Abstracts

Rationale:
Tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) are developmental cortical malformations that represent major causes of pediatric refractory epilepsy. TSC and FCDII are caused by hyperactive mTOR signaling, a master regulator of cell growth and development, but the mechanisms of epilepsy remain unclear. One function of mTOR is to promote mRNA translation by phosphorylating and inactivating the translational suppressor 4EBP1/2. Here, we examined whether expression of constitutively active 4EBP1 to reduce exaggerated translation attenuates epilepsy using an in utero electroporation-based mouse model of TSC and FCDII. We also evaluated whether expression of 4EBP1 decreases the ectopic upregulation of HCN4 channels that we previously identified as a critical driver of hyperexcitability and seizures in this model.
Method:
We expressed constitutively active Rheb (RhebCA), the canonical mTOR activator, in developing mouse neurons via in utero electroporation to activate mTOR and verified 4EBP1/2 phosphorylation by immunostaining. To target mTOR/4EBP1/2-mediated translation, we co-expressed RhebCA and a tamoxifen-inducible, constitutively active form of 4EBP1 (conditional 4EBP1CA, c4EBP1CA) that resists inactivation by mTOR. Symptomatic mice received tamoxifen at 1 month of age and underwent 7-day continuous video-EEG recording at 3 months of age. Brain tissue was collected afterward for evaluation of HCN4 expression by immunostaining. HCN function was assessed by whole-cell slice electrophysiology recording.
Results:
RhebCA mice displayed TSC- and FCDII-like cortical malformations and 4EBP1 hyperphosphorylation compared to control GFP mice (n=3/group, p=0.0071). c4EBP1CA expression attenuated mTOR-induced seizures by 56% in RhebCA+c4EBP1CA (n=18) compared to RhebCA mice (n=11, p=0.0260), with no seizures detected in 8/18 RhebCA+c4EBP1CA mice. c4EBP1CA expression also reduced aberrant HCN4 expression within the cortical malformations by 60% (p=0.0004) and aberrant HCN current amplitudes by 77% (n=14-18 neurons/group, p=0.0001) in RhebCA+c4EBP1CA compared to RhebCA mice and neurons, respectively.
Conclusion:
Our findings support altered mTOR/4EBP1/2-regulated translation as a crucial contributor to epilepsy in TSC and FCDII. Targeting aberrant translation may represent a novel therapeutic strategy for these disorders.
Funding:
:American Epilepsy Society Postdoctoral Fellowship (LHN), NIH-NICHD F32-HD095567 (LHN), NIH-NINDS R01-NS086329 (AB)