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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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Suppression of GluA1 expression by miR-501-3p is required for long-lasting spine remodeling induced by NMDAR activation. Cultured hippocampus neurons (14 DIV) were transfected with designated plasmids or oligonucleotides, treated with NMDA (30 µM for 5 min) at 3–5 d after transfection, and imaged before and at 10, 30, and 90 min after treatment. (A) Representative images; yellow boxes indicate the dendrites in the high magnification images. (B) Quantification of A; n = 6–8 neurons for each group. Data are presented as mean ± SEM; one-way ANOVA was used for comparing spine size and elimination among different groups and P < 0.005 for all time points except for spine elimination at 10 min after NMDA treatment. Mann-Whitney U test was used for comparison between the NMDA treated, venus transfected group versus all other groups at the same time point. The asterisks are color-coded and indicate that the conditions labeled with the same color are significantly different from the NMDA treated, venus transfected group at the same time point. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bars: (A, top) 20 µm; (A, high magnification) 5 µm.
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fig5: Suppression of GluA1 expression by miR-501-3p is required for long-lasting spine remodeling induced by NMDAR activation. Cultured hippocampus neurons (14 DIV) were transfected with designated plasmids or oligonucleotides, treated with NMDA (30 µM for 5 min) at 3–5 d after transfection, and imaged before and at 10, 30, and 90 min after treatment. (A) Representative images; yellow boxes indicate the dendrites in the high magnification images. (B) Quantification of A; n = 6–8 neurons for each group. Data are presented as mean ± SEM; one-way ANOVA was used for comparing spine size and elimination among different groups and P < 0.005 for all time points except for spine elimination at 10 min after NMDA treatment. Mann-Whitney U test was used for comparison between the NMDA treated, venus transfected group versus all other groups at the same time point. The asterisks are color-coded and indicate that the conditions labeled with the same color are significantly different from the NMDA treated, venus transfected group at the same time point. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bars: (A, top) 20 µm; (A, high magnification) 5 µm.

Mentions: The number of AMPAR in synapses positively correlates with the size of dendritic spines (Baude et al., 1995; Matsuzaki et al., 2001, 2004; Passafaro et al., 2003). Having found that miR-501-3p contributes to down-regulation of GluA1 protein by NMDAR activation, we tested whether this regulation is also involved in NMDAR-mediated spine remodeling. To analyze the morphological changes of spines induced by NMDAR activation, neurons (17 DIV; 3–5 d after transfection with the venus, a YFP mutant construct for visualization of dendritic spines) were treated with NMDA (30 µM for 5 min), and the same spines were imaged before and at 10, 30, and 90 min after NMDA stimulation. Consistent with our previous findings (Hu et al., 2014), NMDA treatment caused retraction and rapid, long-lasting shrinkage of spines (Fig. 5). To test whether miR-501-3p is involved in this process, we transfected neurons (14 DIV) with the venus construct along with miR-501-3p antisense or scrambled oligonucleotides. Although transfection of miR-501-3p antisense oligonucleotides caused an increase in dendritic GluA1 protein (Fig. 2), it left the size and density of spines intact and had no effect on spine size and spine elimination in unstimulated neurons during the live imaging experiment (Fig. 5 and Fig. S2). After NMDA treatment, spines in neurons transfected with miR-501-3p antisense oligonucleotides shrunk at 10 min, but they recovered thereafter, reaching the prestimulation size by 90 min after stimulation (Fig. 5). NMDA-induced spine elimination and increase in GluA1 protein expression were also inhibited by transfection of miR-501-3p antisense oligonucleotides (Fig. 5 and Fig. S3). Transfection of scrambled oligonucleotides had no effect on NMDA-induced spine shrinkage, spine retraction, or GluA1 protein increase (Fig. 5 and Fig. S3). These results indicate that miR-501-3p is required for long-lasting spine restructuring induced by NMDAR activation.


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)

Suppression of GluA1 expression by miR-501-3p is required for long-lasting spine remodeling induced by NMDAR activation. Cultured hippocampus neurons (14 DIV) were transfected with designated plasmids or oligonucleotides, treated with NMDA (30 µM for 5 min) at 3–5 d after transfection, and imaged before and at 10, 30, and 90 min after treatment. (A) Representative images; yellow boxes indicate the dendrites in the high magnification images. (B) Quantification of A; n = 6–8 neurons for each group. Data are presented as mean ± SEM; one-way ANOVA was used for comparing spine size and elimination among different groups and P < 0.005 for all time points except for spine elimination at 10 min after NMDA treatment. Mann-Whitney U test was used for comparison between the NMDA treated, venus transfected group versus all other groups at the same time point. The asterisks are color-coded and indicate that the conditions labeled with the same color are significantly different from the NMDA treated, venus transfected group at the same time point. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bars: (A, top) 20 µm; (A, high magnification) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: Suppression of GluA1 expression by miR-501-3p is required for long-lasting spine remodeling induced by NMDAR activation. Cultured hippocampus neurons (14 DIV) were transfected with designated plasmids or oligonucleotides, treated with NMDA (30 µM for 5 min) at 3–5 d after transfection, and imaged before and at 10, 30, and 90 min after treatment. (A) Representative images; yellow boxes indicate the dendrites in the high magnification images. (B) Quantification of A; n = 6–8 neurons for each group. Data are presented as mean ± SEM; one-way ANOVA was used for comparing spine size and elimination among different groups and P < 0.005 for all time points except for spine elimination at 10 min after NMDA treatment. Mann-Whitney U test was used for comparison between the NMDA treated, venus transfected group versus all other groups at the same time point. The asterisks are color-coded and indicate that the conditions labeled with the same color are significantly different from the NMDA treated, venus transfected group at the same time point. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bars: (A, top) 20 µm; (A, high magnification) 5 µm.
Mentions: The number of AMPAR in synapses positively correlates with the size of dendritic spines (Baude et al., 1995; Matsuzaki et al., 2001, 2004; Passafaro et al., 2003). Having found that miR-501-3p contributes to down-regulation of GluA1 protein by NMDAR activation, we tested whether this regulation is also involved in NMDAR-mediated spine remodeling. To analyze the morphological changes of spines induced by NMDAR activation, neurons (17 DIV; 3–5 d after transfection with the venus, a YFP mutant construct for visualization of dendritic spines) were treated with NMDA (30 µM for 5 min), and the same spines were imaged before and at 10, 30, and 90 min after NMDA stimulation. Consistent with our previous findings (Hu et al., 2014), NMDA treatment caused retraction and rapid, long-lasting shrinkage of spines (Fig. 5). To test whether miR-501-3p is involved in this process, we transfected neurons (14 DIV) with the venus construct along with miR-501-3p antisense or scrambled oligonucleotides. Although transfection of miR-501-3p antisense oligonucleotides caused an increase in dendritic GluA1 protein (Fig. 2), it left the size and density of spines intact and had no effect on spine size and spine elimination in unstimulated neurons during the live imaging experiment (Fig. 5 and Fig. S2). After NMDA treatment, spines in neurons transfected with miR-501-3p antisense oligonucleotides shrunk at 10 min, but they recovered thereafter, reaching the prestimulation size by 90 min after stimulation (Fig. 5). NMDA-induced spine elimination and increase in GluA1 protein expression were also inhibited by transfection of miR-501-3p antisense oligonucleotides (Fig. 5 and Fig. S3). Transfection of scrambled oligonucleotides had no effect on NMDA-induced spine shrinkage, spine retraction, or GluA1 protein increase (Fig. 5 and Fig. S3). These results indicate that miR-501-3p is required for long-lasting spine restructuring induced by NMDAR activation.

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