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- W3190991593 abstract "•Learning/memory variation of inbred mice correlates with miR-466f-3p expression•miR-466f-3p regulates neuron morphology, plasticity, and learning/memory capability•MEF2A, a negative regulator of learning/memory, is targeted by miR-466f-3p•Stochastic CREB activation upregulates transcription of miR-466-669 cluster Phenotypic variation is a fundamental prerequisite for cell and organism evolution by natural selection. Whereas the role of stochastic gene expression in phenotypic diversity of genetically identical cells is well studied, not much is known regarding the relationship between stochastic gene expression and individual behavioral variation in animals. We demonstrate that a specific miRNA (miR-466f-3p) is upregulated in the hippocampus of a portion of individual inbred mice upon a Morris water maze task. Significantly, miR-466f-3p positively regulates the neuron morphology, function and spatial learning, and memory capability of mice. Mechanistically, miR-466f-3p represses translation of MEF2A, a negative regulator of learning/memory. Finally, we show that varied upregulation of hippocampal miR-466f-3p results from randomized phosphorylation of hippocampal cyclic AMP (cAMP)-response element binding (CREB) in individuals. This finding of modulation of spatial learning and memory via a randomized hippocampal signaling axis upon neuronal stimulation represents a demonstration of how variation in tissue gene expression lead to varied animal behavior. Phenotypic variation is a fundamental prerequisite for cell and organism evolution by natural selection. Whereas the role of stochastic gene expression in phenotypic diversity of genetically identical cells is well studied, not much is known regarding the relationship between stochastic gene expression and individual behavioral variation in animals. We demonstrate that a specific miRNA (miR-466f-3p) is upregulated in the hippocampus of a portion of individual inbred mice upon a Morris water maze task. Significantly, miR-466f-3p positively regulates the neuron morphology, function and spatial learning, and memory capability of mice. Mechanistically, miR-466f-3p represses translation of MEF2A, a negative regulator of learning/memory. Finally, we show that varied upregulation of hippocampal miR-466f-3p results from randomized phosphorylation of hippocampal cyclic AMP (cAMP)-response element binding (CREB) in individuals. This finding of modulation of spatial learning and memory via a randomized hippocampal signaling axis upon neuronal stimulation represents a demonstration of how variation in tissue gene expression lead to varied animal behavior. Phenotypic variation among individuals endows an evolutionary advantage, since it provides the population diversity on which natural selection acts (Pavlicev et al., 2011Pavlicev M. Cheverud J.M. Wagner G.P. Evolution of adaptive phenotypic variation patterns by direct selection for evolvability.Proc. Biol. Sci. 2011; 278: 1903-1912Crossref PubMed Scopus (64) Google Scholar). Genetic backgrounds and environmental factors contribute to generating such natural variation (Bendesky and Bargmann, 2011Bendesky A. Bargmann C.I. Genetic contributions to behavioural diversity at the gene-environment interface.Nat. Rev. Genet. 2011; 12: 809-820Crossref PubMed Scopus (83) Google Scholar). Experimentally, inbred mice have often been used to study the molecular and cellular basis of average phenotypes and behaviors so to minimize the effects of genetic differences among the tested individuals (Casellas, 2011Casellas J. Inbred mouse strains and genetic stability: a review.Animal. 2011; 5: 1-7Crossref PubMed Scopus (57) Google Scholar). However, variation of tissue transcript abundance has been shown between individual isogenic mice. Genes exhibiting highly variable expression levels are often associated with immune function, stress responses, and hormonal regulation, i.e., processes sensitive to environmental cues (Vedell et al., 2011Vedell P.T. Svenson K.L. Churchill G.A. Stochastic variation of transcript abundance in C57BL/6J mice.BMC Genomics. 2011; 12: 167Crossref PubMed Scopus (16) Google Scholar). Some of these studies have also shown that differences in gene expression or cellular signaling, with or without the influence of environmental factors such as maternal licking, nutrient provision in utero, or stress-induced resilience versus susceptibility (Bale, 2015Bale T.L. Epigenetic and transgenerational reprogramming of brain development.Nat. Rev. Neurosci. 2015; 16: 332-344Crossref PubMed Scopus (273) Google Scholar; Danchin et al., 2011Danchin É. Charmantier A. Champagne F.A. Mesoudi A. Pujol B. Blanchet S. Beyond DNA: integrating inclusive inheritance into an extended theory of evolution.Nat. Rev. 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Genomic copy number variation in Mus musculus.BMC Genomics. 2015; 16: 497Crossref PubMed Scopus (26) Google Scholar; Loos et al., 2015Loos M. Koopmans B. Aarts E. Maroteaux G. van der Sluis S. Verhage M. Smit A.B. Neuro-BSIK Mouse Phenomics ConsortiumWithin-strain variation in behavior differs consistently between common inbred strains of mice.Mamm. Genome. 2015; 26: 348-354Crossref PubMed Scopus (25) Google Scholar; Oey et al., 2015Oey H. Isbel L. Hickey P. Ebaid B. Whitelaw E. Genetic and epigenetic variation among inbred mouse littermates: identification of inter-individual differentially methylated regions.Epigenetics Chromatin. 2015; 8: 54Crossref PubMed Scopus (36) Google Scholar). Spatial learning and memory formation controlled by brain can be separated into two systems: (1) egocentric navigation employing self-movement and internal cues and (2) allocentric navigation primarily involving the hippocampus and nearby brain structures that is stimulated by distal cues outside of the organisms (Ekstrom et al., 2014Ekstrom A.D. Arnold A.E. Iaria G. A critical review of the allocentric spatial representation and its neural underpinnings: toward a network-based perspective.Front. Hum. Neurosci. 2014; 8: 803Crossref PubMed Scopus (129) Google Scholar). The capability of spatial learning and memory allows most animal species progressively adjust their behaviors to adapt in spatially and temporally variable environments, which is critical for their survival. This can be assessed in laboratory animals by applying behavioral paradigms such as the Morris water maze (MWM) (Vorhees and Williams, 2014Vorhees C.V. Williams M.T. Assessing spatial learning and memory in rodents.ILAR J. 2014; 55: 310-332Crossref PubMed Scopus (251) Google Scholar). Notably, genetically identical inbred rodents have been shown to exhibit phenotypic variation in spatial learning and memory (Tsai et al., 2002Tsai K.J. Chen S.K. Ma Y.L. Hsu W.L. Lee E.H. sgk, a primary glucocorticoid-induced gene, facilitates memory consolidation of spatial learning in rats.Proc. Natl. Acad. Sci. USA. 2002; 99: 3990-3995Crossref PubMed Scopus (88) Google Scholar), but the underlying mechanisms of this behavioral variation remained unknown. The formation of new memories is a complex process requiring activity-dependent gene transcription, new protein synthesis, and finely tuned specific neuronal networking as memory engrams to make new neuronal connections and sustained plasticity (Asok et al., 2019Asok A. Leroy F. Rayman J.B. Kandel E.R. Molecular Mechanisms of the Memory Trace.Trends Neurosci. 2019; 42: 14-22Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Hippocampal engrams, which are sparse populations of neurons in the dentate gyrus (DG), represent ensembles of neurons displaying increased activity after memory formation. While the DG engram neurons exhibit a highly distinct pattern of gene expression (Rao-Ruiz et al., 2019Rao-Ruiz P. Couey J.J. Marcelo I.M. Bouwkamp C.G. Slump D.E. Matos M.R. van der Loo R.J. Martins G.J. van den Hout M. van IJcken W.F. et al.Engram-specific transcriptome profiling of contextual memory consolidation.Nat. Commun. 2019; 10: 2232Crossref PubMed Scopus (31) Google Scholar), the engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Various factors and signaling pathways known to either positively or negatively regulate learning and memory process have been identified (Abraham et al., 2019Abraham W.C. Jones O.D. Glanzman D.L. Is plasticity of synapses the mechanism of long-term memory storage?.NPJ Sci. Learn. 2019; 4: 9Crossref PubMed Scopus (69) Google Scholar; Humeau and Choquet, 2019Humeau Y. Choquet D. The next generation of approaches to investigate the link between synaptic plasticity and learning.Nat. Neurosci. 2019; 22: 1536-1543Crossref PubMed Scopus (39) Google Scholar). Among them, the activated form of cyclic AMP (cAMP)-response element binding protein (CREB) stimulates gene transcription in response to activity-dependent increases of intracellular Ca2+ in neurons (Kandel, 2012Kandel E.R. The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB.Mol. Brain. 2012; 5: 14Crossref PubMed Scopus (539) Google Scholar). On the other hand, the cAMP/PKA pathway represses myocyte enhancer factor 2 (MEF2) transcriptional activity by preventing nuclear export of its co-repressor, HDAC5, and nuclear import of co-activator NFAT (Belfield et al., 2006Belfield J.L. Whittaker C. Cader M.Z. Chawla S. Differential effects of Ca2+ and cAMP on transcription mediated by MEF2D and cAMP-response element-binding protein in hippocampal neurons.J. Biol. Chem. 2006; 281: 27724-27732Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Furthermore, unlike CREB, MEF2A activity constrains memory formation (Cole et al., 2012Cole C.J. Mercaldo V. Restivo L. Yiu A.P. Sekeres M.J. Han J.H. Vetere G. Pekar T. Ross P.J. Neve R.L. et al.MEF2 negatively regulates learning-induced structural plasticity and memory formation.Nat. Neurosci. 2012; 15: 1255-1264Crossref PubMed Scopus (91) Google Scholar). Apart from the protein factors, microRNAs (miRNAs) are also involved in regulating neuronal functions, including learning and memory (McNeill and Van Vactor, 2012McNeill E. Van Vactor D. MicroRNAs shape the neuronal landscape.Neuron. 2012; 75: 363-379Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). miRNAs are small (∼22 nt) non-coding RNAs that primarily act as post-transcriptional regulators of gene expression via sequence-specific base-pairing with their recognition sites in the 3′ untranslated regions (3′ UTRs) of specific mRNAs (Daugaard and Hansen, 2017Daugaard I. Hansen T.B. Biogenesis and Function of Ago-Associated RNAs.Trends Genet. 2017; 33: 208-219Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). miRNAs participate in a range of signaling pathways in post-mitotic neurons, and they mediate activity-dependent cellular processes, including dendritic growth and branching, synapse formation, and maturation. By regulating gene expression, they also play important roles in long-lasting forms of synaptic plasticity that underlie memory formation, retrieval, and consolidation (Chen and Shen, 2013Chen Y.L. Shen C.K. Modulation of mGluR-dependent MAP1B translation and AMPA receptor endocytosis by microRNA miR-146a-5p.J. Neurosci. 2013; 33: 9013-9020Crossref PubMed Scopus (37) Google Scholar; Wang et al., 2012bWang W. Kwon E.J. Tsai L.H. MicroRNAs in learning, memory, and neurological diseases.Learn. Mem. 2012; 19: 359-368Crossref PubMed Scopus (144) Google Scholar). In the following, we present evidence for a causative link between stochastic tissue gene expression and individual behavioral variation in animals. In particular, we show that the stochastic activation of a hippocampal CREB-pCREB → miR-466f-3p-MEF2A axis modulates the individual variation of the spatial learning and memory capability in inbred mice. To identify miRNAs involved in the regulation of learning and memory formation, we used the MWM task to distinguish inbred wild-type C57BL/6J mice with good or poor learning and memory capability (Figure 1A). We defined mice that completed the task within 30 s in the last (6th) session as “good learners” (GLN), whereas mice that could not find the platform within 30 s in the last session were considered “poor learners” (PLN). As shown in Figure 1A (left panel), ∼62% of the mice belonged to the GLN group (180 of 289 mice), exhibiting a marked reduction in escape latency from 98.1 s (1st session) to 21.8 s (6th session). In contrast, the escape latency of the remaining ∼38% of the mice, the PLN group (109 of 289 mice), was only moderately reduced between the first and sixth sessions (from 111.8 s in the 1st session to 82.1 s in the 6th session). The probe test, indicating that the mice had indeed learned the task over the six sessions, also showed that GLN mice stayed longer in the platform region than PLN mice (Figure 1A, left pair of bars in the right panel). Next, we analyzed RNAs from the hippocampus of GLN and PLN mice by miRNA microarray hybridization (n = 4 each group). In general, we observed relatively minor differences in the expression profiles of hippocampal miRNAs from GLN and PLN mice. However, we noted that the top 10 miRNAs for which expression levels were higher in GLN mice than PLN mice (data not shown) were all derived from the same rodent-specific miRNA cluster, miR-466-669, located in intron 10 of the mSfmbt2 gene (see below). Based on reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis the expression levels of selected miRNAs in the cluster, we chose miR-466f-3p for further investigation (Figure 1B). Average expression of this miRNA was ∼1.5-fold higher in the hippocampus of GLN mice relative to PLN mice. Notably, average levels of hippocampal miR-466f-3p were similar among the PLN group, home cage (HC) control group, and swimming control group (Figure 1C), thereby excluding exercise-related effect during swimming and further supporting the idea that miR-466f-3p was induced during spatial learning and memory formation. In Figure 1C, the data reveal that expression levels of hippocampal miR-466f-3p of over 42% of GLN mice were at least 1.5-fold higher than the average expression level of HC control mice, whereas the levels in 15% of PLN mice were half that of HC mice. Approximately 25% of GLN mice and 55% of PLN mice had similar levels of miR-466f-3p (∼0.8- to 1.2-fold relative to HC). Pearson correlation scatterplot showing a positive correlation between miR-466f-3p levels and percentage duration on the platform during the probe test is presented in Figure 1D. We also performed miR-466f-3p and U6 small nuclear RNA (snRNA) in situ hybridization (ISH) of the brain slices in both GLN and PLN mice (Figure 1E). Statistical analysis of the miR-466f-3p signals via ISH confirmed that induction of miR-466f-3p was indeed greater in the DG of GLN mice relative to that of PLN mice, whereas no difference was found when the U6 snRNA signals were compared (right histogram, Figure 1E). Although miR-466-3p signals were not equal among the individual granule cells in DG, they still increased significantly and ubiquitously in the hippocampus of GLN mice compared to PLN mice. Therefore, the miR-466f-3p induction did not happen only in a small portion of cells, e.g., the engram neurons. These data indicate that upregulation of miR-466f-3p during the MWM task is closely associated with better spatial learning and memory capability among a significant proportion of GLN mice. We also analyzed the average expression levels of several brain-specific miRNAs by RT-qPCR. Among them, miR-132-3p was upregulated in mouse hippocampus after the MWM task, but we did not observe significant differences between the GLN and PLN groups (bar pair VII, Figure 1B). Expression levels of miR-335-5p and miR-22 were similar among the GLN, PLN, and HC groups (Figure S1A). Thus, unlike miR-466f-3p, hippocampal expression of these miRNAs during MWM training is not correlated with the spatial learning and memory ability of the mice. To confirm that miR-466f-3p was indeed expressed in neurons, we performed miRNA ISH combined with immunofluorescence (IF) staining of NeuN and MAP2 in primary hippocampal neurons. The results showed that, similar to other miRNAs (Cohen et al., 2011Cohen J.E. Lee P.R. Chen S. Li W. Fields R.D. MicroRNA regulation of homeostatic synaptic plasticity.Proc. Natl. Acad. Sci. USA. 2011; 108: 11650-11655Crossref PubMed Scopus (153) Google Scholar; Thomas et al., 2017Thomas K.T. Anderson B.R. Shah N. Zimmer S.E. Hawkins D. Valdez A.N. Gu Q. Bassell G.J. Inhibition of the Schizophrenia-Associated MicroRNA miR-137 Disrupts Nrg1α Neurodevelopmental Signal Transduction.Cell Rep. 2017; 20: 1-12Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), miR-466f-3p was mainly expressed in the neuronal soma region, with some additional signals in the dendrites (Figure S2A). To understand the molecular and cellular basis of the association of upregulated miR-466f-3p with learning and memory formation, we first transiently overexpressed miR-466f-3p together with a dsRed fusion polypeptide under the control of the ubiquitin promoter from the pFUGW-miR-466f-3p-dsRed plasmid into DIV10 primary hippocampal neurons (Figure S2B). Through miRNA ISH, we confirmed that miR-466f-3p signal is stronger in the miR-466f-3p overexpression group relative to vector control or mutant miR-466f-3p group (arrows, Figure S2B). In parallel, we also established a platform to examine the effect of miR-466f-3p loss of function by inhibition miR-466f-3p using miR-sponge located in the 3′ UTR of EGFP mRNA expressed from pFUGW-miR-sponge-EGFP plasmid (Figure S2C). The EGFP reporter in the sponge plasmid served as both an indicator of transfection efficiency and a sensor of cellular miRNA activity (Kluiver et al., 2012Kluiver J. Gibcus J.H. Hettinga C. Adema A. Richter M.K. Halsema N. Slezak-Prochazka I. Ding Y. Kroesen B.J. van den Berg A. Rapid generation of microRNA sponges for microRNA inhibition.PLoS ONE. 2012; 7: e29275Crossref PubMed Scopus (104) Google Scholar). In a significant portion of the transfected HEK293T cells, the EGFP signal decreased upon co-expression with wild-type miR-466f-3p, but not with mutant miR-466f-3p, due to inhibition of EGFP mRNA translation by binding of miR-466f-3p to miR-sponge in the 3′ UTR (compare the left four images and two histogram, Figure S2C). In contrast, we did not observe decreased EGFP signal upon co-expression of the control sponge encoding eight copies of a scrambled sequence from pFUGW-scr-sponge-EGFP plasmid with either miR-466f-3p or its mutant (compare the right four images and two histogram). As shown in Figure 2A, ectopic expression of miR-466f-3p resulted in morphological changes of the neurons, most notably enhanced neurite outgrowth relative to control neurons transfected with the dsRed-expressing vector pFUGW-dsRed or mutant miR-466f-3p-expressing plasmid pFUGW-mut-miR-466f-3p-dsRed. Quantification data showed that average dendritic branch number per neuron was not significantly different between the miR-466f-3p-overexpressing group (bar II) and vector or mutant control (bars I and III) (right upper histogram, Figure 2A). However, the mean total dendrite length and the mean length of primary dendrites increased upon miR-466f-3p overexpression (compare bar II to bars I and III, right lower histogram of Figure 2A). In parallel, we inhibited endogenous miR-466f-3p using miR-sponge. As seen, there was no significant difference in dendritic branch number between miR-466f-3p-inhibition and control groups (compare bar IV to bars I and V, right upper histogram, Figure 2A), but the miR-466f-3p-inhibition group displayed a notable reduction in mean total dendrite length as well as mean lengths of primary and secondary dendrites relative to other groups (compare bar IV to bars I and V, right lower histogram of Figure 2A). Furthermore, IF staining demonstrated that miR-466f-3p overexpression, but not inhibition, also increased the density of dendritic spines (Figure 2B, upper panels and lower left histogram). We also performed co-staining for postsynaptic density protein 95 (PSD-95) and counted the density of colocalized dendritic spines to establish exact numbers of excitatory synapses (Figure 2B, lower right histogram), which revealed that miR-466f-3p overexpression increased the density of PSD-95-positive spines compared to other control groups. However, miR-466f-3p inhibition did not significantly alter the density of PSD-95-positive spines with respect to that of control groups (Figure 2B, lower right histogram). Together, these data indicate that in the absence of neuronal stimulation, miR-466f-3p inhibition alone does not affect the densities of excitatory synapses or total dendritic spines. Given these findings, we next examined if upregulated hippocampal miR-466f-3p expression in mice upon spatial learning and memory formation also affected neuronal morphology in vivo. Significantly, we found that average spine density of the hippocampal neurons of GLN mice was higher than that of PLN mice, as revealed by Golgi impregnation of the pyramidal neurons in the GLN and PLN mouse hippocampus (Figure S3). These findings demonstrate that upregulation of hippocampal miR-466f-3p promotes neurite outgrowth and dendritic spine formation, which is similar to the induction of spine density observed in the hippocampus of GLN mice relative to PLN mice. To investigate whether miR-466f-3p positively regulates mouse spatial learning and memory formation, we used recombinant lentivirus infection approach to overexpress the miRNA in mouse hippocampus. The mice were analyzed 7 days after lentivirus injection into the DG (Figure 3A). Representative images of the hippocampal regions infected with the viral vector (control) or recombinant lentivirus overexpressing miR-466f-3p are shown in the left panels of Figure 3B. As analyzed by RT-qPCR, the hippocampal expression levels of miR-466f-3p was indeed elevated in the miR-466f-3p overexpression group compared to control group (right histogram in Figure 3B). We found that mice with hippocampal overexpression of miR-466f-3p and subjected to the MWM task exhibited escape latencies of 88 ± 5 s in the 1st session and 19 ± 1 s in the 6th session (red dot line, Figure 3C), which were better than for vector control mice (black line) or mutant control mice (red circle line) and similar to the escape latencies of GLN mice described in Figure 1A. In parallel, we examined the effect of miR-466f-3p loss of function on spatial learning and memory by inhibition miR-466f-3p using miR-sponge. Notably, the MWM performance of mice in which hippocampal miR-466f-3p was trapped by the miR-sponge was similar to that of PLN mice, i.e., they did not learn to find the platform even by the last session (solid green square line, Figure 3C). Furthermore, mice injected with lentivirus did not appear to have disrupted homeostatic plasticity, since some mice in each group had learned or at least were in the process of learning during the last session (Figure 3C, right histogram). Given that overexpression of miR-466f-3p in mouse hippocampus enhanced their learning and memory capability, we analyzed the comparative electrophysiology of cultured hippocampal neurons expressing different levels of miR-466f-3p. The miniature excitatory postsynaptic current (mEPSC) from DIV14 hippocampal neurons overexpressing miR-466f-3p or mut-miR-466f-3p, miR-sponge, or control was recorded using whole-cell patch clamps. Whereas there were no significant differences in the mEPSC amplitude, rise Tau, or decay Tau among the four sets of samples, the mEPSC frequency of neurons overexpressing miR-466f-3p was significantly higher compared to other groups (Figure 4A), indicating that postsynaptic glutaminergic receptors were more strongly activated upon miR-466f-3p overexpression. We then measured long-term potentiation (LTP) to directly determine the role of miR-466f-3p in synaptic plasticity in vivo. We injected the hippocampus of mice with recombinant lentivirus as described in Figure 3 and then induced LTP in hippocampal slices by tetanic stimulation (three trains of high-frequency stimulation [3xHFS]) of the Schaffer collateral pathway. We found that our protocol induced LTP in all groups, as evidenced by the persistent increase in field excitatory postsynaptic potentials (fEPSPs) in the cornu ammonis 1 (CA1) region (Figure 4B, left panel). The LTP was stronger in miR-466f-3p-overexpressing slices (188% ± 2% of baseline at 40–50 min after stimulation, mean ± SEM) compared to mutant (169% ± 1% of baseline), scr-sponge (173% ± 3% of baseline), and control-virus-infected slices (159% ± 2% of baseline), as miR-466f-3p inhibition by miR-sponge reduced the LTP (128% ± 1% of baseline) relative to controls (Figure 4B, right panel). We also measured the LTP of the GLN and PLN groups after training. The respective data also revealed significant differences in fEPSP slope in the CA1 region between the GLN and PLN groups (Figure 4C). Thus, elevated level of miR-466f-3p enhances LTP and synaptic plasticity that, in turn, can promote the learning and memory capability of mice. Together, the data presented in Figures 2, 3, and 4 demonstrate that miR-466f-3p plays a critical positive role in spatial learning and memory formation, likely by enhancing spine formation, LTP, and the strength of synaptic plasticity. We conducted a bioinformatics analysis to identity potential target mRNAs regulated by binding of miR-466f-3p to their 3′ UTRs. Among the candidates we identified was the Mef2a mRNA encoding MEF2A. Interestingly, MEF2A expression was previously found to be downregulated after MWM training, and overexpression of this factor exerted a negative effect on the performances of mice (Cole et al., 2012Cole C.J. Mercaldo V. Restivo L. Yiu A.P. Sekeres M.J. Han J.H. Vetere G. Pekar T. Ross P.J. Neve R.L. et al.MEF2 negatively regulates learning-induced structural plasticity and memory formation.Nat. Neurosci. 2012; 15: 1255-1264Crossref PubMed Scopus (91) Google Scholar). Therefore, we used a luciferase reporter assay to examine if miR-466f-3p regulates the translation of Mef2a mRNA by binding to its 3′ UTR. We inserted wild-type or mutant Mef2a 3′ UTR sequences downstream of an SV40-promoter-driven luciferase cDNA (Luc), resulting in the reporter plasmid psiCHECK2-MEF2A 3′ UTR or psiCHECK2-mut-MEF2A 3′ UTR (Figure 5A). As shown in the left histogram of Figure 5B, co-expression of miR-466f-3p attenuated the expression of luciferase directed by Mef2a 3′ UTR. This effect appeared to be dependent on the interaction between miR-466f-3p and Mef2a 3′ UTR, since mutation of either the predicted binding site of miR-466f-3p on the Mef2a 3′ UTR (5′-UGUGUAU-3′) or the seed region of miR-466f-3p (5′-AUACACA-3′) recognizing Mef2a 3′ UTR abrogated the inhibitory effect of miR-466f-3p on luciferase activity (Figure 5B, middle histogram). Furthermore, co-expression of the miR-sponge, but not the scr-sponge, also eliminated the repressive effect of miR-466f-3p on luciferase activity (Figure 5B, right histogram). Notably, neither overexpression of miR-466f-3p nor miR-sponge had any effect on the levels of Mef2a mRNA in primary hippocampal neurons (Figure 5C), but they did reduce or increase, respectively, the level of MEF2A protein (Figure 5D). We also found that overexpression of miR-466f-3p or miR-sponge, respectively, downregulated or upregulated the mRNA level of activity-regulated cytoskeletal associated protein (Arc) in primary hippocampal neurons (Figure 5C), which was a known downstream target positively regulated by MEF2A (Flavell et al., 2006Flavell S.W. Cowan C.W. Kim T.K. Greer P.L. Lin Y. Paradis S. Griffith E.C. Hu L.S. Chen C. Greenberg M.E. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number.Science. 2006; 311: 1008-1012Crossref PubMed Scopus (425) Google Scholar). We also performed fluorescence in situ hybridization (FISH) to detect miR-466f-3p and combined it with IF labeling of MEF2A in DIV14 primary hippocampal neurons without or with forskolin treatment. Forskolin is known to induce chemical LTP and activate adenylyl cyclase, thus raising intracellular cAMP levels. We observed nuclear colocalization of MEF2A with miR-466f-3p, as illustrated by the representative images in Figure 5E. After forskolin stimulation, however, the miR-466f-3p signal increased in both soma and dendrites, whereas the MEF2A signal in the nucleus diminished, as shown by the reciprocal changes of the intensities of the MEF2A and Fast Red signals, respectively, of the individual neuronal cells (Figure 5E). In sum, the data in Figures 5A–5E indicate that miR-466f-3p negatively regulates the expression of MEF2A protein by binding to the 3′ UTR of Mef2a mRNA and consequently repressing its translation. Consistent with the above-described results, we found that hippocampal levels of" @default.
- W3190991593 created "2021-08-16" @default.
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- W3190991593 date "2021-08-01" @default.
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- W3190991593 title "Activation of a hippocampal CREB-pCREB-miRNA-MEF2 axis modulates individual variation of spatial learning and memory capability" @default.
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