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. 2008 Jul 1;105(26):9093-8.
doi: 10.1073/pnas.0803072105. Epub 2008 Jun 24.

An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP

Gary A Wayman  1 , Monika Davare, Hideaki Ando, Dale Fortin, Olga Varlamova, Hai-Ying M Cheng, Daniel Marks, Karl Obrietan, Thomas R Soderling, Richard H Goodman, Soren Impey
Affiliations

An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP

Gary A Wayman et al. Proc Natl Acad Sci U S A. .

Abstract

Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synapse growth and plasticity remain largely uncharacterized. Here, we show that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway. Introduction of miR132 into hippocampal neurons enhanced dendrite morphogenesis whereas inhibition of miR132 by 2'O-methyl RNA antagonists blocked these effects. Furthermore, neuronal activity inhibited translation of p250GAP, a miR132 target, and siRNA-mediated knockdown of p250GAP mimicked miR132-induced dendrite growth. Experiments using dominant-interfering mutants suggested that Rac signaling is downstream of miR132 and p250GAP. We propose that the miR132-p250GAP pathway plays a key role in activity-dependent structural and functional plasticity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
miR132 is an activity- and CREB-regulated rapid response gene. (A) Hippocampal neurons were treated with 20 μM bicuculline for the indicated times. RNA was isolated, reverse-transcribed, and analyzed by real-time PCR with premiR132 cDNA primers (black squares) or mature-miR132 primers (red squares) by using the ABI TaqMan miRNA real-time PCR. The data were normalized to GAPDH cDNA levels also determined by real-time PCR (±SEM, n = 5–6). Error is SEM (**, P < 0.01). (B) Hippocampal neurons were treated with bicuculline for 1 h ±50 μM D-APV. PremiR132 levels were analyzed by real-time PCR and normalized to GAPDH. (C) Hippocampal neurons were pretreated with 10 μM UO126 or 10 μM KN-62 for 90 min and stimulated with 20 μM bicuculline ± inhibitors, as indicated, for 2 h. PremiR132 levels were analyzed by real-time PCR and normalized to GAPDH. (D) Hippocampal neurons were transfected with vector control or dominant-negative CREB (ACREB) and then treated 36 h later with KCl for 60 min. RNA was reverse-transcribed, analyzed by real-time PCR with premiR132 primers, and normalized to GAPDH cDNA levels.
Fig. 2.
Fig. 2.
Activity and CREB-regulated dendritic growth require miR132. (A–C) Hippocampal neurons were transfected with plasmids encoding MAP2B-EGFP ± miR132 or 2′OM-132 and treated with ±20 μM bicuculline for 48 h. Dendritic length (B) and branch number (C) were quantified at 9 DIV (***, P < 0.001). (D–F) Hippocampal neurons were transfected with plasmids encoding MAP2B-EGFP ± caCREB ± 2′OM-miR132. Dendritic length (E) and branch number (F) were quantified at 9 DIV.
Fig. 3.
Fig. 3.
Synaptic activity regulates p250GAP via miR132. (A) The MRE in p250GAP is required for miR132 regulation. Hippocampal neurons were transfected with pRL-TK reporter plasmid (pRL) or a reporter containing the p250GAP MRE in the correct or reverse orientation. An excess of empty vector (EV) or miR132 plasmid was cotransfected. Renilla luciferase activity was normalized to cotransfected firefly luciferase activity. Data are expressed as percentage of wild-type pRL signal, and error is SEM. (B) miR132 inhibits p250GAP expression. Hippocampal neurons were transfected by electroporation with plasmids encoding either GFP-WT-p250GAP (wild type) or GFP-mt-p250GAP (miR132 MRE mutant) ± empty vector (EV) or miR132. After transfection, the neurons were cultured for 48 h. p250GAP expression was analyzed by Western blotting using an anti-GFP antibody. (C) MiR132 inhibits expression of p250GAP. Hippocampal neurons were transfected by Amaxa electroporation with either empty vector (EV) or plasmid encoding miR132. After transfection, the neurons were cultured for 48 h, and p250GAP expression was analyzed by Western blotting using an anti-p250GAP antibody. (D) Hippocampal neurons were treated with and without 20 μM bicuculline for 24 h and immunostained with anti-p250GAP. Representative images of control and stimulated neurons are shown. (E) Hippocampal neurons were treated ±20 μm bicuculline ± 50 μM D-APV for 24 h. Endogenous p250GAP and ERK2 expression was analyzed by Western blotting. (F) Hippocampal neurons were pretreated ±KN-93 and UO126 for 2 h, then stimulated with ±20 μM bicuculline for 24 h. Endogenous p250GAP and ERK2 expression was analyzed by Western blotting in triplicate samples. (G) Primary hippocampal DIV 7 neurons were treated with antisense miR132 antagomir or control sense antagomir for 24 h. Cells were then treated with KCl for 24 h and immunoblotted for endogenous p250GAP and ERK2. Quantitation of p250GAP levels in G is shown at the bottom of the figure (n = 6). Statistical analyses used ANOVA and Tukey's post test (±SEM, ***, P < 0.001).
Fig. 4.
Fig. 4.
miR132 regulation of p250GAP is required for activity- and CREB-dependent dendritic growth. (A and B) Hippocampal neurons were transfected with plasmid encoding MAP2B-EGFP ± miR132 si-p250GAP (short-hairpin siRNA targeting p250GAP) and treated with ±20 μM bicuculline for 48 h. Dendritic length and branching were quantified at 9 DIV. (C and D) Expression of miR132-insensitive p250GAP (mt-p250GAP), but not wild-type p250GAP (WT-p250GAP), blocks bicuculline- and miR132-stimulated dendritic growth. Hippocampal neurons (7 DIV) were transfected with plasmids encoding MAP2B-EGFP ± either WT- or mt-p250GAP ± miR132 then treated with ±20 μM bicuculline. (E and F) Hippocampal neurons were transfected with plasmid encoding MAP2B-EGFP ± miR132 or si-p250GAP ± dnRac or dnCdc42 for 48 h. Dendritic growth was quantified at 9 DIV. Statistical analyses used ANOVA and Tukey's post test (±SEM, **, P < 0.01; ***, P < 0.001).
Fig. 5.
Fig. 5.
miR132 regulates dendritic morphology in organotypic hippocampal slices. (A) Hippocampal slices were cultured for 3 days, subjected to biolistic transfections with TFP ± other plasmids as indicated, allowed to recover for 1 day, then treated for 2 days with ±20 mM bicuculline. (B) Synaptic activity, miR132, and p250GAP regulate dendritic length of CA1 pyramidal neurons in slice culture. Representative examples of control or stimulated CA1 pyramidal neurons are depicted. Statistical analyses used ANOVA and Tukey's post test (±SEM, ***, P < 0.001).
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