KCC2 Dysfunction Enhances Synaptic Excitability of Cytomegalic Neurons in TSC

More than 50% of tuberous sclerosis complex (TSC) patients develop seizures that are resistant to anti-epileptic medications. Seizures are neurological conditions characterized by abnormal electrical activity in the brain and decreased inhibition mediated by type A gamma-aminobutyric acid (GABA) receptors (GABAARs). The polarity and efficacy of GABAARs are regulated by the neuronal specific chloride (Cl-) transporter KCC2, which extrudes Cl- and maintains low intraneuronal Cl- concentration essential for GABA mediated inhibition. KCC2 has been recently found to be severely diminished in the brains of people suffering from drug-resistant epilepsy. In TSC patients, KCC2 levels are low in cortical tubers, and tuber neurons show excitatory GABAR responses in contrast to hyperpolarizing GABA inhibition in neurons from non-TSC case controls. It remains elusive whether altered KCC2 levels and GABAergic transmission may arise as a cause for or a consequence of seizure activities in TSC.

This project will address if impaired KCC2 function underlies the hyperexcitability of tuber neurons and epileptogenesis in TSC. We have generated a novel TSC1KO mouse model in which Tsc1 inactivation in late embryonic radial glial cells produces TSC tuber like cytomegalic neurons (CMNs), leading to spontaneous behavioral seizures. We found that KCC2 was dysregulated in cytomegalic neurons, leading to increased intraneruonal Cl- concentration and impaired GABA-mediated hyperpolarization. Therefore, we hypothesize that impaired KCC2-mediated Cl- extrusion and GABA inhibition in cytomegalic neurons may contribute to synaptic hyperexcitability and the relative refractoriness of GABAergic antiepileptic drugs in TSC, e.g., vigabatrin. To test this hypothesis, we propose three project aims. Aim 1 will determine the functional dysregulation of KCC2 in CMNs at a range of ages, before and after the onset of seizures. Aim 2 will examine whether GABA paradoxically depolarizes and excites CMNs. Aim 3 will address whether enhancing KCC2 in CMNs rescues impaired GBAB inhibition and ameliorates synaptic hyperexcitability and refractoriness to AEDs. These experiments will be the first demonstration in TSC model mice that KCC2 dysfunction underlies epileptogenesis and the development of drug-resistant seizures. Our findings will suggest that rescuing KCC2 function could restore the therapeutic effectiveness of current anti-epileptic drugs, and lay the foundation for future assessment of KCC2 enhancers as a novel therapeutic strategy for TSC patients suffering from drug-resistant seizures.