Scientific Abstract: A common heterogeneous non-inherited condition, focal cortical dysplasia (FCD), is the most common cause of refractory focal epilepsy. While FCD is presumably a stable developmental malformation, epilepsy onset is variable and cases of non-epileptogenic FCD are reported. What factors initiate epilepsy in FCD are unknown. Nevertheless, in pediatric epilepsy most therapeutic resections are performed for FCD patients with severely medically refractory seizures. The tissue collected from this procedure allowed us to study epileptogenesis in these cortical malformations. To discover molecular pathways and to identify potential therapeutic targets in epilepsy, we banked resections and performed a transcriptome analysis of the epileptogenic tissue from FCD and a related disorder Tuberous Sclerosis Complex (TSC). Our preliminary transcriptome analysis on surgical samples from intractable focal epilepsy surgical cases included patients with focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC). The statistical analysis of gene expression in that study identified a decrease in the mRNA levels of the transcription factor, Circadian Locomotor Output Cycles Kaput (Clock), expression in epileptogenic tissue from both FCD and TSC compared with non-epileptic brain. This result was confirmed by Western analysis in a larger cohort of FCD cases. We found a reduction of Clock in both excitatory and inhibitory neurons. We created mouse lines with selective deletion of Clock in either excitatory neurons in the cortex or inhibitory neurons. We found that mice with specific deletion of the Clock gene in excitatory neurons have spontaneous seizures. Based on these results we hypothesize loss of Clock disrupts downstream gene expression leading to circuit dysfunction and epilepsy. Conversely, maintenance of Clock function prevents circuit and molecular changes causative for epilepsy. We will test this hypothesis in three aims: 1) We will determine the effect of Clock loss of function on cortical microcircuits. 2) We will determine a molecular mechanism for Clock by studying its targets, the PARbZip transcription factors. 3) We will use small molecule modifiers of circadian transcription genetic techniques to rescue the effects of decreased Clock. This approach has the potential to improve epilepsy care by developing new therapeutic strategies and refining epilepsy biomarkers.
It is not totally understood how a seizure focus differs from normal brain. Working with epileptologists and neurosurgeons, we have collected tissue samples from patients who are having surgery to remove abnormal brain regions that generate seizures for treatment of their epilepsy. In this epileptogenic tissue we have found altered gene expression of the circadian transcription factor, Clock. Deletion of the Clock gene in excitatory neurons of the cortex causes spontaneous seizures in mice and our goals in this proposal are 1) to identify the circuitry defects in neurons deficient in Clock, 2) molecular mechanisms of Clock in epilepsy and 3) test whether rescue of circadian defects raises seizure threshold.
