MicroRNA regulation in dentate gyrus subregions following induction of long-term potentiation in vivo

Abstract

MicroRNAs (miRNAs) are short, endogenous RNAs that are non-coding; that is, they are not translated into protein. Instead, they function primarily as post-transcriptional inhibitors of protein synthesis by base-pairing with specific target messenger RNAs (mRNAs). Recently, miRNAs have been implicated in the maintenance of long-term potentiation (LTP), a biological substrate for memory. The late phase of LTP requires new protein synthesis, both rapidly via translation of extant synaptic mRNA, and more slowly via transcription. Our recent genome-wide studies have shown that mRNA levels are dramatically regulated in a temporally-specific manner between 20 minutes and 24 hours after LTP induction in vivo, and indicate that miRNAs may contribute to the regulation of these complex LTP-related gene networks. Therefore, the aim of the current study was to profile global miRNA expression in subregions of the dentate gyrus after LTP induction in vivo and investigate the functional significance of regulated miRNAs. Five hours after LTP induction in awake rats, microarrays and RNA sequencing were used to quantify global miRNA expression in laser-microdissected dentate gyrus middle molecular layer and granule cell layer, respectively. Differential expression was validated using the reverse-transcription quantitative polymerase chain reaction (RT-qPCR). I identified two miRNAs that were up-regulated specifically in the dentate gyrus middle molecular layer, containing activated synapses: miR-151-3p and miR-23a-3p. This result suggests that miR-23a-3p and miR-151-3p are ideally suited to couple synaptic activation, translational regulation, and LTP persistence. One miRNA, miR-132-3p, was up-regulated specifically in the dentate gyrus granule cell layer. To understand how miRNA regulation contributes to LTP maintenance, I used bioinformatics to identify potential targets of regulated miRNAs, and validated predicted miRNA:mRNA interactions using luciferase assays. Hn1 and Klhl11 were identified as novel targets of miR-132-3p. In addition, bioinformatic analysis identified nine putative novel miRNAs in the dentate gyrus granule cell layer, one of which was validated using RT-qPCR, and is predicted to target genes that function at the synapse. These data provide the first evidence of miRNA regulation in subcellular compartments after LTP induction in awake rats and support the hypothesis that miRNAs regulate LTP-related gene expression. For the first time, I have demonstrated regulation of miRNAs in isolated neuropil following LTP induction in vivo, indicating that miRNAs may regulate local dendritic protein synthesis.