Abstracts

Rationale: The most common neurological symptom of tuberous sclerosis complex (TSC) is early-life epilepsy. TSC-related seizures are often refractory to conventional antiepileptic drugs (AED) and require surgical excision. Although previous human and animal model studies have shown that excitatory glutamatergic neurotransmission is enhanced in TSC brain, the possible role of altered GABA-ergic signaling in seizure pathogenesis in these patients has not been fully evaluated. Hyperpolarizing GABA receptors (GABARs) can become depolarizing, depending on the intracellular chloride concentration, regulated by the chloride-importer NKCC1 and the chloride-exporter KCC2. In addition, altered GABAR subunit composition may result in lack of modulation by benzodiazepine. Here we hypothesized that dysplastic neurons in cortical tubers would demonstrate higher NKCC1 and lower KCC2 expression compared to control neurons, a profile that is consistent with the presence of depolarizing GABARs. We also hypothesized that GABAR subunit expression would be altered in tuber vs. control tissue. Methods: Cortical tubers (n=5) were obtained following brain surgery for pharmacoresistant epilepsy. Autopsy cortical samples from cases with normal neurologic history were used as controls (n=4). Tissue was collected and handled in accordance with the Clinical Research Committee at Children’s Hospital, Boston. Paraformaldehyde-fixed tissue was cut at 50 μm and immunocytochemically double labeled with neuronal or glial markers and NKCC1 (1:100) or KCC2 (1:500) antibodies. For western blotting, frozen cortex was used to separate the membrane fraction. Blots were incubated with NKCC1 (1:200), KCC2 (1:500), and GABAR subunits α 1 (1:500) and α 2 (1:500). All primary antibodies were purchased from Millipore. Results: Double labeling of TSC and control tissue demonstrated that, relative to control neurons, there was a marked increase in NKCC1 levels, associated with a decrease in KCC2 expression in most normal-sized and enlarged dysplastic neurons within the tubers. Notably, strong NKCC1 immunoreactivity was also observed in tuber giant cells and dysplastic astroglia. NKCC1 and KCC2 protein quantification demonstrated significantly higher NKCC1 (217% of control) and lower KCC2 (39% of control) levels in TSC samples vs. controls (t-test, p<0.05). The expression of GABAR subunits was also significantly altered in tubers vs. controls, with lower α 1 (26% of control) and higher α 2 (284% of control) expression levels (t-test; p<0.05). Conclusions: Our results demonstrate an imbalance in neuronal NKCC1 and KCC2 expression (increased NKCC1: KCC2 ratio) in human cortical tubers that may render GABARs depolarizing, as well as altered GABAR subunit expression (decreased α 1: α 2 ratio), consistent with decreased benzodiazepine sensitivity. These mechanisms may play a critical role in the seizure susceptibility and relative seizure refractoriness to GABAR agonists, and may represent new targets for therapeutic intervention in TSC. Funded by: TSA, Epilepsy Fdn, Lombroso Trust,Neurosurgical Fdn, NIH NS31718.