Abstracts

Rationale: Focal cortical dysplasia (FCD), a malformation of brain development containing dysplastic neurons and glia, is the most common cause of intractable focal epilepsy in children. Cancer-associated mosaic mutations in kinases comprising the intracellular PI3K/AKT/MTOR pathway have been identified in a significant proportion of FCD brain specimens. In this study, we analyzed the functional consequences of neuronal expression of these activated kinases.Methods: Neuronal cultures were prepared from wild-type embryonic day 16 C57BL/6 mouse cortex. These cultures were transfected with plasmids expressing constitutively active forms of PIK3CA, AKT3, or MTOR along with GFP. At defined time points after transfection, neuronal size, morphology, and expression of phosphorylated PI3K/AKT/MTOR pathway members were assessed. In some experiments, cultures were treated with 10 nM of the mTORC1 inhibitor rapamycin after transfection.Results: Expression of the PIK3CA, AKT3, and MTOR mutant kinases induced changes in neuronal morphology and size which were most prominent with constitutively-active AKT3. These findings mirrored our previous studies of dysplastic human cortex containing mosaic mutations. While all of the activated kinases caused increases in expression of phosphorylated ribosomal protein S6 (phospho-S6), activated PIK3CA additionally caused prominent PDK1 phosphorylation while activated AKT3 promoted its own phosphorylation. The clinically-identified MTOR S2215Y mutation induced selective upregulation of mTORC1 signaling without significant effect on mTORC2. Treatment of transfected cultures with rapamycin decreased phosho-S6 expression and cell size but did not reverse increases in AKT phosphorylation.Conclusions: Expression of activated kinases of the PI3K/AKT/MTOR pathway in neuronal cultures recapitulates many of the morphologic and functional phenotypes identified in epileptogenic cortical dysplasia. While some of these phenotypes are common to all pathway members, some diverge based on the specific protein and mutation. Treatment with the mTORC1 inhibitor rapamycin can reverse some, but not all, of these phenotypes. These findings have significant implications for potential targeting of therapies for epilepsy due to focal cortical dysplasia. Funding for this study was provided to L.A.J. by the CURE Pediatric Epilepsies Award and by NIH NINDS award K02 NS072162.