Abstract
The genome of humans and other eukaryotes contain multiple non-coding elements including microRNA, which are now recognised as important negative regulators of gene expression. Indeed, microRNA are likely to be critical regulators of the biological processes underlying memory and its dysfunction in Alzheimer’s disease. Long-term potentiation (LTP) is the preeminent model of memory widely used to understand its underlying molecular and cellular processes. LTP induced at hippocampal perforant path synapses of awake freely moving animals shows remarkable persistence, which is underpinned by dynamic regulation of gene networks. Our recent bioinformatic analysis predicted microRNA to be important contributors in regulating LTP-related gene expression. Further, our microarray analysis demonstrated concurrent with rapid up-regulation of mRNA expression post-LTP induction was rapid down-regulation of microRNA expression. Therefore, it was hypothesised that a release of microRNA-mediated inhibition on gene expression may contribute to LTP persistence.
To explore the significance of microRNA regulation following LTP, the expression of two mature microRNA (miR-34a-5p and miR-132-3p), down-regulated in the microarray analysis, were assessed with RT-qPCR in our in vivo LTP model in awake male Sprague-Dawley rats. These microRNA, previously linked to multiple LTP-related processes, including neurotransmission, transcription, and dendritic spine morphology, were confirmed to be down-regulated 20 min post-LTP induction in a N-methyl-D-aspartic acid receptor-dependent manner. While levels of both microRNA returned to baseline by 24 h, miR-34a-5p remained down-regulation at 5 h. Further, no temporal regulation of pri-miR-34a was found, while pri-miR-132/212 showed a dramatic up-regulation at 20 min, attenuated by 5 h, and down-regulated at 24 h. Therefore, multiple regulatory mechanisms appear to underlie the expression of these microRNA following LTP induction.
To fully understand the potential contribution of miR-34a-5p and miR-132-3p to the persistence of LTP, a number of bioinformatic tools were utilised to identify novel targets of these microRNA. A Weighted Gene Co-Expression Network Analysis demonstrated these microRNA did not target clusters of genes temporally co-expressed post-LTP. Next, evaluation of nine microRNA targeting algorithms showed no one algorithm, or their intersection, predicted sufficiently previously validated targets. Their union, however, covered the majority of this microRNA-target interactome, and was filtered for genes annotated as involved in LTP-related processes. This identified several novel targets of miR-34a-5p and miR-132-3p. Of interest were predicted interactions between miR-132-3p and the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor subunit gene Gria2, and the important signalling molecule gene Mapk1. Luciferase assays demonstrated miR-132-3p does interact with Mapk1, but not Gria2. Further investigation of Mapk1 using RT-qPCR and Western blots found that the mRNA and associated p42-MAPK protein were up-regulated 5 and 24 h following LTP induction. These results support the hypothesis that miR-132-3p is involved in regulating Mapk1 post-LTP induction.
In summary, this work demonstrated that microRNA are dynamically regulated following LTP induction and that they can target genes involved in the processes underlying its persistence. Further, with careful consideration, microRNA targeting algorithms have the potential to identify microRNA targets for further investigation. Thus, microRNA are likely to play an important role in regulating gene expression underlying LTP persistence and memory.