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. 2014 Jul;61:1-12.
doi: 10.1016/j.mcn.2014.04.006. Epub 2014 Apr 28.

GluA2 mRNA Distribution and Regulation by miR-124 in Hippocampal Neurons

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Free PMC article

GluA2 mRNA Distribution and Regulation by miR-124 in Hippocampal Neurons

Victoria M Ho et al. Mol Cell Neurosci. .
Free PMC article

Abstract

AMPA-type glutamate receptors mediate fast, excitatory neurotransmission in the brain, and their concentrations at synapses are important determinants of synaptic strength. We investigated the post-transcriptional regulation of GluA2, the calcium-impermeable AMPA receptor subunit, by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in cultured mouse hippocampal neurons. Using computational approaches, we identified a conserved microRNA-124 (miR-124) binding site in the 3'UTR of GluA2 and demonstrated that miR-124 regulated the translation of GluA2 mRNA reporters in a sequence-specific manner in luciferase assays. While we hypothesized that this regulation might occur in dendrites, our biochemical and fluorescent in situ hybridization (FISH) data indicate that GluA2 mRNA does not localize to dendrites or synapses of mouse hippocampal neurons. In contrast, we detected significant concentrations of miR-124 in dendrites. Overexpression of miR-124 in dissociated neurons results in a 30% knockdown of GluA2 protein, as measured by immunoblot and quantitative immunocytochemistry, without producing any changes in GluA2 mRNA concentrations. While total GluA2 concentrations are reduced, we did not detect any changes in the concentration of synaptic GluA2. We conclude from these results that miR-124 interacts with GluA2 mRNA in the cell body to downregulate translation. Our data support a model in which GluA2 is translated in the cell body and subsequently transported to neuronal dendrites and synapses, and suggest that synaptic GluA2 concentrations are modified primarily by regulated protein trafficking rather than by regulated local translation.

Keywords: AMPAR; Dendritic mRNA localization; Local translation; MicroRNA; Post-transcriptional regulation; miR-124.

Figures

Figure 1
Figure 1
The GluA2 3' untranslated region (UTR) has a functional miR-124 target site. (A) Schema showing the position and conservation of the fully complementary miR-124 target site at the 5' end of the mouse GluA2 3' UTR. The seed region of miR-124 is shown in bold. Vertical bars depict Watson-Crick base pairs. (B–D) Wild type and mutant reporter constructs were transfected into HEK293T cells with miR-124 mimics or mutant miR-124 mimics with two point mutations in the seed region (underlined). Luciferase activities are reported relative to reporter-only controls. (B) Transfection of the wild type reporter (“WT”) with miR-124 resulted in robust knockdown of luciferase activity while transfection with mutant miR-124 did not. Significance determined by one-way analysis of variance (ANOVA) with Bonferroni correction. (C) Transfection of miR-124 with a reporter lacking the miR-124 target site (“TD”) did not reduce luciferase activity. ^ site of deletion. Significance determined by two-tailed t-test. (D) Transfection of a mutant reporter with two point mutations in the miR-124 target site (“PM”) did not show reduced translation by miR-124. Translational reduction was restored when the mutant reporter was transfected with the complementary mutant miR-124. Significance determined by one-way ANOVA with Bonferroni correction. ***p<0.001; error bars show standard error of the mean (S.E.M.); N = 4 independent experiments per group.
Figure 2
Figure 2
miR-124 is enriched at synapses while GluA2 mRNA is de-enriched. (A) Synaptosome fractions were prepared from adult forebrain. Immunoblotting shows enrichment for PSD-95 in the synaptosome fraction (5 μg of total protein was loaded in each lane). (B) Comparative RT-qPCR on RNA extracted from total and synaptosome fractions of mouse forebrain. Relative ratios are plotted on the y-axis. Significance was determined by one-way ANOVA with Bonferroni correction. Pairwise comparisons between the ratios for Camk2α vs. GluA2 and GluA2 vs. miR-124 were significantly different (p<0.01 and p<0.001 respectively). GluA2 and miR-124 were not significantly different (p>0.05). Error bars show S.E.M. N = 3 independent experiments.
Figure 3
Figure 3
GluA2 mRNA and miR-124 have different patterns of distribution. (A) Representative fluorescence in situ hybridization (FISH) images of straightened dendrites. Top: red, FISH puncta; cyan, MAP2; blue, Hoechst. Bottom: FISH puncta in gray scale. Scale bar = 20 μm. (B) Group data for mean puncta distance from the center of the cell body. Error bars show 95% confidence intervals. (C) Puncta were classified into three subcompartments and the percentage of puncta in each subcompartment is shown. “Soma” represents the cell body; “proximal” represents from 0 to 20 μm from the cell body; and “distal” represents greater than 20 μm from the cell body. Error bars show 95% confidence intervals. (D) Table showing the absolute number of puncta per micron in each subcompartment, along with number of dendrites measured for each gene. Dendrites were imaged from 4 to 7 independent experiments.
Figure 4
Figure 4
Control experiments demonstrate the specificity of the FISH signals. (A) miR-124 signal is drastically reduced in the presence of a fully complementary competitive inhibitor at 10X concentration. Red, miR-124; green, GFAP; cyan, MAP2; blue, Hoechst. Scale bar = 20 μm. (B) miR-124 signal is absent from astrocytes. Red, miR-124; green, GFAP; cyan, MAP2; blue, Hoechst. (C) GluA2 mRNA signal is absent from astrocytes. Red, GluA2 mRNA; green, GFAP; cyan, MAP2; blue, Hoechst. (D) Camk2α mRNA signal is absent from inhibitory neurons. Red, Camk2α mRNA; green, GAD67; white, MAP2.
Figure 5
Figure 5
Overexpression of miR-124 down-regulates total GluA2 protein in neurons. (A) Overexpression and control lentiviral constructs. (B) Comparative RT-qPCR measurement of miR-124 and GluA2 mRNA fold changes in neurons transduced with pMIRNA1-124 relative to control. Error bars show S.E.M. N = 3 independent experiments. (C) Immunoblot analysis of protein lysates from transduced cultures. Band intensities were quantified and normalized to Tuj1. The differences relative to controls are shown below with standard error. N = 4 independent experiments.
Figure 6
Figure 6
Overexpression of miR-124 down-regulates cytoplasmic but not synaptic GluA2 protein levels. (A–C) Measurement of total GluA2 protein expression by immunocytochemistry (ICC). N = 5 independent experiments, 3 to 5 fields per experiment. Quantification of the cell body and dendritic signals were performed on the same neurons. (A) GluA2 expression in the cell body. (i) Representative images of cell bodies of transduced neurons. Red/grayscale, GluA2 protein; green, copGFP. (ii) Mean GluA2 intensity per pixel. (iii) GluA2 intensity per cell body. (B) GluA2 expression in proximal dendrites (i) Representative images. Red/grayscale, GluA2 protein; cyan, MAP2 (ii) Mean GluA2 intensity per pixel. (C) GluA2 expression distal dendrites (i) Representative images. Red/grayscale, GluA2 protein; cyan, MAP2 (ii) Mean GluA2 intensity per pixel. (D) Measurement of synaptic GluA2 protein expression by surface labeling of GluA2 in live neurons followed by fixation and staining for synapsin. (i) Representative images of surface expressed GluA2. Red, GluA2; cyan, synapsin. (ii) Fraction of synapsin puncta that appose GluA2 puncta. (iii) Integrated intensity of GluA2 puncta that appose synapsin puncta. N = 3 independent experiments, each with dendrites from 5 different neurons quantified. P values were determined by one-tailed t-tests. *P<0.05, **P<0.01, ***P<0.001. Error bars show S.E.M.

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