Abstracts

Rationale: Focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC) are characterized by developmental brain malformations leading to intractable epilepsy and cognitive impairments. FCD and TSC are associated with increased activation of the mTOR pathway, a key regulator of cell growth and survival. Although clinical trials have shown efficacy of mTOR inhibitors in the treatment of FCD- and TSC-related seizures, a number of patients do not respond to mTOR inhibitors. Thus, identification of novel treatment targets is needed. The MEK/ERK (MAPK) pathway is another critical regulator of cell growth and development that is aberrantly activated in human FCD and TSC and in mouse models. Similarly to, but independently of mTOR, MEK/ERK hyperactivation causes abnormal dendritic morphology that may contribute to seizures. We previously found that inhibition of the MEK/ERK pathway is sufficient to rescue dendritic defects in a mouse model of TSC. However, the effects on seizures are unknown. Here, we evaluated the efficacy of MEK/ERK inhibition on seizures in two mouse models of FCD and TSC. Methods: Cortical malformations, such as those observed in FCD and TSC, are generated by persistent activation of mTOR in a subset of developing neurons during embryonic life. We modeled this event in mice by expressing constitutively active Rheb (Rheb^CA), the canonical activator of mTOR, in mouse embryos via in utero electroporation. Adult Rheb^CA mice were recorded with continuous video-EEG for 5 days to establish pre-treatment baseline activity. Mice were then treated with the MEK/ERK inhibitor PD0325901 (5 mg/kg) or vehicle for 10 days. Video-EEG activity was continuously recorded during treatment. In a parallel study, TSC1 conditional knockout (cKO: TSC1^flox/flox; GFAP-Cre⁺) mice were treated with PD0325901 (6 mg/kg) or vehicle daily for 4 weeks, starting at 3 weeks of age. Video-EEG activity was continuously recorded during treatment. Brain tissue from treated Rheb^CA and TSC1 cKO mice was collected and evaluated with western blotting to assess the effects of PD0325901 on MEK/ERK activity, using ERK1/2 phosphorylation level as readout of MEK/ERK activation. Results: PD0325901-treated Rheb^CA mice displayed significantly lower seizure frequency compared to pre-treatment baseline (p=0.02, n=5 mice). In comparison, no differences in seizure frequency were observed in vehicle-treated Rheb^CA mice before or during treatment (p=0.40, n=5 mice). Similarly, PD0325901-treated TSC1 cKO mice displayed significantly fewer seizures compared to vehicle-treated TSC1 cKO mice (p=0.01, n=14-16 mice/group). Brain protein levels of phosphorylated ERK1/2 in PD0325901-treated Rheb^CA and TSC1 cKO mice were significantly decreased compared to their respective vehicle-treated controls (Rheb^CA: p=0.01, n=5 mice/group; TSC1 cKO: p<0.001, n=7-14 mice/group), indicating inhibition of MEK/ERK activity. Additionally, there was a significant correlation between the level of MEK/ERK activation and seizure frequency (p=0.03). Conclusions: Our studies support inhibition of the MEK/ERK pathway as an effective treatment for seizures associated with FCD and TSC. Future studies aim to investigate the efficacy of combined MEK/ERK-mTOR inhibitor therapy for these disorders. Funding: NIH-R01 NS086329, Tuberous Sclerosis Alliance