Abstracts

Rationale: Tuberous sclerosis (TS) and cortical dysplasia (CD) are associated with hyperexcitable cytomegalic neurons which are implicated in the drug resistant epilepsy. Microarray studies on cytomegalic neurons found aberrant regulation of the extracellular regulated kinase (ERK) and mammalian target of rapamycin (mTOR) growth pathways. The interaction between ERK and mTOR is well characterized in cancer biology, however it is not well-understood in the central nervous system. In the study presented, we wanted to evaluate the ERK and mTOR pathway crosstalk in the normal and cytomegalic mouse cortical neurons using various genetic and pharmacologic techniques. Methods: Wildtype C57BL6/J mouse cortical neurons were transfected with constitutively active mitogen activated protein kinase kinase 1 / green fluorescent protein (caMEK1/GFP) to increase ERK activation. Immunocytochemistry was performed to evaluate phosphorylation (p) and activation of ERK and the ribosomal protein S6 (pS6), an mTOR-downstream target. Neurons and dendrites were traced using the Neurolucida software to assess morphology. In parallel, (tuberous sclerosis complex 1) TSC1fl/fl neurons were transduced with iCre-RFP lentivirus to knock out TSC1 in mouse cortical neurons to increase mTOR activation and generate an in vitro model of cytomegalic neurons. The TSC1 and pS6 protein staining was used to identify TSC1 knockout (TSC1-/-) neurons. Map2 staining was used to assess morphology in these cells. Rapamycin, an mTOR inhibitor, was used to manipulate mTOR activity. Studies are underway to evaluate the effects of ERK inhibitors in these models. Results: Wildtype C57Bl6/J mouse cortical neurons transfected with caMEK1/GFP showed an increase in pERK and pS6 staining, indicating increased ERK and mTOR signaling, respectively. Rapamycin decreased the levels of pS6 in caMEK1 transfected cells. The number of dendritic branches and cell size significantly decreased in the transfected neurons compared to controls (p<0.05, n=5). The soma area of TSC1-/- mouse cortical neurons significantly increased as compared to wildtype neurons (p<0.001, n=4). The effects of increased ERK and mTOR activation on morphology were reversed by rapamycin. Conclusions: ERK pathway activation in caMEK1/GFP transfected cortical neurons is associated with increased mTOR activation and decreased somatic size and dendritic branching. mTOR inhibition in these neurons reverses the aberrant phenotypes, suggesting an interplay between the ERK and mTOR signaling cascades in neurons. In addition, knocking out TSC1 in mouse cortical neurons increases mTOR activation and cell size as expected. Thus, this culture system will provide a model for screening ERK inhibitors and other compounds targeting these pathways.