Abstracts

Rationale:
Neuronal chloride concentration ([Cl-]i) is an important determinant of both post-synaptic GABAA-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 first tested the conditions under which WNK-SPAK inhibition might promote neuronal Cl- extrusion. We demonstrated previously (SFN-2015) in a model of acute  traumatic brain injury and epileptogenesis in vitro, that STOCK1S-50699 (Kikuchi et al., 2014), the inhibitor of WNK-SPAK signaling, significantly reduced seizure frequency and power  but exhibited neurotoxicity, highlighting the need for novel non-toxic strategies of targeting the WNK-SPAK pathway for therapeutic benefit.
Method:
We determined the acute anticonvulsive and neuro-protective efficacy of 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). 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. We found that: (i) WNK463  strongly reduced the duration and power of recurrent ictal-like epileptiform discharges; (ii) the anti-ictal  effect of WNK463 existed for at least one hour after washing out  without any immediate visible evidence of cytopathological deterioration suggestive of neurotoxicity; (iii) the anti-ictal effect was consistent with an increased neuronal chloride extrusion rate during ictal-like discharges and reduced base-line chloride concentration and cell volume in  sub-population of injured neurons.
Conclusion:
Our results demonstrate that WNK463, the allosteric inhibitor of WNK-SPAK signaling, significantly reduced the duration and power of spontaneous ictal-like epileptiform discharges in a chronically epileptic in vitro preparation.  In contrast to STOCK1S-50699, WNK463 did not exhibit immediate evidence of neurotoxicity, although we will be investigating this issue in more detail.  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:
:This study was supported by NIH grant R01 NS040109 (NINDS).