Abstracts

Rationale: Medically refractory cases of focal epilepsy, regardless of etiology, may be treated by resection of the region of epileptogenic tissue, which is often curative indicating the importance of the seizure focus in pathogenesis of epilepsy. Nevertheless, the understanding of the structural and molecular underpinnings of the epileptogenic focus is largely absent. To study how focal seizures arise, we have studied human samples directly. We conducted transcriptome analysis of samples obtained from surgical resection of focal epilepsy cases as a basis for understanding pathogenesis. Identification of gene expression changes may allow us to identify novel pathways and therapeutic targets for common causes of epilepsy. Methods: Our strategy is to combine transcriptome analysis of human samples with functional studies in mouse models to determine the significance of changes that we observe in focal epilepsy samples. To this end, we have collected human surgical samples from two different etiologies of epilepsy: 1) Focal cortical dysplasia (FCD) and 2) tuberous sclerosis complex (TSC). In addition, we have control tissue from non-epileptic regions of brain that was removed in the course of resection of epileptogenic tissue. These would ordinarily be discarded. From these samples we performed transcriptome profiling using microarray (MA). We identified many changes in gene expression, but chose promising candidates identifying Circadian Locomotor Output Cycles Kaput (Clock). We validated expression changes at the protein level using Western analysis and then performed functional validation in animal models. 1) We performed expression studies of Clock in human and mouse brain by immunohistochemistry. 2) Using RNA interference we decreased expression of Clock in mouse neurons and assessed the effect on neuronal morphology.Results: Our preliminary data using microarray demonstrate clear differences between control brain, FCD, and TSC. In addition we show a group of 911 genes regulated in the same way in FCD and TSC. Statistical analysis of gene expression, has identified significant downregulation of the transcription factor, Circadian Locomotor Output Cycles Kaput (Clock), expression in both TS and FCD cases compared with non-epileptic brain. In normal brain our expression data in cortex demonstrate expression of Clock in a laminar specific manner with Clock expression highest in layer 5. Using RNA interference technology, we have knocked down expression of Clock resulting in simplification of neuronal morphology, including a decrease in spine density.Conclusions: Based on these results we hypothesize that alterations in gene expression of Clock maybe important for pathogenesis of epilepsy. Decreased expression of Clock may affect the normal formation and stability of neuronal circuits, resulting in aberrant neuronal activity and seizures. Thus, epilepsy pathogenesis may involve similar mechanisms to those observed in the oscillatory plasticity governing circadian rhythms, perhaps explaining the propensity of seizures occurring during sleep- wake transitions.