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. 2010 Jun;28(6):1060-70.
doi: 10.1002/stem.431.

MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase mind bomb-1

Richard D Smrt  1 , Keith E Szulwach, Rebecca L Pfeiffer, Xuekun Li, Weixiang Guo, Manavendra Pathania, Zhao-Qian Teng, Yuping Luo, Junmin Peng, Angelique Bordey, Peng Jin, Xinyu Zhao
Affiliations

MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase mind bomb-1

Richard D Smrt et al. Stem Cells. 2010 Jun.

Abstract

The maturation of young neurons is regulated by complex mechanisms and dysregulation of this process is frequently found in neurodevepmental disorders. MicroRNAs have been implicated in several steps of neuronal maturation including dendritic and axonal growth, spine development, and synaptogenesis. We demonstrate that one brain-enriched microRNA, miR-137, has a significant role in regulating neuronal maturation. Overexpression of miR-137 inhibits dendritic morphogenesis, phenotypic maturation, and spine development both in brain and cultured primary neurons. On the other hand, a reduction in miR-137 had opposite effects. We further show that miR-137 targets the Mind bomb one (Mib1) protein through the conserved target site located in the 3' untranslated region of Mib1 messenger RNA. Mib1 is an ubiquitin ligase known to be important for neurodevelopment. We show that exogenously expressed Mib1 could partially rescue the phenotypes associated with miR-137 overexpression. These results demonstrate a novel miRNA-mediated mechanism involving miR-137 and Mib1 that function to regulate neuronal maturation and dendritic morphogenesis during development.

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Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
miR-137 is enriched in neurons and is expressed in the dentate gyrus and the molecular layer of the hippocampus. (A) Identification of lineage specific miRNAs in A94-NSCs. Plotted are the ratios of RQ values determined by comparing A94-NSCs differentiated toward the neuron lineage to undifferentiated A94-NSCs over RQ values determined by comparing A94-NSCs differentiated toward the astrocyte lineage to undifferentiated A94-NSCs (Figure S1A and S1B). Ratios ≥8 were set to a value of 8. (B) miR-137 expression during neuronal differentiation of A94-NSCs for 0.5, 1, 2, 3, and 4 days (miR-137 expression calibrated to undifferentiated A94-NSCs, n=3, mean ± 95% CI). (C) Enrichment of miR-137 in E17 neurons as compared to mouse primary aNSCs (miR-137 expression calibrated to mouse primary aNSCs, n=3, mean ± SEM). (D–E) Hybridization with a miR-137-specific probe showed an enrichment of miR-137 within the DG and molecular layer of the hippocampus compared with miR-1, which is expressed at low levels in the CNS (h, hilus; g, dentate gyrus; m, molecular layer).
Figure 2
Figure 2
miR-137 regulates dendritic development and phenotypic maturation of new neurons in vivo. (A) A schematic diagram showing the retroviral vector used for in vivo miR-137 expression. miR-137 (sh-miR-137) or control miR (sh-Con) was expressed as a short hairpin under U6 RNA Polymerase III promoter while eGFP was expressed under a chicken β-actin (CAG) promoter. (B) Schematic diagram showing that control virus (sh-Con) was injected into the left hemisphere, and retrovirus expressing miR-137 (sh-miR-137) was injected into the right hemisphere. (C, D) Confocal z-stacks showing eGFP-expressing neurons at 4 weeks post-injection (4 wpi) with representative traces from both the sh-Con (C) and sh-miR-137 condition (D) (scale bar = 50 μm). (E) Neurons overexpressing sh-miR-137 show reduced dendritic complexity compared with controls, as determined by Scholl analysis. (F–H) Neurons overexpressing sh-miR-137 show reduced dendritic length (F), number of nodes (branch points, G), and dendritic ends (H). (I) Neurons overexpressing sh-miR-137 show reduced dendritic spine density. (J) Confocal z-stacks showing eGFP-expressing dendrites (scale bar = 20 μm). (K) A representative dendritic segment used for spine density analysis (* = p < 0.05)
Figure 3
Figure 3
Overexpression of miR-137 leads to altered neuronal maturation of new neurons in vivo. (A) Illustration showing the stage-specific neuronal markers that can be used to identify the maturation state of developing DG granule neurons. (B, C) Confocal images showing two representative eGFP-expressing neurons in the DG: a relatively immature eGFP neuron (B) expressed DCX (immature marker) but not NeuN (mature neuron) and a relatively mature eGFP+ neuron (C) expressed NeuN but not DCX. (D) The miR-137-overexpressing neuron population had decreased proportions of NeuN+ only (blue) mature neurons and of DCX+/NeuN+ (yellow) transitioning neurons, but increased proportion of DCX+ only (red) immature neurons compared with control (* = p < 0.05).
Figure 4
Figure 4
miR-137 is important for dendritic development in vitro. (A) A schematic diagram showing the lentiviral vector used for miR-137 expression. miR-137 (sh-miR-137) or control miR (sh-Con) was expressed as a short hairpin under U6 RNA Polymerase III promoter while eGFP was expressed under a CMV promoter. (B, C), E17 primary hippocampal neurons were transfected with lentiviral vectors expressing either control Con (B) or miR-137 (C), as well as eGFP. Single eGFP-expressing neurons were shown next to their representative traces (scale bar = 50 μm; 20x/oil). (D), Scholl analysis showing neurons overexpressing sh-miR-137 had reduced dendritic complexity compared with neurons overexpressing sh-Control. (E), Neurons overexpressing sh-miR-137 had reduced total dendritic length compared with controls. (F), Scholl analysis showing that neurons overexpressing miR-137 had reduced dendritic complexity compared with controls, whereas neurons transfected with an anti-miR-137 had opposite effect. (G), Neurons overexpressing miR-137 had reduced total dendritic length compared with neurons overexpressing miR-Control. On the other hand, neurons transfected with anti-miR-137 showed increased dendritic length compared to neurons transfected with anti-miR-Control. (*, p < 0.05, **, p < 0.01)
Figure 5
Figure 5
Mib1 is a functional target of miR-137. (A) A miR-137 target site was found in the Mib1 3′ untranslated region (3′UTR) as predicted by TargetScan software. The Mutant Mib1 3′UTR used in B-E with miR-137 site deleted is shown. (B) Schematic diagram showing the predicted seed region where miR-137 is expected to bind the rLuc-Mib1 3′-UTR (upper), and the mutated version lacking the binding site for miR-137. (C) Mib1-3′-UTR–dependent expression of a Renilla luciferase reporter gene (R-luc) was suppressed by miR-137 over 50% in DIV6 primary neurons at 48 hours post-transfection (n = 7, p < 0.001). The 3′-UTR-dependent Renilla luciferase (R-Luc) activities were normalized to control firefly luciferase (f-Luc) activities in the result of miR-137 coexpression was calculated relative to the miR-Con in C–E. (D) The mutant Mib1-3′-UTR alleviated the miR-137-mediated suppression of luciferase activity, suggesting that the action of miR-137 is specific to the miR-137 seed region within the Mib1-3′-UTR (n = 5, p < 0.001). (E) Mib1-3′-UTR–dependent expression of R-Luc was enhanced 28% by anti-miR-137 (n = 3, p < 0.05). (F), 10x fluorescence and bright field images showing high infection efficiency of lentivirus expressing sh-miR-137 (also eGFP) in E17 primary cortical neurons. (G) Primary neurons infected with lentivirus expressing sh-miR-137 had reduced Mib1 protein expression compared with neurons infected with lentivirus expressing sh-Con at 48 hours post-infection. (*, p < 0.05; ***, p < 0.001)
Figure 6
Figure 6
Mib1 could rescue the neuronal maturation deficits associated with miR-137 overexpression in vitro. (A) Overexpression of Mib1 enhanced dendritic length, whereas acute knockdown of Mib1 (Mib1 shRNA) reduced dendritic length. Mib1 expression partially rescued the miR-137-mediated reduction in dendritic length in cultured primary neurons. (B) Overexpression of Mib1 enhanced the dendritic complexity of cultured neurons, whereas acute knockdown of Mib1 reduced dendritic complexity. (C) Mib1 could rescue the miR-137-mediated reduction in dendritic complexity (F(1,35) = 18.51, p < 0.001). (D) A hypothetic model illustrating that miR-137 may regulate dendritic morphogenesis in developing neurons, at least in part, by translational regulation of Mib1. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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