In this study, we have defined a protective role of Clock gene in KA-induced seizures in mice (Figure 8). More importantly, we have uncovered that Clock regulates epileptic seizures through inhibiting ferroptosis in the hippocampus. The evidence for the links between Clock, ferroptosis and epileptic seizures is strong. First, DFO, an inhibitor of ferroptosis, significantly alleviated KA-induced seizures in both wild-type and Clock−/− mice (Figure 2). Second, Clock ablation promoted ferroptosis in mice and in N2a cells, whereas Clock overexpression protected against ferroptosis in N2a cells (Figure 5). Third, Clock positively regulated the transcription and expression of both GPX4 and PPAR-γ, two ferroptosis-inhibiting factors, through specific binding to an E-box element in target gene promoters (Figure 7). Therefore, Clock gene protests against KA-induced seizures through promoting expression of GPX4 and PPAR-γ and inhibiting ferroptosis. Our findings provide increased understanding of the complex pathways for regulation of epileptic seizures by circadian clock.
Previous studies have shown that epileptic seizures in rodents display circadian rhythms, and seizures tend to occur more frequently in the later light than in the dark phase35,37. It is consistent to find that herein in wild-type mice the seizures were more severe when KA was injected at ZT10 and less severe when KA was injected at ZT22 (Figure 4). Interestingly, the circadian time-dependency of seizure severity was considerably attenuated in Clock-/- mice (Figure 4), indicating involvement of Clock in circadian regulation of epileptic seizures. Therefore, the circadian rhythm in seizures appears to arise from the rhythmicity in the extent of ferroptosis caused by circadian expression of GPX4 and PPAR-γ that are directly driven by the CLOCK protein. This is supported by the fact that GPX4 and PPAR-γ share similar diurnal patterns with that of the CLOCK protein (Figure 4).
Considering that Clock gene has a protective role in epileptic seizures and is down-regulated in epileptic tissues, it is most likely involved in epileptogenesis. We have previously shown that REV-ERBα (a direct target of CLOCK) drives the expression of GABA transporters and enhances GABA reuptake, thereby alleviating GABA-mediated inhibition and promoting epileptic seizures30. Li et al found that deletion of Clock in pyramidal cells causes seizures during sleep in mice39. This phenotype was linked to the alteration of cortical circuits39. Therefore, although we have shown that Clock gene protests against KA-induced seizures through inhibiting ferroptosis, there is a possibility that other mechanisms are involved, such as disruption of GABAergic function and alteration of cortical circuits.
KA is an excitatory amino acid. Treatment with KA can cause epileptic seizures in the hippocampus. These seizures propagate to other limbic structures and the induced neuropathlogical changes in the hippocampus are comparable to those of patients with TLE5. Thus, the Clock gene may be a promising drug target for treating TLE as small molecules targeting CLOCK such as CLK8 are being identified and synthesized. We found that KA-induced seizures were associated with ferroptosis. This support the notion that ferroptosis contributes to epileptic seizures33,34. We showed that the ferroptosis inhibitor DFO reduced (a ~50% reduction) the severity of seizure in wild-type mice (Figure 2). The reducing effect of DFO was also observed in Clock knockout mice. However, the reduction (~30%) is less evident (Figure 2). This was probably because a higher level of ferroptosis caused by Clock deletion was more difficult to be repressed by DFO.
We have identified CLOCK as a positive regulator of PPAR-γ based on several lines of evidence. First, Clock ablation reduces hippocampal expression of PPAR-γ at both mRNA and protein levels in mice (Figure 3). Second, Clock knockdown reduces, whereas Clock overexpression increases, the expression of PPAR-γ in N2a cells (Figure 6). Third, based on a combination of luciferase reporter, mobility shift and ChIP assays, CLOCK protein trans-activated Ppar-γ through specific binding to an E-box element in gene promoter (Figure 7). However, Reitz et al found that Ppar-γ mRNA expression was up-regulated in the heart in Clock▲19/▲19 (exon 19 deletion of Clock gene and a 51-amino-acid deletion of CLOCK protein) mice, suggesting a negative regulatory effect of Clock on Ppar-γ25. Although the exact reason for this contradiction was unknown, there was a possibility that the positive versus negative action of CLOCK was tissue dependent. This is because the activity of CLOCK is strongly affected by the cellular microenvironments such as the redox state and the types of cofactors.
It was noteworthy that mouse Ppar-γ gene generates two different mRNA transcripts (i.e., Ppar-γ1 and Ppar-γ2) using two distinct promoters. The primer for qPCR assay of Ppar-γ in current study cannot discriminate these two transcripts. Thus, mRNA expression of Ppar-γ here measures the total level of Ppar-γ1 and Ppar-γ2. Although both transcripts are expressed in mouse hippocampus, Ppar-γ1 is the dominant form as the expression ratio of Ppar-γ1 over Ppar-γ2 is more than 7 and a specific antibody fails to detect Ppar-γ2 protein in the hippocampus. Therefore, the western blots most likely detected the Ppar-γ1 protein in the hippocampus, and the diurnal pattern may be the authentic rhythm of Ppar-γ1 protein (Figure 4).
In summary, we have demonstrated that Clock gene protests against KA-induced seizures through promoting expression of GPX4 and PPAR-γ and inhibiting ferroptosis. Our findings enhance a deeper understanding of the crosstalk between circadian clock and epileptic seizures. Targeting CLOCK protein may provide a promising approach for management of epileptic seizures.