Focal Epilepsy: When the Brakes on the Network Go Kaput

Commentary

Loss of CLOCK Results in Dysfunction of Brain Circuits Underlying Focal Epilepsy.

Li P, Fu X, Smith NA, Ziobro J, Curiel J, Tenga MJ, Martin B, Freedman S, Cea-Del Rio CA, Oboti L, Tsuchida TN, Oluigbo C, Yaun A, Magge SN, O'Neill B, Kao A, Zelleke TG, Depositario-Cabacar DT, Ghimbovschi S, Knoblach S, Ho CY, Corbin JG, Goodkin HP, Vicini S, Huntsman MM, Gaillard WD, Valdez G, Liu JS. 2017;96:387–401.

Because molecular mechanisms underlying refractory focal epilepsy are poorly defined, we performed transcriptome analysis on human epileptogenic tissue. Compared with controls, expression of Circadian Locomotor Output Cycles Kaput (CLOCK) is decreased in epileptogenic tissue. To define the function of CLOCK, we generated and tested the Emx-Cre; Clock^flox/flox and PV-Cre; Clock^flox/flox mouse lines with targeted deletions of the Clock gene in excitatory and parvalbumin (PV)-expressing inhibitory neurons, respectively. The Emx-Cre; Clock^flox/flox mouse line alone has decreased seizure thresholds, but no laminar or dendritic defects in the cortex. However, excitatory neurons from the Emx-Cre; Clock^flox/flox mouse have spontaneous epileptiform discharges. Both neurons from Emx-Cre; Clock^flox/flox mouse and human epileptogenic tissue exhibit decreased spontaneous inhibitory postsynaptic currents. Finally, video-EEG of Emx-Cre; Clock^flox/floxmice reveals epileptiform discharges during sleep and also seizures arising from sleep. Altogether, these data show that disruption of CLOCK alters cortical circuits and may lead to generation of focal epilepsy.

Circadian rhythms are endogenous rhythms that control behavioral and physiological processes, such as body temperature, hormonal secretion, and sleep–wake cycles. These rhythms are controlled by both external factors (light/dark cycle) and internal cues (molecular clocks). In addition to controlling these normal homeostatic functions, there is also evidence that circadian clocks may play a role in pathophysiological processes, such as contributing to neurodegeneration and the circadian regulation of seizures in patients with epilepsy. In fact, focal epilepsies often have a distinct circadian pattern of seizures that varies depending on the location of the seizure focus and the type of epilepsy (1).

It has been long appreciated that there are circadian patterns of seizures in patients with epilepsy (2). Approximately two-thirds of patient with epilepsy exhibit a circadian pattern in their seizure presentation (2). There are different types of patterns of circadian seizures: nocturnal, diurnal, and diffuse. Nocturnal seizures predominantly occur during the dark phase, whereas diurnal seizures typically occur during wakefulness. The diffuse pattern refers to seizures that occur randomly without a specific circadian pattern. Despite the appreciation of the circadian pattern of seizures in epilepsy, research into the underlying mechanisms has not been extensively explored; this issue deserves further attention (3).

A further complication is the interplay among sleep, seizures, and circadian rhythms. Anecdotally, it is well accepted that sleep deprivation is a seizure trigger in patients with epilepsy and, conversely, seizures can disrupt sleep (4). Recently, more complex interactions between sleep and seizures have been elucidated (for review, see [5]) insomuch as sleep state impacts seizures and vice versa. Further, sleep apnea has been demonstrated to influence seizures and impact quality of life in patients with epilepsy. Likely, the most tragic is the role of sleep in sudden unexpected death in epilepsy, which primarily occurs during sleep. Thus, the interplay among sleep, circadian rhythms, and seizures is complex, but this currently highlighted study by Li and colleagues takes an important step in attempting to unravel these complicated interactions.

The circadian pattern of focal seizures has been well documented and is thought to be mediated by molecular Circadian Locomotor Output Cycles Kaput (CLOCK) genes that control circadian rhythms (6). Interestingly, CLOCK genes have already demonstrated to alter neuronal excitability and seizure susceptibility. Previous studies demonstrated that mice lacking specific genes—those in the proline and acidic amino acid-rich basic leucine zipper (PAR bZip) transcription factor family such as albumin D-site-binding protein (DBP), hepatic leukemia factor (HLF), and thyrotroph embryonic factor (TEF)—exhibit spontaneous seizures and audiogenic seizures (7). These findings implicate CLOCK-regulated transcription factors in altering neuronal excitability and seizure susceptibility.

So, what is novel about the Li et al. study? The highlighted study performed an unbiased, transcriptome analysis of tissue resected from the epileptogenic focus from patients with focal epilepsy and then identified a decrease in the expression of CLOCK transcripts in both epilepsy patients with focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC). These findings are not definitive since sleep abnormalities are common in patients with epilepsy, which can also influence molecular CLOCK expression (8), as can caveats regarding tissue collection and appropriate controls for human studies. To delve further into these findings, the authors directly examined the role of CLOCK in neuronal excitability and seizure susceptibility using mice which lack CLOCK in excitatory neurons (Emx-Cre; Clock^flox/flox mice). Emx-Cre; Clock^flox/flox mice exhibit epileptiform bursts of spontaneous excitatory postsynaptic currents (sEP-SCs), decreased seizure threshold, and spontaneous seizures that arise during sleep. It is arguable whether the bursts of sEP-SCs constitute epileptiform activity; however, the observation of spontaneous seizures in these animals is more convincing. These studies demonstrate that the loss of CLOCK in excitatory neurons is sufficient to induce spontaneous seizures in mice. that said, it does not demonstrate that the loss of CLOCK in the epileptic foci in tissue resected from patients is causally related to the focal epilepsy.

These findings are provocative; however, the current study falls short of identifying a mechanism through which CLOCK deficits result in increased neuronal excitability, decreased seizure threshold, and spontaneous seizures. It is tempting to think that the CLOCK deficits are epiphenomenon associated with epilepsy, which could result from sleep disruption commonly associated with epilepsy. However, the evidence that the loss of CLOCK in excitatory neurons is sufficient to increase seizure susceptibility and induce spontaneous seizures argues against this being merely an epiphenomenon. Thus, the current study leaves us wondering…how? How does the loss of CLOCK in excitatory neurons alter neuronal excitability and seizure susceptibility? A potential clue to the underlying mechanism may be the decreased number of spines and the decreased spine density in the apical dendrite and primary dendritic branches. Additional studies are required to determine whether these observed changes contribute to the increased excitability in the Emx-Cre; Clockflox/flox mice.

CLOCK genes exert a myriad of functions in the central nervous system, regulating nearly half of the genes in the mammalian genome and controlling numerous physiological processes. CLOCK genes in the suprachiasmatic nuclei (SCN) in the hypothalamus are thought to be the “master regulators” of circadian rhythms. Much less is known about the function of CLOCK genes outside the SCN, although they are widely expressed throughout the brain. Circadian rhythms have been shown to impact numerous processes which may impact excitability. For example, ion channels and membrane excitability have been shown to be circadian regulated (1), which can directly impact neuronal excitability. Relevant to the current commentary, PARbZip proteins have been shown to control neurotransmitter metabolism (7), which may underlie changes in synaptic transmission, neuronal excitability, and seizure susceptibility associated with dysregulation of CLOCK proteins. Sleep deprivation or disruption of circadian rhythms can cause elevations in stress hormones and proinflammatory mediators, which are known to be proconvulsant and can also impact neuronal excitability and seizure susceptibility. CLOCK genes have also been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which has been commonly demonstrated to be hyperactivated in epilepsy. Relevant to the currently highlighted study, excessive mTOR signaling has been implicated in both FCD and TSC (for review, see [1]). Thus, given the broad influence of CLOCK genes, the mechanisms which may mediate the impact on epilepsy are likely numerous. Although this is not the first time that CLOCK signaling has been implicated in epilepsy, there is still much to be learned about the effects of CLOCK on neuronal excitability and seizure susceptibility and this study is an important contribution to that effort.