A significant reduction in circadian locomotor output cycles kaput expression results in dysfunction of brain circuits in pediatric patients with focal epilepsy, according to research presented at the 2015 American Epilepsy Society Annual Meeting in Philadelphia.
Judy Liu, MD, PhD, of the Children's National Medical Center, Washington DC, and fellow researchers enrolled pediatric patients based on their referral for therapeutic surgical resection for focal epilepsy. Researchers used high-resolution 3T magnetic resonance imaging to determine epileptogenic foci and collected samples directly from the operating room for transcriptome analysis.
A mice study was also conducted for histological and gene expression analyses, whole cell patch-clamp electrophysiology, and pentylenetetrazole (PTZ) seizure induction.
Research indicated that compared with normal brain tissue, there was a significant reduction of circadian locomotor output cycles kaput (Clock) expression in epileptogenic tissue. Specifically, 20 out of 25 patients demonstrated decreased Clock.
Among patients with decreased Clock, there were several types of focal cortical dysplasias, tuberous sclerosis complex, Sturge-Weber syndrome, and Rasmussen's encephalitis, according to Dr. Liu and colleagues.
In the mice study, deletion of Clock in excitatory neurons was associated with loss of spines in the apical dendrite and primary branches, a phenotype the researchers also witnessed in epileptogenic tissue of human pyramidal neurons.
“We demonstrate that mouse excitatory neurons lacking Clock have an imbalance in excitation vs inhibition due to a severe defect in spontaneous [inhibitory postsynaptic currents] as compared with [excitatory postsynaptic currents] ascertained by whole cell patch-clamp electrophysiology,” Dr. Liu and colleagues wrote in the abstract. “This imbalance is associated with a selective reduction of inhibitory synaptic proteins and a reduction in seizure threshold.”
The researchers concluded that circadian genes play a key role in the pathogenesis of pediatric focal epilepsy.
(Abst. 3.019|A.01), 2015Loss of Clock results in dysfunction of brain circuits that underlie pediatric focal epilepsyAuthors: J. Liu, X. Fu, P. Li, M. Tenga, J. Curiel, B. Martin, C. Oluigbo, A. Yaun, T. Tsuchida, M. M. Huntsman, G. Valdez, W. Gaillard
Rationale: To study how focal seizures arise, we conducted transcriptome analysis of epileptogenic tissue obtained from surgical resection of pediatric focal epilepsy cases. Identification of gene expression changes may define novel pathways and therapeutic targets for common causes of epilepsy. We found reduced levels of the circadian transcription factor, Circadian Locomotor Output Cycles Kaput (Clock), in epileptogenic tissue and we modeled the effect of reduced Clock using mice with developmental deletions of the Clock gene in excitatory neurons of the cortex and hippocampus.
Methods: Patients were enrolled in this study based on their referral for therapeutic surgical resection for focal epilepsy. Informed consent was obtained from parents or guardians of pediatric patients and assent obtained when appropriate. Epileptogenic foci were identified and defined by high resolution 3T MRI consistent with standard epilepsy protocols and standard intraoperative electrocorticography. No tissue was removed for research purposes only and control tissue were obtained only when removal was necessary to reach deeper regions of epileptogenic tissue. Samples were collected directly from the operating room and transcriptome analysis was perfomed by microarray using Illumina® Gene Expression BeadChip Array technology (Illumina, Inc., San Diego, CA). Animal modeling: All animal use procedures were carried out in strict accordance with NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC at CNMC. Emx Cre +/-: Clock-/- and Emx Cre+/-;Clock+/+ animals were generated for histological and gene expression studies, whole cell patch-clamp electrophysiology, and pentylenetetrazole (PTZ) seizure induction.
Results: In epileptogenic tissue, the Clock expression is significantly decreased compared with normal brain tissue. We found decreased Clock in 20 out of 25 patient samples. Cases with decreased Clock included all types of focal cortical dysplasias, tuberous sclerosis complex, Sturge Weber, and Rassmussen’s encephalitis. To determine the functional significance of diminished Clock in epilepsy, we created mice with conditional deletions of Clock in both excitatory and inhibitory neurons in the cortex. In mice, deletion of Clock in excitatory neurons results in loss of spines in the apical dendrite and primary branches, a phenotype also observed in human pyramidal neurons from epileptogenic tissue. We demonstrate that mouse excitatory neurons lacking Clock have an imbalance in excitation vs. inhibition due to a severe defect in spontaneous IPSCs as compared with EPSCs ascertained by whole cell patch-clamp electrophysiology. This imbalance is associated with a selective reduction of inhibitory synaptic proteins and a reduction in seizure threshold.
Conclusions: Thus, circadian genes play a key role in the pathogenesis of pediatric focal epilepsy. This study provides a link between the sleep-wake cycle and seizure threshold.Furthermore, these functional analyses suggest that the circadian pathway may be a promising target for therapeutic intervention. -
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