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

Abstract

Abstract 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.