INTRODUCTION: Epilepsy is a chronic neurological disorder featuring recurrent and unprovoked seizures. GABA inhibition in adult CNS play an important role in epileptogenesis. GABA AR-mediated inhibition depends on the maintenance of the low intracellular [Cl-] concentration, which is mainly regulated by both Na+-K+-Cl- cotransporter-1 (NKCC1) and K+-Cl- cotransporter-2 (KCC2) in neurons. Previous studies have shown that both co-transporters are regulated by the WNK-SPAK-KCC2 signaling pathway and play important role in pathological states. ZT-1a is a selective SPAK inhibitor, supposed to negatively modulate NKCC1 and positively modulate KCC2 expression.

AIM(S): This study aims to explore whether ZT-1a can interrupt the epileptogenesis through inhibiting WNK-SPAK-NKCC1/KCC2 signaling pathway.

METHOD(S): We applied ZT-1a in either in vivo, pentylenetetrazol and pilocarpine models or in vitro cyclothiazide- induced cultured hippocampal neuron model. The changes of correlated signaling pathway proteins and GABAAR-mediated inhibition were investigated by a series of experiment such as Western Blots, electrophysiology recordings, immunostaining, etc. to provide theoretical basis for the potential therapeutic effect of ZT-1a.

RESULTS: We discovered that ZT-1a, (1) attenuated CTZ-induced epileptiform bursting activities in primary cultured hippocampal neurons, (2) decreased the seizure susceptibility to PTZ stimulation and alleviated pilocarpine-induced chronic spontaneous seizures by reversing seizure induced KCC2 down regulation as well as NKCC1 upregulation, and (3) the suppressive effect of ZT-1a on epilepsy attributed to the inhibiting of the WNK-SPAK phosphorylation during seizure activities.

CONCLUSIONS: Our current study demonstrated that ZT-1a inhibition of WNK-SPAK pathway capable of simultaneously preventing KCC2 deficit and NKCC1 enhancement during pathological stimulation related epileptogenesis. In conclusion, WNK-SPAK inhibitor ZT-1a may serve as a lead compound for future anti-seizure drug development.