Authors :
Presenting Author: Naziha Bakouh, PhD – Imagine Institute
reyes castano, Master Degree – PhD Student, IHU Imagine; Loredana Dan, PhD – Transsylvanian Institute for Neuroscience; Alice Metais, MD – MD, Histo Pathology, GHU Sainte-Anne, Paris; Edor Kabashi, Md-PhD – Lab Director, Translational Research in neurological Disorders lab, IHU Imagine; Pascale Varlet, MD-PhD – Professor of Neuropathology, Histo Pathology, GHU Sainte Anne; Rima Nabbout, MD-PhD – Pediatric Neurology – Hôpital Necker; Chiara Guerrera, Lab Director – INEM; Sorana Ciuria, PhD – Senior Researcher, Translational Research in Neurological Disorders Lab, IHU Imagine; Thomas Blauwblomme, MD-PhD – Professor, Head of Pediatric Neurosurgery, Pediatric neurosurgery, Hôpital Necker
Rationale:
Focal cortical dysplasia type II (FCDII) are emblematic malformations of cerebral cortical development associated with epilepsy in children. Somatic mutations in the PI3/AKT/mTOR pathways have been identified in FCD II, and recently we showed GABAergic dysfunction secondary to abnormal expression of chloride co-transporters (CCC) NKCC1 and KCC2. However, the pathways linking the genetic alterations to functional abnormalities and altered physiological excitability are missing. Here, we investigate a possible link between mTOR activation, and CCC deregulation through abnormal control of the WNK1/SPAK/OSR pathway.
Methods:
Ex vivo electrophysiological recordings were performed with Microelectrode arrays (MEA) in acute human cortical slices from pediatric patients operated of FCDII. Pharmacological tests on spontaneous activities were carried out using inhibitors of the WNK1/SPAK/OSR pathway (N-EthylMalmeide :NEM and Staurosporines) and those of the PI3/AKT/mTOR pathway (Alpelisib, Everolimus and Rapamycin). The physical interactions between the partners of the mTOR pathway and those of the WNK1 pathway were determined by co-immunoprecipitation. Also the expression and phosphorylation levels of KCC2 and NKCC1 were analyzed by western blot.
Results:
First, we recorded spontaneous interictal spikes from human FCDII slices incubated with artificial cerebrospinal fluid (ACSF). They were abolished after administration of mTOR inhibitors demonstrating the involvement of mTOR pathway in the occurrence of epileptic activity. Second, we revealed a physical interaction between mTOR and mSIN1 of mTORC2 with WNK1 and SPAK/OSR1 respectively. Consequently mTORC2 increases the activation and the degree of phosphorylation of WNK1/SPAK/OSR1 and CCC. Third, western blot analysis showed reduced levels of KCC2/NKCC1 ratio in FCDII cortical slices as compared to the controls. Interestingly, inhibition of WNK/SPAK/OSR1 kinases with Staurosporine and N-ethylmalemide (dephosphorylate the Thr233 and Ser373 of SPAK) induced an increased KCC2 expression and rescued the KCC2/NKCC1 ratio in FCDII, along with blocage of epileptic activities in FCDII cortical slices on MEA recordings. Eventually, treatment with mTOR inhibitors restored the KCC2/NKCC1 ratio along with a decreased activity of WNK/SPAK/OSR.
Conclusions:
This study shows that mutations of mTOR pathway activates WNK/SPAK/OSR kinases resulting in deregulation of cation chloride cotransporters; KCC2 and NKCC1 in FCDII, supporting epileptogenesis through dysfunction of GABAa inhibition.
Funding: French National Agency for Research: ANR 20- CE 17- 0003 "DYSCLO project"