Abstracts

Rationale: There are multiple changes in mRNA and protein expression reported following a prolonged seizure. MicroRNAs are small noncoding transcripts that function to control gene expression at a post-transcriptional level and offer another level of protein expression regulation. The role of microRNAs in epileptogenesis has not been investigated to date. Here we identify microRNA changes in the hippocampus after status epilepticus (SE). Methods: Adult male rats underwent pilocarpine induced SE. Hippocampi were harvested 4 hours, 48 hours and 3 weeks later. Total RNA was extracted and used to probe an exiqon microRNA array. We used a low stringency cutoff for defining change, a simple T test P value less than 0.05. Real time PCRs were carried out using Applied Biosystems probes and primers. Subcellular fractionation of the hippocampus using a ficoll gradient was carried out to fractionate the nuclear and synaptoneurosome population. HEK293 cells were transfected with pMirReport vector with luciferase and a neurotrophin-3 (NT3) 3’tail. MiR21 RNA oligo and scrambled oligo were co-transfected, and 24 hours later cells were lysed and luciferase activity was measured. Results: Multiple microRNAs are altered following SE. Approximately 81 microRNAs increased at 4, 48 hours and/or 3 weeks. A large number of microRNAs ~182 decrease specifically at 48 hours. Only 8 microRNAs increased at 4 or 3 weeks but also decreased at 48 hours. Real time PCRs confirmed 80% of the array results tested. A second group of animals at 48 hours were studied and the decrease in microRNAs at 48 hours is most prominent in the synaptoneurosome enriched population of microRNAs. To begin to identify mRNA targets of microRNAs altered following SE, we have focused on miR21, a microRNA shown to be important in numerous cellular processes including stress response. MiR21 does not change at 4 hours and increases 2.5 fold at 48 hours and 3 weeks following SE. Putative mRNA targets of miR21 include NT3, which contains a highly conserved miR21 binding site and is altered following SE. NT3 mRNA levels decrease 50% at 48 hours and 3 weeks following SE. Using a luciferase reporter plasmid with the 3’ tail of the NT3 we found that co-transfection with miR21 causes >75% reduction in luciferase activity. Conclusions: Multiple microRNAs are expressed in the hippocampus and approximately 250 appear to change following SE. The pattern of change is interesting with one subset of microRNAs increasing at 4, 48 hours and 3 weeks and a second mostly distinct set of microRNAs decreasing at 48 hours. The decrease in microRNAs at 48 hours was prominent in the synaptoneurosome population of microRNAs suggesting a loss of microRNAs in cell processes such as dendrites. MiR21 increases following SE, and preliminary studies suggest it is a candidate for down regulation of neurotrophin-3 mRNA following SE. MicroRNAs are candidates for regulation of multiple proteins following SE and may contribute to the development of epilepsy. This work was supported by NINDS.