Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia

Objective: The most common neurological symptom of tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) is early life refractory epilepsy. As previous studies have shown enhanced excitatory glutamatergic neurotransmission in TSC and FCD brains, we hypothesized that neurons associated with these lesions may also express altered γ-aminobutyric acid (GABA) A receptor (GABA AR)-mediated inhibition. Methods: Expression of the GABA AR subunits α1 and α4, and the Na +-K +-2Cl - (NKCC1) and the K +-Cl - (KCC2) transporters, in human TSC and FCD type II specimens were analyzed by Western blot and double label immunocytochemistry. GABA AR responses in dysplastic neurons from a single case of TSC were measured by perforated patch recording and compared to normal-appearing cortical neurons from a non-TSC epilepsy case. Results: TSC and FCD type IIb lesions demonstrated decreased expression of GABA AR α1, and increased NKCC1 and decreased KCC2 levels. In contrast, FCD type IIa lesions showed decreased α4, and increased expression of both NKCC1 and KCC2 transporters. Patch clamp recordings from dysplastic neurons in acute slices from TSC tubers demonstrated excitatory GABA AR responses that were significantly attenuated by the NKCC1 inhibitor bumetanide, in contrast to hyperpolarizing GABA AR-mediated currents in normal neurons from non-TSC cortical slices. Interpretation: Expression and function of GABA ARs in TSC and FCD type IIb suggest the relative benzodiazepine insensitivity and more excitatory action of GABA compared to FCD type IIa. These factors may contribute to resistance of seizure activity to anticonvulsants that increase GABAergic function, and may justify add-on trials of the NKCC1 inhibitor bumetanide for the treatment of TSC and FCD type IIb-related epilepsy. Copyrigh

Mechanistic target of rapamycin complex 1 and 2 in human temporal lobe epilepsy

Objective: Temporal lobe epilepsy (TLE) is a chronic epilepsy syndrome defined by seizures and progressive neurological disabilities, including cognitive impairments, anxiety, and depression. Here, human TLE specimens were investigated focusing on the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and complex 2 (mTORC2) activities in the brain, given that both pathways may represent unique targets for treatment. Methods: Surgically resected hippocampal and temporal lobe samples from therapy-resistant TLE patients were analyzed by western blotting to quantify the expression of established mTORC1 and mTORC2 activity markers and upstream or downstream signaling pathways involving the two complexes. Histological and immunohistochemical techniques were used to assess hippocampal and neocortical structural abnormalities and cell-specific expression of individual biomarkers. Samples from patients with focal cortical dysplasia (FCD) type II served as positive controls. Results: We found significantly increased expression of phospho-mTOR (Ser2448), phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), and phospho-Akt (Ser473) in TLE samples compared to controls, consistent with activation of both mTORC1 and mTORC2. Our work identified the phosphoinositide 3-kinase and Ras/extracellular signal-regulated kinase signaling pathways as potential mTORC1 and mTORC2 upstream activators. In addition, we found that overactive mTORC2 signaling was accompanied by induction of two protein kinase B–dependent prosurvival pathways, as evidenced by increased inhibitory phosphorylation of forkhead box class O3a (Ser253) and glycogen synthase kinase 3 beta (Ser9). Interpretation: Our data demonstrate that mTOR signaling is significantly dysregulated in human TLE, offering new targets for pharmacological interventions. Specifically, clinically available drugs that suppress mTORC1 without compromising mTOR2 signaling, such as rapamycin and its analogs, may represent a new group of antiepileptogenic agents in TLE patients. Ann Neurol 2018;83:311–327