MicroRNA-212/132 Family is Involved in the Regulation of Long-Term Spatial Memory and Synaptic Remodeling

Abstract

MicroRNAs are noncoding transcripts with crucial functions in probably every organ system. The roles of miRNAs in neuronal processes and cognitive functions are recently being appreciated. The discovery of neuronal activity regulated miR-212/132 family with their relatively high expression in brain, functional roles in synaptic remodeling and altered expression in disease states oriented special focus on their function in neuronal systems. In order to elucidate the in vivo functions of the miR-212/132, a mutant mouse line was generated via deletion of the genomic regions encoding pre-miR-212 and pre-miR-132 sequences. The β-galactosidase reporter gene knocked-in into the targeted locus in this mouse line allowed us to study the spatial expression pattern of miR-212/132. ß-galactosidase expression was detected in distinct layers of the cerebral cortex, CA3, CA1 and DG regions of the hippocampus in varying levels through its dorsal to ventral axis, as well as in lateral amygdala and striatum. The miR-212/132 null mutant mice did not show any overt structural alterations in the brain, which led us to investigate possible phenotypes in the cognitive and electrophysiological functions of this mouse line. Hidden platform water maze assay results revealed alterations in the spatial memory of the miR-212/132-/- mice when compared with their WT littermates. In the open field test miR-212/132-/- mice were observed to be slightly hyperactive although they exhibited normal anxiety levels. The baseline excitatory synaptic transmission on Schaffer collateral synapses of the miR-212/132 null hippocampus was normal. On the other hand the paired pulse ratios at different stimulus-intervals were higher in the miR-212/132 null hippocampus indicating alteration in the short-term synaptic facilitation. Moreover, long-term potentiation (LTP) on Schaffer collateral pathway, which is widely accepted as an assay modeling learning and memory, was enhanced in miR-212/132-/- mice. To elucidate the molecular mechanisms leading to the observed defects in the behavior and synaptic transmission of miR-212/132-/- mice, temporal regulation of immediate early genes (IEG) were studied ex vivo. Arc, c-Fos and BDNF, which are the mediators of neuronal activity dependent changes in neurons, were downregulated in miR-212/132-/- hippocampal neurons. Kainic acid treatment of neuronal cultures, which results in a robust neuronal activity, revealed a phenotypical difference in the induction of IEG expression in miR-212/132-/- neurons. The relative levels of c-Fos, Arc and BDNF were similar in miR-212/132-/- and WT neurons upon incubation with kainic acid for 30 min or 3 hrs. In contrary the upregulation of c-Fos and Arc were significantly higher in miR-212/132-/- neurons after 6 hrs of kainic acid treatment, which indicates that the loss-of-function of miR-212/132 alters the regulation of c-Fos and Arc upon longer neuronal induction. Cyclic AMP response element binding protein (CREB) is a transcription factor that is phosphorylated after neuronal activation and subsequently mediates the expression of many downstream effector genes including c-Fos, Arc and BDNF. My results in this thesis have demonstrated that the loss of miR-212/132 function attenuates the phosphorylation of CREB in hippocampal neurons, and thereby providing evidence that miR-212/132 family is involved in the regulation of synaptic remodeling in hippocampal networks and consequently long-term spatial memory via regulating the CREB signaling which in turn influences the IEG expression.