GluA2 mRNA distribution and regulation by miR-124 in hippocampal neurons

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.

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Single-molecule insights into mRNA dynamics in neurons

“…In live neurons, single molecule approaches have expanded our ability to explore the precise mechanism of RNA motility and localization [13,14]. Recent work highlighted here, utilizing single molecule fluorescence in situ hybridization (FISH) to endogenous mRNAs and live imaging of single mRNA in neurons has resulted in novel and at times even reformed insights into how mRNAs are localized in the brain [15–17]. For the sake of simplicity, technologies which are capable of yielding single molecule resolution are discussed as single molecule imaging without focusing on whether this sensitivity was achieved in each study.…”

“…AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, a type of glutamate receptor, may be composed of various combinations of subunits (GluA 1–4) and inclusion of the GluA2 subunit selectively diminishes the receptor’s permeability to extracellular calcium. Branched DNA labeling of endogenous mRNA showed that the GluA2 subunit mRNA of the AMPA receptor was not present in dendrites but instead was largely restricted to the soma [17]. In contrast, earlier studies reported dendritic localization of AMPA receptor subunit mRNAs (GluA1–4) and that subsequent synthesis of AMPA receptors could be modulated locally in an activity-dependent manner [71–75].…”

“…Quantitative amplification of fluorescent signal using branched oligo FISH labeling can facilitate detection of sequences as short as 20 nucleotides in length. In the study of single molecule GluA2 mRNA in dendrites, single transcripts of miR-124 were observed along dendrites, offering novel in situ insight into the regulation of local translation via microRNAs [17]. Similarly, Ube3a mRNA, which produces a seemingly non-functional protein, localizes to dendrites and plays a role in regulating the availability of miRNA-134, is localized to dendrites [82].…”