microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus

Abstract

Newborn neurons in the dentate gyrus of the adult hippocampus rely upon cAMP response element binding protein (CREB) signaling for their differentiation into mature granule cells and their integration into the dentate network. Among its many targets, the transcription factor CREB activates expression of a gene locus that produces two microRNAs, miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activity-dependent dendritic growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, miR-132 and -212 and their reverse strands miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Generation and validation of floxed miR-212/132 mice. (A) Schematic of miR-212/132 targeting vector before and after Cre-mediated excision. The floxed mice have a PGK-Neo cassette upstream of the miR-212/132 locus. A REST binding site is located between CRE1 and CRE2 and between miR-212 and CRE3. (B) PCR analysis showing that the floxed allele is excised only when Cre is present (lane 1) in heterozygous floxed mice. (C) Mature miR-132 and miR-212 are decreased in the cortex of floxed mice compared with C57Bl6/J mice and are absent in the knockout mice generated by crossing the floxed mouse with a Deleter-Cre mouse (miR-132: f2,15 = 20.3, P < 0.001; Tukey's post hoc test, P < 0.01 for all comparisons; miR-212: f2,15 = 98.8, P < 0.001; Tukey's post hoc test, P < 0.01 for all comparisons; error bars show SEM). There was no change in the level of miR-16, an unrelated microRNA.

Fig. 2.

Loss of miR-212/132 decreases dendritic length and branching in newborn neurons in the adult hippocampus. Representative neurons are shown 21 d postinjection. (A) C57Bl6/J mouse injected with mCherry virus. (C and E) Floxed miR-212/132 mouse injected with mCherry and GFP-Cre. (C shows a neuron from a floxed mouse that did not coexpress GFP-Cre.) (B, D, F, and H) Dendritic branching is decreased in Flox-Ctrl and Flox-Cre mice (f2,79 = 68.0, P < 0.001; Tukey's post hoc test, P < 0.001 for all comparisons). (G) Total dendritic length is decreased in Flox-Ctrl and Flox-Cre neurons (f2,79 = 83.9, P < 0.001; Tukey's post hoc test, P < 0.001 for all comparisons; error bars show SEM).

Fig. 3.

Ratiometric sensors for microRNA activity. (A) A bidirectional promoter (Bi-CMV) simultaneously drives expression of AcGFP whose 3′ UTR contains three MREs. DsRedEx1 is expressed in the antisense direction as an internal control. Lentiviral packaging elements include psi and self-inactivating (SIN) LTRs. (B) SH-SY5Y neuroblastoma cells expressing the 132-MRE sensor were transfected with increasing amounts (up to 10 nM total) of miR-132 mimic and a miR-Scrm mimic (pink = 0 nM; cyan = 0.1 nM; orange = 1.0 nM; green = 10 nM). The G/R ratio in 1 × 104 individual cells per condition was assessed using flow cytometry and graphed as a cumulative frequency distribution. Black and blue lines represent the response of the negative control No-MRE sensor to the same concentrations of miR-132 mimic. (C) The 132-MRE sensor responded to the exogenous (0.1 nM) miR-132 mimic (blue) but not to the highly related miR-212 mimic (red). (D) 132-MRE and 212-MRE sensors were expressed in SH-SY5Y cells under growth (white bars) and differentiation (black bar) conditions (1% serum + 10 ng/mL retinoic acid for 2 d). The G/R ratios were normalized to that of the Scrm-MRE negative control. (E) Addition of 2′O-methyl inhibitors specific to miR-132 confirmed that the 132-MRE sensor detected endogenous miR-132 in SH-SY5Y cells. (F) The same concentrations of 2′O-methyl inhibitors did not affect expression of the No-MRE control vector.

Fig. 4.

miR-132 is the predominant functional product of the miR-212/132 locus in primary hippocampal neurons. (A) Taqman assays from DIV 0 and 4 dissociated hippocampal cultures measuring the relative abundance of mature miR-132 and miR-212. (B–E) Sensors (black bars) for each of the putative microRNAs from the miR-212/132 locus were expressed in dissociated hippocampal cultures and assessed with flow cytometry after DIV 1 or DIV 3–5. The median G/R ratios of 5 × 103 neurons were normalized to that of control sensors (white bars) in the same experiment, and the SEM from seven cultures is shown. **P < 0.01; ANOVA P < 0.001. (F) Representative confocal images of the sensors in DIV 4 neurons. Scrm-MRE control corresponds to C. elegans miR-239b.

Fig. 5.

miR-132 is the primary functional product of the miR-212/132 locus in immature granule neurons in vivo. The dentate gyrus of WT mice was stereotactically injected with lentiviral microRNA sensors, and immature granule neurons were detected with DCX staining (Alexa Fluor 647). (A) The mean percentage of DCX-positive neurons that also express GFP (f4,106 = 23.4; Dunnett's multiple comparison post hoc test **P < 0.01; Error bars show SEM). (B) Representative confocal images (n = 15 sections from two or three mice per condition).