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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: 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.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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The expression of miR-501-3p and GluA1 is inversely correlated during development. Proteins and total RNAs were isolated from the hippocampus of rats and cultured hippocampal neurons at indicated ages for immunoblotting (A, B, D, and E) or qRT-PCR (B and F). (A and D) Representative blots. (B and E) Quantification of A and D; n = 3–8 rats or cultures for each age. (B and F) qRT-PCR analysis of miR-501-3p expression in the rat hippocampus (B) and cultured hippocampal neurons (F); n = 3–8 rats or cultures for each age group. (C) Correlation between expression levels of miR-501-3p and GluA1 at different ages in the rat hippocampus. Data are presented as mean ± SEM; one-way ANOVA was used for statistical analysis among different groups; Mann-Whitney U test was used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005.
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fig3: The expression of miR-501-3p and GluA1 is inversely correlated during development. Proteins and total RNAs were isolated from the hippocampus of rats and cultured hippocampal neurons at indicated ages for immunoblotting (A, B, D, and E) or qRT-PCR (B and F). (A and D) Representative blots. (B and E) Quantification of A and D; n = 3–8 rats or cultures for each age. (B and F) qRT-PCR analysis of miR-501-3p expression in the rat hippocampus (B) and cultured hippocampal neurons (F); n = 3–8 rats or cultures for each age group. (C) Correlation between expression levels of miR-501-3p and GluA1 at different ages in the rat hippocampus. Data are presented as mean ± SEM; one-way ANOVA was used for statistical analysis among different groups; Mann-Whitney U test was used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Mentions: GluA1 protein expression increases during the postnatal development of neurons (Zhu et al., 2000). Because miR-501-3p targets Gria1, it might contribute to GluA1’s developmental change. To test this possibility, we analyzed miR-501-3p expression in developing hippocampal neurons. Small RNAs were isolated from the hippocampus of rats (1–56 d old) and cultured hippocampal neurons (7–28 DIV), and analyzed by quantitative PCR. In the rat hippocampus, the level of GluA1 protein increased between postnatal days 1–35 and then remained stable thereafter (Fig. 3, A and B). Likewise, cultured hippocampal neurons also progressively increased their GluA1 protein expression between 7 and 28 DIV (Fig. 3, D and E). The level of miR-501-3p, in contrast, decreased while that of GluA1 protein became higher (Fig. 3, B, C, and F). The inverse correlation between miR-501-3p and GluA1 protein expression suggests that miR-501-3p might be involved in the developmental increase in GluA1 protein expression. To test this possibility, we transfected cultured hippocampal neurons with miR-501-3p mimic (double-stranded RNAs functionally mimicking endogenous miR-501-3p) at 4, 11, 18, and 25 DIV and analyzed GluA1 proteins at 3 d after each transfection. The developmental increase in GluA1 protein expression was mitigated by miR-501-3p overexpression (Fig. 3, D and E). Transfection of antisense oligonucleotides against miR-501-3p, in contrast, augmented the developmental increase in GluA1 protein expression (Fig. 3, D and E). Hence, the developmental change in GluA1 protein expression is, at least in part, a result of diminishing miR-501-3p expression. Collectively, these results demonstrate that Gria1 is a physiological target of miR-501-3p.


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)

The expression of miR-501-3p and GluA1 is inversely correlated during development. Proteins and total RNAs were isolated from the hippocampus of rats and cultured hippocampal neurons at indicated ages for immunoblotting (A, B, D, and E) or qRT-PCR (B and F). (A and D) Representative blots. (B and E) Quantification of A and D; n = 3–8 rats or cultures for each age. (B and F) qRT-PCR analysis of miR-501-3p expression in the rat hippocampus (B) and cultured hippocampal neurons (F); n = 3–8 rats or cultures for each age group. (C) Correlation between expression levels of miR-501-3p and GluA1 at different ages in the rat hippocampus. Data are presented as mean ± SEM; one-way ANOVA was used for statistical analysis among different groups; Mann-Whitney U test was used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The expression of miR-501-3p and GluA1 is inversely correlated during development. Proteins and total RNAs were isolated from the hippocampus of rats and cultured hippocampal neurons at indicated ages for immunoblotting (A, B, D, and E) or qRT-PCR (B and F). (A and D) Representative blots. (B and E) Quantification of A and D; n = 3–8 rats or cultures for each age. (B and F) qRT-PCR analysis of miR-501-3p expression in the rat hippocampus (B) and cultured hippocampal neurons (F); n = 3–8 rats or cultures for each age group. (C) Correlation between expression levels of miR-501-3p and GluA1 at different ages in the rat hippocampus. Data are presented as mean ± SEM; one-way ANOVA was used for statistical analysis among different groups; Mann-Whitney U test was used for statistical analysis; *, P < 0.05; **, P < 0.01; ***, P < 0.005.
Mentions: GluA1 protein expression increases during the postnatal development of neurons (Zhu et al., 2000). Because miR-501-3p targets Gria1, it might contribute to GluA1’s developmental change. To test this possibility, we analyzed miR-501-3p expression in developing hippocampal neurons. Small RNAs were isolated from the hippocampus of rats (1–56 d old) and cultured hippocampal neurons (7–28 DIV), and analyzed by quantitative PCR. In the rat hippocampus, the level of GluA1 protein increased between postnatal days 1–35 and then remained stable thereafter (Fig. 3, A and B). Likewise, cultured hippocampal neurons also progressively increased their GluA1 protein expression between 7 and 28 DIV (Fig. 3, D and E). The level of miR-501-3p, in contrast, decreased while that of GluA1 protein became higher (Fig. 3, B, C, and F). The inverse correlation between miR-501-3p and GluA1 protein expression suggests that miR-501-3p might be involved in the developmental increase in GluA1 protein expression. To test this possibility, we transfected cultured hippocampal neurons with miR-501-3p mimic (double-stranded RNAs functionally mimicking endogenous miR-501-3p) at 4, 11, 18, and 25 DIV and analyzed GluA1 proteins at 3 d after each transfection. The developmental increase in GluA1 protein expression was mitigated by miR-501-3p overexpression (Fig. 3, D and E). Transfection of antisense oligonucleotides against miR-501-3p, in contrast, augmented the developmental increase in GluA1 protein expression (Fig. 3, D and E). Hence, the developmental change in GluA1 protein expression is, at least in part, a result of diminishing miR-501-3p expression. Collectively, these results demonstrate that Gria1 is a physiological target of miR-501-3p.

Bottom Line: 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.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