In the present study, we found that Bmal1 protein was reduced in the hippocampal DG and CA1 of mice with TLE. Neuron-specific knockout of bmal1 in DG of Bmal1flox/flox mice lowers the threshold of pilocarpine-induced seizures. With high-throughput sequencing and western blotting, the downstream gene PCDH19 regulated by Bmal1 was firstly identified and then detected in the hippocampus of epileptic mice. Furthermore, the expression of Bmal1 and PCDH19 were detected in HS type I, HS type III, and no HS. The levels of Bmal1 and PCDH19 protein in DG of HS type I and HS type III were decreased, compared with no HS group. These results suggest that these changes in Bmal1 and PCDH19 expression may be strongly associated with epileptogenesis and play an important role in the pathogenesis of TLE.
Circadian rhythm genes, as the important transcription factors, not only control rhythmic physiological activities such as sleep and hormone secretion, but also Clock-controlled genes (CCGs) are involved in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease [25, 26]. In previous studies, the levels of Bmal1, CLOCK, Cry and Per mRNA have been confirmed to be decreased after drug-induced and electrically stimulated seizures in animal models [15]. The threshold of seizures induced by electrical stimulation in Bmal1 knockout (KO) mice is lower compared with the control group [16]. In the study, the knockout of clock genes is systemic, so it is unclear which organs or tissues of clock genes are knocked-out directly affect seizures. Here, the role of Bmal1 in the epileptogenesis and seizures for TLE was examined in Bmal1flox/flox mice with conditional deletion of Bmal1 gene in hippocampal neurons. Our results show that Bmal1 was decreased in DG of mice and patients, and neuron-specific knockout of Bmal1in DG of Bmal1flox/flox mice significantly shortened the latency for seizures. This suggests that bmal may be involved in the epileptogenesis of TLE. In our study, the changes in Bmal1 expression mainly occurred in the CA1 and DG neurons. Therefore, we did not perform knockout and functional verification of Bmal1 in hippocampal astrocytes. The changed functions of astrocytes caused by Bmal1 deficiency may be involved in the pathogenesis of TLE. Astrocyte-specific Bmal1 deletion induces astrocyte activation and inflammatory gene expression in vitro and in vivo and alters circadian locomotor behavior and cognition through GABA signaling in mice [17, 18]. Astrocyte activation and gliosis are one of the common pathological symptoms of TLE [27].
At present, the molecular mechanism of Bmal1 involved in epileptogenesis has not been reported. In the present study, by using high-throughput sequencing, 25 up-regulated or 19 down-regulated mRNAs in Bmal1 cKO mice were screened (FDR ≤ 0.05). As one of the candidate genes, PCDH19 is a cell adhesion molecule belonging to the cadherin family. Its prominent expression is in the nervous system especially in limbic areas and cortex [24]. PCDH19 mutations result in an epileptic syndrome known as EIEE9 (OMIM # 300088). A mechanism of cellular interference has been suggested, wherein the coexistence of neurons expressing wild-type (WT) or mutant PCDH19 disrupts cell-cell interactions [28]. PCDH19 downregulation has been proved to bind and regulate GABAARs kinetics, and increase the frequency of action potential firing [23]. PCDH19 downregulation in rat hippocampal neurons also affects the dendrite morphology [29]. Interestingly, Clockflox/flox mice with conditional deletion of the Clock gene in excitatory neurons also show specific spine defects and increased excitability [12]. Considering that Bmal1 and Clock are involved in transcription in the form of Bmal1:Clock complex, these indicate that the abnormal expression of Bmal1 and Clock in neurons may cause similar phenotypes and affect the epileptogenesis.
In epilepsy, DG cells formed excessive de novo excitatory connections and recurrent excitatory loops, leading to the amplification and propagation of excessive recurrent excitatory signals [30]. The granule cells' aggregate excitability has the potential to provide a therapeutic target [31]. In the present study, the expression of Bmal1 was significantly reduced in DG of patients and mice with TLE and lowered the seizure threshold via PCDH19. Therefore, DG was chosen as the target for Bmal cKO with AAV.
Clinical and animal experiments find that patients and animals with TLE show a 24-hour non-uniform distribution of seizure occurrence. These suggest that the epileptogenesis and seizures of TLE may be associated with the circadian rhythms. The two hypotheses have been proposed in seizures of TLE: (1) Rhythmic activity of molecules causes an increase in excitability periodically exceed the seizure threshold, displaying the behavioral seizures. (2) Oscillation of neuronal excitability in the suprachiasmatic nucleus (SCN) modulates the rhythmic excitability in the hippocampus via neural projections [32]. Previous studies have found that Bmal1 expression level in hippocampus still presents the circadian rhythmic oscillation in epilepsy [15]. Although the connection of nerve fibers between the suprachiasmatic nucleus and the dentate gyrus is not clear, the circadian rhythmic activity of DG has been reported in TLE [33, 34]. These suggest that the rhythmic activity of Bmal1 may cause increased excitability via PCDH19, and then periodically exceed the seizure threshold.
However, one of the disadvantages of this study is that it cannot artificially reduce the expression of Bmal1 while maintaining its periodic expression oscillation characteristics in Bmal1flox/flox mice with conditional deletion of the Bmal1 gene. We did not study the effect of Bmal1 KO in SCN on the epileptogenesis and seizures of TLE. Because we have not yet determined the expression changes of circadian rhythm molecules in SCN of TLE. The role of Bmal1 in the SCN of TLE will continue to be explored in the future.
In conclusion, we have disclosed a new biological function of Bmal1 in the epileptogenesis and seizures of TLE and found a downstream gene regulated by Bmal1. Our research findings may help in developing chronotherapy for mTLE, based on the chronobiology of spontaneous seizures. More detailed understandings of the role of Bmal1 and other Clock genes in the brain are required and may give novel insights into the mechanism underlying epileptogenesis and seizures of TLE.