The Clock gene regulates kainic acid-induced seizures through inhibiting ferroptosis in mice

Objectives: Temporal lobe epilepsy (TLE) is a common and intractable form of epilepsy. There is a strong need to better understand molecular events underlying TLE and to find novel therapeutic agents. Here we aimed to investigate the role of Clock and ferroptosis in regulating TLE.

Methods: TLE model was established by treating mice with kainic acid (KA). Regulatory effects of the Clock gene on KA-induced seizures and ferroptosis were evaluated using Clock knockout (Clock-/-) mice. mRNA and protein levels were determined by quantitative real-time PCR and western blotting, respectively. Ferroptosis was assessed by measuring the levels of iron, GSH and ROS. Transcriptional regulation was studied using a combination of luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays.

Key findings: We found that Clock ablation exacerbated KA-induced seizures in mice, accompanied by enhanced ferroptosis in the hippocampus. Clock ablation reduced the hippocampal expression of GPX4 and PPAR-γ, two ferroptosis-inhibitory factors, in mice and in N2a cells. Moreover, Clock regulates diurnal expression of GPX4 and PPAR-γ in mouse hippocampus and rhythmicity in KA-induced seizures. Consistent with this finding, Clock overexpression up-regulated GPX4 and PPAR-γ and protected against ferroptosis in N2a cells. In addition, luciferase reporter, mobility shift and ChIP assays showed that CLOCK trans-activated Gpx4 and Ppar-γ through direct binding to the E-box elements in the gene promoters.

Conclusion: CLOCK protects against KA-induced seizures through increased expression of GPX4 and PPAR-γ and inhibition of ferroptosis.