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Published on: June 12, 2018
The Clock gene regulates kainic acid-induced seizures through inhibiting ferroptosis in mice
Fei Wang1,2, Lianxia Guo1,2, Zhengping Wu3
1College of Pharmacy, Jinan University, Guangzhou, China.
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.
Insights
The Clock gene protects against temporal lobe epilepsy (TLE) by upregulating ferroptosis inhibitors GPX4 and PPAR-γ, reducing seizure severity and ferroptosis. Clock gene ablation worsens TLE by increasing ferroptosis.
Area of Science:
- Neuroscience
- Molecular Biology
- Epilepsy Research
Background:
- Temporal lobe epilepsy (TLE) is a prevalent and treatment-resistant neurological disorder.
- Understanding the molecular mechanisms of TLE is crucial for developing new therapies.
- The roles of the Clock gene and ferroptosis in TLE pathogenesis require further investigation.
Purpose of the Study:
- To investigate the involvement of the Clock gene and ferroptosis in the regulation of TLE.
- To elucidate the molecular pathways through which Clock influences TLE.
- To explore Clock as a potential therapeutic target for TLE.
Main Methods:
- A kainic acid (KA)-induced mouse model was used to establish TLE.
- Clock knockout mice were employed to assess the gene's regulatory effects on seizures and ferroptosis.
- Quantitative real-time PCR, western blotting, and biochemical assays were used to measure gene/protein expression and ferroptosis markers (iron, GSH, ROS).
- Molecular techniques including luciferase reporter, mobility shift, and ChIP assays were utilized to study transcriptional regulation.
Main Results:
- Clock gene ablation aggravated KA-induced seizures and hippocampal ferroptosis in mice.
- Clock deficiency led to decreased expression of the ferroptosis inhibitors GPX4 and PPAR-γ.
- CLOCK was found to directly trans-activate Gpx4 and Ppar-γ by binding to E-box elements in their promoter regions, regulating their diurnal expression and seizure rhythmicity.
Conclusions:
- The Clock gene plays a protective role in TLE by inhibiting ferroptosis.
- CLOCK upregulates GPX4 and PPAR-γ, thereby mitigating seizure severity and ferroptosis.
- Targeting the Clock-GPX4/PPAR-γ pathway may offer a novel therapeutic strategy for TLE.
