTOR signaling in these cells was determined using immunohistochemistry (IHC) and western blotting (WB) with antibodies against the phosphorylated forms of ERK (pERK), TSC2 (ERK phosphorylation site S664; pTSC2), and S6 (phosphorylation site S235/236; pS6). Since these pathways are implicated in dendritic remodeling, dendritic morphological analysis of transfected mouse cortical neurons also was performed using Neurolucida software. Human brain tissue sections obtained after resection from epilepsy surgery were stained with these antibodies and IHC was correlated with the neuropathological and clinical history. Results: IHC 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. 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. Furthermore, IHC analysis of brain sections obtained from individuals undergoing epilepsy surgery showed aberrant labeling with pS6, pTSC2 and pERK antibodies in areas of cortical dysplasia. Conclusions: Our findings suggest that there is ERK and mTOR pathway cross-talk at the level of TSC2 and S6 in neurons under physiologic conditions and in epilepsy. Future studies will elucidate the role of interplay between these two pathways in the CNS and may provide support for targeting both pathways in epilepsy. FUNDING SOURCES: Vivian L Smith Foundation Grant, Texas Children s Hospital; RO1NS039943; 5RO1NS49427; and 5T32NS043124