Rationale:
Hyperpolarizing Cl
- currents, mediated by γ-aminobutyric acid type A receptors (GABAARs), are critical for fast neuronal inhibition in the adult brain. The GABAAR currents are critically dependent on Cl
- gradient, which is generated by the Cl
- extrusion activity mediated by the neuronal K
+/Cl
- co-transporter KCC2. Proper activity of KCC2 is essential for the anticonvulsant efficacy of benzodiazepines (BDZ), which allosterically potentiates GABAARs. In line with its role in supporting inhibitory neurotransmission, mutations in KCC2 and compromised KCC2 activity are implicated in Status Epilepticus (SE), a medical emergency that often rapidly becomes refractory to BDZ (BDZ-RSE). We have shown that a KCC2 activator, OV350, restores efficacy of BDZs in terminating ongoing SE and decreases the seizure-associated neuronal cell death in kainic acid-induced seizure mouse model. With a complementary approach, we aim to confirm our findings with an additional phospho-mutant knock-in KCC2 T906/T1007A mouse model, in which threonine residues are mutated to alanine to prevent phospho-dependent KCC2 inactivation, resulting in enhanced KCC2 activity.
Methods:
Utilizing cortical EEG recordings, we assessed the efficacy of diazepam (DZP) to terminate SE in KCC2 T906/1007A animals, in which KCC2 activity is enhanced. Effects of KCC2 potentiation on neuronal injury and death were examined in animals that underwent kainic acid-induced SE by TUNEL, Fluoro-Jade C, and immunohistochemical stainings
Results:
Similar to animals treated with OV350, KCC2 T906/1007A animals restored the efficacy of BDZs in attenuating KA-induced SE. Reduction of Fluoro-Jade C positive neurons and microglia immunoreactivity were observed in animals administered with OV350, 48 hours following KA-induced SE, indicating its efficacy in limiting neurodegeneration and neuroinflammation. KCC2 T906/1007A animals also exhibited a lower level of microglia expression post-SE.
Conclusions:
Our results provide evidence that potentiating KCC2 activity via pharmacological intervention or genetic manipulation is efficacious in terminating BDZ-resistant seizures and in limiting neuronal injury and inflammation.
Funding:
Ovid Therapeutics and NIH NINDS (NS108378, NS111064, NS111338).