WNK signaling as A new Therapeutic Target in Epilepsy – WATT

Ionotropic GABAA receptors selectively conduct chloride (Cl-) through their pore. The direction of Cl- flux through the channel depends on transmembrane Cl- gradients. Therefore, Cl- homeostasis critically determines the polarity and efficacy of GABAergic transmission in the brain.
Epilepsy is often associated with altered neuronal Cl- homeostasis. The accumulation of intra-neuronal Cl- as a result of down-regulation of the neuronal Cl- extruders KCC2 and KCC3 and up-regulation of the Cl- importer NKCC1 may weaken inhibitory GABA signaling and thereby promote the emergence of epileptic seizures. Drugs aimed at reducing intracellular Cl- concentration thus represent promising therapeutic strategies against epilepsy and other diseases with altered inhibition such as neuropathic pain and psychiatric disorders. It is therefore crucial to discover novel mechanisms of regulation of KCCs and NKCC1 transporters that may help to develop new treatments.

We have recently discovered that the WNK1 signaling pathway rapidly tunes Cl- homeostasis by controlling KCC2 membrane diffusion and stability in hippocampal neurons. Furthermore, we showed that this signaling is massively activated in the epileptic brain. This signaling operates by regulating the phosphorylation of key KCC2 Threonine (Thr) residues. Knowing this pathway also promotes KCC3 and NKCC1 Thr phosphorylation, resulting in KCCs inhibition and NKCC1 activation, the regulation of the WNK signaling must be an efficient mechanism to restore neuronal Cl- homeostasis in epilepsy and limit seizure occurrence/severity.

We aim to elucidate the mechanisms underlying WNK activation in epilepsy and to determine whether interfering with this pathway is beneficial for epilepsy. The manipulation of this pathway is particularly promising toward the development of novel treatment as a WNK inhibitor is already available for oral administration in humans. Our project thus holds the potential to help uncover novel therapeutic strategies for epilepsy, and other diseases with impaired inhibition. This project will also shed light on the virtually unknown role of a widespread signaling pathway in the nervous system.

Our first am is to decipher the mechanisms of regulation of KCC2, KCC3 and NKCC1 in Parvalbumin (PV) Interneurons and Principal cells of the hippocampus, and to determine whether KCC2/KCC3 and NKCC1 membrane trafficking in these cells is altered in mouse and human epileptic tissues. Our second aim is to determine whether a genetic/pharmacological inhibition of the WNK signaling protects against epilepsy. We will inhibit the WNK signaling and examine its effect on KCC2/KCC3/NKCC1 Thr phosphorylation status, membrane dynamics, stability and activity, as well as its impact on epileptic activities and seizure susceptibility/severity. Finally, we will reveal the contribution of KCC2, KCC3 and NKCC1 Thr residues in chloride homeostasis and epilepsy in Pv Interneurons and Principal cells.

To address these questions, we have designed a multidisciplinary approach, spanning different levels of analysis (from molecules to functional studies in mouse and human chronic epileptic tissues) using biochemistry and electrophysiology, as well as state-of-the-art imaging techniques. This project will also lead to technological breakthrough by developing 3D dual color Single Particle Tracking in live hippocampal tissue and by imaging/recording human epileptic tissue.

The consortium gathers three leading experts in their respective fields, including a foreign partner.