Abstracts

Rationale: Epileptogenesis is a process of molecular and cellular changes leading to the development of chronic epilepsy after an initial insult such as status epilepticus (SE), mechanisms of which are still not completely understood. Several studies explored changes in gene expression following SE. Although these transcriptome analyses yield a plethora of information on changes in SE-triggered gene expression, they do not reveal which mRNAs are being actively translated into proteins. This information is critical for our understanding of the molecular mechanisms of the cellular response. Here we used a polysome profiling technique, based on the isolation of mRNAs simultaneously bound to multiple ribosomes, to identify which genes are actively synthesized into proteins following SE.  Methods: SE was induced in male Sprague Dawley rats (32-45 days old) by i.p. injection of scopolamine (1 mg/kg) followed by pilocarpine (320 mg/kg; i.p.) or vehicle (saline; i.p.) 30 minutes later. SE was terminated by pentobarbital (30mg/Kg; i.p.) after 1 hour. To obtain polysome, cytosolic extracts (90% of Vinitial) of hippocampus were collected at 24h after SE and layered on top of 15-45% sucrose gradients, centrifuged at 35,000 rpm for 2 hours at +4°C, and fractionated using a gradient elution with real time optical detection (254nm). Following fractionation, polysome RNA was extracted using Trizol-LS. Actively translated mRNAs obtained from polysomal fraction represented translatome. The transcriptome was obtained by extracting RNA from the remaining initial cytosolic extract. Transcriptome and translatome mRNAs were then profiled with RNA-next generation sequencing and differential gene expression analysis was done. Enriched gene ontology and pathway for the differential expressed genes were investigated on GSEA based on hypergeometric tests (Broad Institute, MIT) [ broadinstitute.org/gsea/msigdb/annotate.jsp]. Results: Differential expression analysis revealed 1838 differentially expressed genes (DEGs, 0.5 < fold change < 1.5 and false discovery rate < 1%) from the translatome and 1210 from the transcriptome between SE and Sham (n=3 each group). Out of these DEG 42% were specific for translatome and 12% were specific for transcriptome. Gene ontology analysis showed the increased translation is prevalent in intracellular processes such as enzyme binding, signal transduction, response to stress, and cell motility. Immune system, extracellular matrix, transmembrane transport, axon guidance, and actin cystoskeleton were among over-represented pathways in translatome of SE, while genes related to RNA-DNA metabolism and protein synthesis machinery were seen in the transcriptome of SE. Conclusions: Our data demonstrate that there are greater changes in the translatome compared to the transcriptome following SE, suggesting that acute SE response predominantly modifies translation of already transcribed genes. These translational programs merit further investigation as they may shed light on mechanisms of epileptogenesis. Our results strongly emphasize the importance of comparative studies of the translatome versus transcriptome in understanding acute and long-term changes related to epileptogenesis.    Funding: NIH RO1 NS811053