Abstracts

Rationale:
Tuberous Sclerosis Complex (TSC) is a genetic disorder characterized by brain malformations and severe epilepsy in up to 90% of patients. Mutations in TSC1 or TSC2 genes that encode the proteins hamartin (TSC1) and tuberin (TSC2), respectively, result in dysregulation of the mTOR pathway. Brain malformations are associated with areas of aberrant neuronal hyperexcitability and thus are highly epileptogenic. mTOR inhibitors are promising therapeutic agents and are predicted to function as anti-epileptogenic agents. However, clinical studies demonstrate that mTOR inhibitors function as anti-convulsants or epileptostatics by reducing seizure frequency, indicating seizure generating mechanisms remain intact despite targeting mTOR activity. Here we treated a mouse model of TSC with the mTOR inhibitor RAD001 (Everolimus) and tracked EEG activity during treatment. Furthermore, to explore potential mechanisms of mTOR inhibitor resistance in circuit behavior we have begun pilot experiments using the octuplet patch technique in cortical slices from KO mice.
Method:
Littermate mouse pups with conditional forebrain deletion of the Tsc2 gene in excitatory neurons [Wild type, (WT) NEX-Cre+/Tsc2WT/WT; Knockout (KO), NEX-Cre+/Tsc2flox/flox] were treated with vehicle or 6mg/kg RAD001 by intraperitoneal route every other day starting on postnatal day 8 (P8). Video electroencephalopgraphy (vEEG) activity was recorded (Natus Nicolet). Offline EEG analysis of epileptiform and seizure activity was carried out using LabChart v8 (ADInstruments). Slices from these mice were prepared and octuplet patch recordings obtained. Quadro EPC 10 amplifiers, PatchMaster software (HEKA), and custom-written Matlab-based programs were used to operate the recording system and perform online and offline analysis of the electrophysiology data to determine connectivity and synchronicity.
Results:
vEEG recordings revealed Tsc2 KO mice treated with the mTOR inhibitor RAD001 beginning early in postnatal development exhibit a significant delay in development of seizures from P12 to approximately P50. During inhibitor treatment there is a seizure-free interval of normal EEG activity followed by an incremental rise in epileptiform activity leading up to the appearance of seizures by P50 which is markedly delayed compared to the untreated group. In multi-whole cell patch recordings we found excitatory neurons from Tsc2 KO mice have increased connectivity and inhibitory neurons have excitatory properties, which is different from WT.
Conclusion:
Our results in NEX-Tsc2 mutant mice suggest that, as in TSC humans, RAD001 is suppressing seizures without a long-lasting disease modifying effect. Examination of aberrant circuit behavior may help reveal mechanisms of drug resistance and seizure recurrence.
Funding:
:Ruth L. Kirschstein NRSA Training Grant for Multidisciplinary Training in Brain Disorders and Development