Enhancing KCC2 function reduces interictal activity and prevents seizures in mesial temporal lobe epilepsy

The neuronal K/Cl cotransporter KCC2 controls intraneuronal chloride and subsequently the efficiency of GABA signaling. In many neurological disorders, including mesial temporal lobe epilepsy (mTLE), reduced KCC2 expression or function may lead to depolarizing GABA signaling, which may then contribute to pathological activity and seizures. Compensating for the dysregulation of chloride transport in the pathology therefore appears to be a promising therapeutic strategy. Two small molecules - prochlorperazine (PCPZ) and CLP-257 - recently identified from library screening as candidate KCC2 enhancers, appear to improve symptoms in animal models of disorders associated with KCC2 extinction. However, their mode of action in cortical neurons and their therapeutic potential in epilepsy remain elusive and even controversial. Here, we show in rat hippocampal neurons that PCPZ and CLP-257 act by increasing KCC2 function and clustering while decreasing its membrane diffusion, independent of phosphorylation of canonical regulatory residues. Consistently, both compounds suppress spontaneous interictal-like discharges in postoperative tissue from patients with mTLE. Chronic administration of PCPZ also reduces seizure frequency and interictal activity in a mouse model of mTLE. These results unravel the mechanism of action of two KCC2 enhancers and validate their therapeutic potential in mesial temporal lobe epilepsy. ### Competing Interest Statement JCP, FD and ZA are co-inventors on the patent: Methods and Pharmaceutical Compositions for Treating Refractory Epilepsy. WO2022219021. 2022 October. All other authors declare that they have no competing interests. * CCCs : cation-chloride cotransporters CNS : central nervous system DMSO : dimethylsulfoxide D2R : dopamine receptor D2 ECoG : electrocorticography FDA : Food and Drug Administration GABA : γ-Aminobutyric acid HFOs : high-frequency oscillations HS : hippocampal sclerosis ILAE : International League Against Epilepsy KCC2 : K/Cl cotransporter 2 IID : interictal discharge IILDs : interictal-like discharges MEA : multi-electrode arrays mIPSCs : miniature inhibitory postsynaptic currents mTLE : mesial temporal lobe epilepsy NKCC1 : N/K/Cl cotransporter 1 OSR1 : oxidative stress-responsive kinase 1 PCPZ : prochlorperazine SE : status epilepticus SPAK : SPS1-related proline/alanine-rich kinase WNK : with-no-lysine kinase