Anticonvulsant and antiepileptogenic effects of system xc- inactivation in chronic epilepsy models

Abstract

Objective: The cystine/glutamate antiporter system $x_c^{-}$ could represent a new target for antiepileptogenic treatments due to its crucial roles in glutamate homeostasis and neuroinflammation. To demonstrate this, we compared epilepsy development and seizure susceptibility in xCT knockout mice (xCT^-/- ) and in littermate controls (xCT^+/+ ) in different chronic models of epilepsy.

Methods: Mice were surgically implanted with electrodes in the basolateral amygdala and chronically stimulated to develop self-sustained status epilepticus (SSSE); continuous video-electroencephalography monitoring was performed for 28 days after SE and hippocampal histopathology was assessed. Corneal kindling was induced by twice daily electrical stimulation at 6 Hz and maintenance of the fully kindled state was evaluated. Next, messenger RNA (mRNA) and protein levels of xCT and of the proteins involved in the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase 3β (GSK-3β)/eukaryotic initiation factor 2α (eIF2α)/activating transcription factor 4 (ATF4) signaling pathway were measured at different time points during epileptogenesis in NMRI mice treated with pilocarpine. Finally, the anticonvulsant effect of sulfasalazine (SAS), a nonselective system $x_c^{-}$ inhibitor, was assessed against 6 Hz-evoked seizures in pilocarpine-treated mice.

Results: In the SSSE model, xCT^-/- mice displayed a significant delayed epileptogenesis, a reduced number of spontaneous recurrent seizures, and less pronounced astrocytic and microglial activation. Moreover, xCT^-/- mice showed reduced seizure severity during 6 Hz kindling development and a lower incidence of generalized seizures during the maintenance of the fully kindled state. In pilocarpine-treated mice, protein levels of the PI3K/Akt/GSK-3β/eIF2α/ATF4 pathway were increased during the chronic phase of the model, consistent with previous findings in the hippocampus of patients with epilepsy. Finally, repeated administration of SAS protected pilocarpine-treated mice against acute 6 Hz seizure induction, in contrast to sham controls, in which system $x_c^{-}$ is not activated.

Significance: Inhibition of system $x_c^{-}$ could be an attractive target for the development of new therapies with a potential for disease modification in epilepsy.

Keywords: xCT; cystine/glutamate antiporter; disease modification; epilepsy; kindling; status epilepticus.