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miR-501-3p mediates the activity-dependent regulation of the expression of AMPA receptor subunit GluA1.

Hu Z, Zhao J, Hu T, Luo Y, Zhu J, Li Z - J. Cell Biol. (2015)

Bottom Line: Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA.We confirmed that miR-501-3p targets Gria1 and regulates its expression under physiological conditions.These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unit on Synapse Development and Plasticity, National Institute of Mental Health, and Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
The number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in synapses determines synaptic strength. AMPAR expression can be regulated locally in dendrites by synaptic activity. The mechanisms of activity-dependent local regulation of AMPAR expression, however, remain unclear. Here, we tested whether microRNAs (miRNAs) are involved in N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent AMPAR expression. We used the 3' untranslated region of Gria1, which encodes the AMPA receptor subunit GluA1, to pull down miRNAs binding to it and analyzed these miRNAs using next-generation deep sequencing. Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA. We confirmed that miR-501-3p targets Gria1 and regulates its expression under physiological conditions. The expression of miR-501-3p and GluA1, moreover, is inversely correlated during postnatal brain development. miR-501-3p expression is up-regulated locally in dendrites through the NMDAR subunit GluN2A, and this regulation is required for NMDA-induced suppression of GluA1 expression and long-lasting remodeling of dendritic spines. These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

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Local regulation of dendritic GluA1 by miR-501-3p. In A and B, primary neurons were treated with NMDA (30 µM for 5 min) and collected at 90 min after treatment to test for miR-501-3p. (A) The level of miR-501-3p in whole cell lysates; n = 3–5 experiments. (B) miR-501-3p associated with RISC; n = 3–4 experiments. In C and D, transfected hippocampal neurons (17 DIV; 3 d after transfection) were treated with NMDA, and then stained for GluA1. (C) Representative images of transfected neurons. (D) Quantification of C; n = 14–29 cells for each group; AP5 was added 10 min before treatment and present during and after NMDA treatment. In E–G, hippocampal slices in which cell bodies of CA1 pyramidal neurons were removed or intact hippocampal slices were treated with NMDA (30 µM for 5 min). (E) Representative immunoblots. (F) Quantification of E; n = 4 rats for the intact slice group and 6 rats for the neuropil group. (G) miR-501-3p expression normalized to U6; n = 5 rats for the intact slice group and 6 rats for the neuropil group. Data are presented as mean ± SEM; Mann-Whitney U test is used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bar, 5 µm.
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fig4: Local regulation of dendritic GluA1 by miR-501-3p. In A and B, primary neurons were treated with NMDA (30 µM for 5 min) and collected at 90 min after treatment to test for miR-501-3p. (A) The level of miR-501-3p in whole cell lysates; n = 3–5 experiments. (B) miR-501-3p associated with RISC; n = 3–4 experiments. In C and D, transfected hippocampal neurons (17 DIV; 3 d after transfection) were treated with NMDA, and then stained for GluA1. (C) Representative images of transfected neurons. (D) Quantification of C; n = 14–29 cells for each group; AP5 was added 10 min before treatment and present during and after NMDA treatment. In E–G, hippocampal slices in which cell bodies of CA1 pyramidal neurons were removed or intact hippocampal slices were treated with NMDA (30 µM for 5 min). (E) Representative immunoblots. (F) Quantification of E; n = 4 rats for the intact slice group and 6 rats for the neuropil group. (G) miR-501-3p expression normalized to U6; n = 5 rats for the intact slice group and 6 rats for the neuropil group. Data are presented as mean ± SEM; Mann-Whitney U test is used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bar, 5 µm.

Mentions: Having validated that Gria1 is a target gene of miR-501-3p, we next examined whether miR-501-3p contributes to the expression change in GluA1 protein induced by NMDAR activation (Grooms et al., 2006). We first tested whether NMDAR activation also affects miR-501-3p expression. Primary hippocampal neurons (17 DIV) were treated with NMDA (30 µM for 5 min) and harvested 90 min after stimulation for RNA isolation and quantitative RT-PCR (qRT-PCR). We detected more miR-501-3p in NMDA-treated than in control cells, and the increase in miR-501-3p was blocked by the NMDAR antagonist (2R)-amino-5-phosphonovaleric acid (AP5; 100 µM; added 10 min before NMDA treatment and present during and after NMDA treatment until the collection of cells; Fig. 4 A). Hence, NMDA treatment enhances miR-501-3p expression.


miR-501-3p mediates the activity-dependent regulation of the expression of AMPA receptor subunit GluA1.

Hu Z, Zhao J, Hu T, Luo Y, Zhu J, Li Z - J. Cell Biol. (2015)

Local regulation of dendritic GluA1 by miR-501-3p. In A and B, primary neurons were treated with NMDA (30 µM for 5 min) and collected at 90 min after treatment to test for miR-501-3p. (A) The level of miR-501-3p in whole cell lysates; n = 3–5 experiments. (B) miR-501-3p associated with RISC; n = 3–4 experiments. In C and D, transfected hippocampal neurons (17 DIV; 3 d after transfection) were treated with NMDA, and then stained for GluA1. (C) Representative images of transfected neurons. (D) Quantification of C; n = 14–29 cells for each group; AP5 was added 10 min before treatment and present during and after NMDA treatment. In E–G, hippocampal slices in which cell bodies of CA1 pyramidal neurons were removed or intact hippocampal slices were treated with NMDA (30 µM for 5 min). (E) Representative immunoblots. (F) Quantification of E; n = 4 rats for the intact slice group and 6 rats for the neuropil group. (G) miR-501-3p expression normalized to U6; n = 5 rats for the intact slice group and 6 rats for the neuropil group. Data are presented as mean ± SEM; Mann-Whitney U test is used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bar, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4384731&req=5

fig4: Local regulation of dendritic GluA1 by miR-501-3p. In A and B, primary neurons were treated with NMDA (30 µM for 5 min) and collected at 90 min after treatment to test for miR-501-3p. (A) The level of miR-501-3p in whole cell lysates; n = 3–5 experiments. (B) miR-501-3p associated with RISC; n = 3–4 experiments. In C and D, transfected hippocampal neurons (17 DIV; 3 d after transfection) were treated with NMDA, and then stained for GluA1. (C) Representative images of transfected neurons. (D) Quantification of C; n = 14–29 cells for each group; AP5 was added 10 min before treatment and present during and after NMDA treatment. In E–G, hippocampal slices in which cell bodies of CA1 pyramidal neurons were removed or intact hippocampal slices were treated with NMDA (30 µM for 5 min). (E) Representative immunoblots. (F) Quantification of E; n = 4 rats for the intact slice group and 6 rats for the neuropil group. (G) miR-501-3p expression normalized to U6; n = 5 rats for the intact slice group and 6 rats for the neuropil group. Data are presented as mean ± SEM; Mann-Whitney U test is used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bar, 5 µm.
Mentions: Having validated that Gria1 is a target gene of miR-501-3p, we next examined whether miR-501-3p contributes to the expression change in GluA1 protein induced by NMDAR activation (Grooms et al., 2006). We first tested whether NMDAR activation also affects miR-501-3p expression. Primary hippocampal neurons (17 DIV) were treated with NMDA (30 µM for 5 min) and harvested 90 min after stimulation for RNA isolation and quantitative RT-PCR (qRT-PCR). We detected more miR-501-3p in NMDA-treated than in control cells, and the increase in miR-501-3p was blocked by the NMDAR antagonist (2R)-amino-5-phosphonovaleric acid (AP5; 100 µM; added 10 min before NMDA treatment and present during and after NMDA treatment until the collection of cells; Fig. 4 A). Hence, NMDA treatment enhances miR-501-3p expression.

Bottom Line: Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA.We confirmed that miR-501-3p targets Gria1 and regulates its expression under physiological conditions.These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Unit on Synapse Development and Plasticity, National Institute of Mental Health, and Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

ABSTRACT
The number of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in synapses determines synaptic strength. AMPAR expression can be regulated locally in dendrites by synaptic activity. The mechanisms of activity-dependent local regulation of AMPAR expression, however, remain unclear. Here, we tested whether microRNAs (miRNAs) are involved in N-methyl-D-aspartate (NMDA) receptor (NMDAR)-dependent AMPAR expression. We used the 3' untranslated region of Gria1, which encodes the AMPA receptor subunit GluA1, to pull down miRNAs binding to it and analyzed these miRNAs using next-generation deep sequencing. Among the identified miRNAs, miR-501-3p is also a computationally predicted Gria1-targeting miRNA. We confirmed that miR-501-3p targets Gria1 and regulates its expression under physiological conditions. The expression of miR-501-3p and GluA1, moreover, is inversely correlated during postnatal brain development. miR-501-3p expression is up-regulated locally in dendrites through the NMDAR subunit GluN2A, and this regulation is required for NMDA-induced suppression of GluA1 expression and long-lasting remodeling of dendritic spines. These findings elucidate a miRNA-mediated mechanism for activity-dependent, local regulation of AMPAR expression in dendrites.

Show MeSH
Related in: MedlinePlus