Abstracts

Rationale: Mesial temporal lobe epilepsy is the most common form of partial epilepsy, and also the most drug-resistant. Hippocampal sclerosis is observed in the majority of drug-resistant mesial temporal lobe epilepsy patients, making it an important pathological biomarker in this disease, and possibly a valuable tool for deciphering disease pathophysiology. In this study, we evaluated the gene expression profiles in dentate granule cells of mesial temporal lobe epilepsy patients with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from a small number of cells, and to begin to understand hippocampal pathology in this form of epilepsy. Methods: Dentate gryus specimens were collected and flash-frozen from four mesial temporal lobe patients with hippocampal sclerosis and five without, matched as closely as possible for gender, age, and ethnicity. Dentate granule cells from the granule cell layer were collected using laser capture microdissection in duplicate. RNA was extracted using the ARCTURUS PicoPure RNA Isolation Kit and amplified using the Clontech SMARTer Ultra Low RNA Kit. Sequencing libraries were then prepared using the TruSeq RNA Sample Preparation Kit. RNAseq was performed by multiplexing two samples, one with and one without hippocampal sclerosis, across one lane of a HiSeq 2000. After removal of sequencing adaptors, the sequencing reads were aligned using TopHat2, and the transcripts were assembled with Cufflinks. Results: High quality RNA was obtained from each of the dentate granule cell populations. We obtained ~8 million sequencing reads, of which >90% aligned. We observed highly concordant transcriptional profiles between RNA samples collected and processed in duplicate. Principal component analysis identified the presence or absence of hippocampal pathology as the largest source of variation in the dataset. Among the expression changes associated with hippocampal pathology, we observed gene expression changes associated with hippocampal sclerosis enriched in the RAP1 and PI3K-AKT signaling pathways, although larger samples sizes will be needed to confirm these profiles. Conclusions: Analysis of the expression data from these specimens revealed clear, reproducible, and likely biologically relevant expression changes associated with the hippocampal pathology, suggesting that RNAseq from homogenous cellular populations in larger sample sizes may be a powerful approach to understanding the pathophysiology of hippocampal sclerosis. Funding:NINDS-5R21NS078657