Molecular mechanisms underlying the ferroptosis-induced epileptiform activity in mouse cortical slices

Ferroptosis, a newly defined iron-dependent programmed cell death, characterized by excessive accumulation of lipid peroxides and reactive oxygen species, is involved in epilepsy, particularly in those forms resistant to drugs. In a previous study, we have demonstrated that exposure of mouse cortical slices to the ferroptosis inducer RSL-3, induces interictal epileptiform discharges. To investigate the mechanisms underlying ferroptosis-induced epileptic activity in RSL-3-treated cortical slices, we analysed the expression of the main contributors to ferroptosis susceptibility in cells.

The expression of proteins involved in RSL-3 induced ferroptosis pathways were analysed on mouse cortical slices by Western blot and qRT-PCR. The epileptogenic response was investigated by electrophysiological patch clamp recordings, in current clamp mode, from layer 2/3 mouse cortical slices.

In cortical neurons, the ferroptosis induction by RSL-3 was associated with a reduced expression of the GPX4/GSH redox pathway, responsible for the clearance of lipid peroxides, and an upregulation of 15-LOX, which promotes the formation of lipid peroxides. Furthermore, the cysteine/glutamate antiporter Xc^-, a modulator of excitotoxicity in the brain, was up-regulated either after RSL-3-treatment or by incubating neurons with 4-HNE, the bioactive product of lipid peroxidation. Interestingly, 4-HNE was able to generate spontaneous interictal bursts.

Our findings establish a direct link between lipid peroxidation and Xc^- activity. These results suggest that the xCT antiporter may represent a promising therapeutic target for treating ferroptosis-mediated drug-resistant epilepsy.