Abstracts

Rationale: Neuronal chloride concentration ([Cl^-]_i) is an important determinant of both post-synaptic GABA_A-receptor mediated signaling and cell volume regulation. Restoration of [Cl^-]_i equilibrium after synaptic activity is achieved by the net regulated activities of the cation-Cl^- co-transporters (CCC) NKCC1 and KCC2. Depending on the ionic and osmotic gradients across the neuronal cytoplasmic membrane, either antagonizing or stimulating CCC activity might reduce swelling and [Cl^-]_i in injured neurons, restore GABAergic inhibition, suppress seizures, and prevent epileptogenesis. NKCC1 and KCC2 are stimulated and inhibited, respectively, by direct phosphorylation mediated by the Cl^--sensitive WNK (lysine-deficient protein kinase)-activated SPAK (proline/alanine-rich kinase) kinase complex (Alessi et al., 2014). We demonstrated previously (AES-2020), that WNK463, a recently identified and an orally bioavailable pan-With-No-Lysine (WNK)-kinase inhibitor that reduces phosphorylation of the WNK1 downstream targets SPAK/OSR1 (Yamada et al., 2017) and thereby inhibits NKCC1 and KCC2 phosphorylation (de Los et al., 2014), significantly reduced the frequency, duration and power of recurrent seizures without any immediate visible evidence of cytopathological deterioration suggestive of neurotoxicity.


Methods: We determined now the net effects of WNK463 on cytoplasmic neuronal chloride elevation and Cl^- extrusion rates during spontaneous recurrent seizures, as well as the correlation between ionic and electrographic effects. Organotypic hippocampal slice cultures from mice expressing the Cl^- sensitive fluorescent protein Clomeleon (CLM-1) or Super Clomeleon (S-CLM) (Grimley et al., 2013) were studied as a model of acute traumatic brain injury and epileptogenesis in vitro.


Results: After a 1-week latent period, slice cultures developed spontaneous ictal-like epileptiform discharges (ILDs) and electrical status epilepticus (Dzhala and Staley, 2021). We found that: (i) WNK463 strongly reduced the frequency, duration, and power of recurrent ILDs; (ii) WNK463 progressively increased the Cl^- accumulation rate and increased Cl^- extrusion rate during recurrent ILDs; (iii) WNK463 abolished neuronal Cl^- transients and reduced cell volume; (iv) antagonists of GABA(A) receptor as well as NKKC1 and KCC2 transport activity prevented or significantly reduced the anti-ictal effect of WNK463.

Conclusions: Our results demonstrate that anti-ictal effect of WNK463 is consistent with an increased neuronal chloride extrusion rate during ictal-like discharges. The increased rate of neuronal chloride accumulation may reflect reduced base line chloride concentration and cell volume in a sub-population of injured neurons. Our data validate WNK463 as a promising target of investigation for antiepileptic therapies and highlight the potential utility of specific activators of CCC co-transport.


Funding: Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number R01NS120973 (V.D.) and R35 NS116852 (K.S.).