Abstracts

Rationale: Focal cortical dysplasia (FCD) is the most common cause of intractable focal epilepsy in children. Genetic mutations resulting in increased activity of the intracellular PI3K/AKT/MTOR pathway have been identified in a significant proportion of individuals with FCD. In this study, we utilized a cortical neuronal culture model to investigate the efficacy of different selective PI3K/AKT/MTOR pathway inhibitors in correcting the morphologic and functional phenotypes induced by expression of constitutively active forms of AKT3. Methods: Neuronal cultures were prepared from wild-type embryonic day 16 C57BL/6 mouse cortex. These cultures were transfected with plasmids expressing myristolated AKT3 (myr-AKT3) or AKT3 E17K (mutation identified in multiple FCD/HMEG patients). Expression of either mutant form of AKT3 causes constitutive activation of the MTOR pathway. At defined time points after transfection, neuronal size, morphology, excitability, and expression of phosphorylated PI3K/AKT/MTOR pathway members were assessed. Cultures were treated with either the mTORC1 inhibitor rapamycin, the PI3K inhibitor BKM-120, the allosteric AKT inhibitor MK 2206, or the ATP-competitive AKT inhibitor AZD5363. Results: Neurons transfected with myr-AKT3 or AKT3 E17K demonstrated morphologic features similar to dysmorphic neurons present in epileptogenic human cortical dysplasia specimens, including enlarged cell size, irregular cell borders, and increased dendritic complexity. Increased expression of phosphorylated PI3K/AKT/MTOR pathway members was detected in these cells, including p-S6, p-GSK3?, T308 p-AKT, and S473 p-AKT. The excitability of activated AKT-transfected and control neurons after exposure to a low magnesium solution was compared via imaging using the fluorescent calcium indicator GCaMP6f. We found that the mTORC1 inhibitor rapamycin and the ATP-competitive AKT inhibitor AZD5363 reversed both the cell size phenotype and upregulation of p-S6 in neurons expressing myr-AKT3 or AKT3 E17K. However, these parameters were not affected by treatment with either the allosteric AKT inhibitor MK 2206 or the PI3K inhibitor BKM-120. The inhibitors also had differential effects on expression levels of other phosphorylated PI3K/AKT/MTOR pathway members, as well as on dendritic complexity and neuronal excitability. Conclusions: Expression of activated AKT3 in neuronal cultures recapitulates many of the morphologic and functional phenotypes identified in epileptogenic cortical dysplasia. We have found that the response to treatment of these cultures with PI3K/AKT/MTOR pathway inhibitors depends not only on the kinase targeted by the inhibitor, but also on the specific mechanism of action of the inhibitor. These findings have significant implications for potential targeting of therapies for epilepsy due to focal cortical dysplasia, and also provide a rapid, in vitro means by which these therapies can be tested on neurons expressing the specific mutation identified in each individual patient. Funding: Funding for this study was provided to L.A.J. by the CURE Pediatric Epilepsies Award.