Abstracts

Rationale: Hyperactivation of mTOR signaling due to mutations in regulatory genes is associated with malformations of cortical development and intractable epilepsy. mTOR regulates many key functions involved in cellular growth but it is unclear which downstream processes contributes to epilepsy. Since current mTOR inhibitors such as rapamycin can have side effects, do not fully block all of mTOR's functions, and display limited efficacy, a more specific understanding of the downstream mechanisms by which mTOR contribute to epilepsy is crucial to inform and improve treatment options. mTOR complex 1 (mTORC1) regulates many cellular processes, with the best-studied function being cap-dependent translational control. mTORC1 activation promotes cap-dependent translation via inactivation of the translational suppressor 4EBP. In pathological conditions of constitutive mTORC1 hyperactivation, proper translation is disrupted. We previously showed that normalizing cap-dependent translation via expression of constitutive active 4EBP1 (4EBP1CA) prevents mTORC1-induced cytoarchitectural abnormalities in mice, but the effects on seizures are unknown. Here, we evaluated whether reducing mTORC1-mediated increases in translation suppresses seizures in mice with hyperactive mTOR signaling.  Methods: Our lab previously developed an in utero electroporation-based mouse model in which constitutively active Rheb (RhebCA), the canonical activator of mTORC1, is expressed in developing cortical neurons, leading to persistent mTORC1 hyperactivation, focal cortical malformations, and seizures. To evaluate the effects of reducing translation in this model, we performed in utero electroporation at embryonic day (E) 15.5, targeting layer 2/3 cortical neurons. Half of the litter received a mixture of plasmids encoding RhebCA, conditional 4EBP1CA(c4EBP1CA), and CreER, while the other half (control) received conditional GFP (cGFP) instead of c4EBP1CA. Mice were treated with tamoxifen from P28 to P32 to induce expression of the conditional plasmids. The resulting effects on seizures were monitored with continuous video-EEG recording for 7 days starting at 12 weeks of age. The expression and function of the 4EBP1CA plasmid were validated by co-transfecting RhebCA and 4EBP1CA plasmids in HEK cells and performing dual luciferase reporter assays and western blots to measure cap-dependent translation and protein levels of mTOR pathway markers, respectively.  Results: 4EBP1CA expression decreased mTORC1-induced cap-dependent translation in HEK cells without altering phospho-S6 (S240/244) or phospho-AKT (S473) levels, markers of mTORC1 and mTORC2 activity, respectively (n=3 replicates, p=0.0025 by Student's t-test). Reducing translation via 4EBP1CA expression significantly reduced mTOR-induced seizures by 60% in adult RhebCA mice compared to control (n=8-14 mice/groupm, p=0.0212 by Mann-Whitney test).  Conclusions: Our findings indicate that targeting cap-dependent translation is sufficient to reduce seizures in mTOR-related epilepsy. These findings support altered cap-dependent translation as a crucial contributor to mTOR-induced epilepsy and targeting translation may be a more specific therapeutic strategy. Future studies aim to identify distinct molecules regulated by the mTORC1-4EBP1 pathway that contribute to the seizure mechanisms.  Funding: American Epilepsy Society Postdoctoral Fellowship and National Institute of Child Health and Human Development NRSA (LN).