MicroRNA-451a overexpression induces accelerated neuronal differentiation of Ntera2/D1 cells and ablation affects neurogenesis in microRNA-451a-/- mice

Abstract

MiR-451a is best known for its role in erythropoiesis and for its tumour suppressor features. Here we show a role for miR-451a in neuronal differentiation through analysis of endogenous and ectopically expressed or silenced miR-451a in Ntera2/D1 cells during neuronal differentiation. Furthermore, we compared neuronal differentiation in the dentate gyrus of hippocampus of miR-451a-/- and wild type mice. MiR-451a overexpression in lentiviral transduced Ntera2/D1 cells was associated with a significant shifting of mRNA expression of the developmental markers Nestin, βIII Tubulin, NF200, DCX and MAP2 to earlier developmental time points, compared to control vector transduced cells. In line with this, accelerated neuronal network formation in AB.G.miR-451a transduced cells, as well as an increase in neurite outgrowth both in number and length was observed. MiR-451a targets genes MIF, AKT1, CAB39, YWHAZ, RAB14, TSC1, OSR1, POU3F2, TNS4, PSMB8, CXCL16, CDKN2D and IL6R were, moreover, either constantly downregulated or exhibited shifted expression profiles in AB.G.miR-451a transduced cells. Lentiviral knockdown of endogenous miR-451a expression in Ntera2/D1 cells resulted in decelerated differentiation. Endogenous miR-451a expression was upregulated during development in the hippocampus of wildtype mice. In situ hybridization revealed intensively stained single cells in the subgranular zone and the hilus of the dentate gyrus of wild type mice, while genetic ablation of miR-451a was observed to promote an imbalance between proliferation and neuronal differentiation in neurogenic brain regions, suggested by Ki67 and DCX staining. Taken together, these results provide strong support for a role of miR-451a in neuronal maturation processes in vitro and in vivo.

Fig 1. Endogenous miR-451a expression increases during neuronal differentiation.

Endogenous miR-451a expression was upregulated time-dependently during neuronal differentiation of NT2 cells (A). (B-F) show neuronal microscopic images of NT2 cells during retinoic acid-induced differentiation in vitro. The statistical significance of changes in miR-451a expression was tested using Friedman‘s test. *p≤0.05 after Bonferroni correction. n = 4 biological replicates; Scale bars: 100 μm. Error bars show standard error of the mean (SEM).

Fig 2. Differential expression of neural precursor and neuronal differentiation markers upon miR-451a overexpression.

Expression of neural stem/precursor cell marker Nestin (A), was downregulated at days 22 and 28 in AB.G.miR-451a transduced cells, compared to control cells. Expression profiles of neuronal differentiation markers GFAP (B), βIII Tubulin (C), NF200 (D), DCX (E) and MAP2 (F) were shifted significantly to an earlier developmental time point in AB.G.miR-451a transduced cells compared to the control (AB.G.ct). The statistical significance of the observed changes was assessed using the Mann-Whitney U-test. *p≤0.05. n = 3 biological replicates. Error bars show standard error of the mean (SEM).

Fig 3. MiR-451a overexpression promotes neurite outgrowth from neurospheres.

AB.G.ct neurospheres showed less neurite outgrowth from adherent neurospheres at day 22 of differentiation (A) as compared to AB.G.miR-451a neurospheres (B). G-0 neurospheres (G-0: control for miR-451a silencing vector) (C) showed more neurite outgrowth than G-U6-451PT neurospheres (miR-451a silenced) (D). Most branched and densest neurite networks with longer neurites were observed in AB.G.miR-451a neurospheres (E-H). On the other hand, remarkably shorter neurites and lower network density were observed upon miR-451a knockdown (D, H). Neurospheres were immunostained for Neurofilament heavy chain (NF200). Quantification of mean neurite length (n≥3 neurospheres per group) (I) confirmed the statistical significance of longer neurite observation in miR-451a overexpressing neurospheres. The mean ratio of the length of single neurites to neurosphere diameter at day 22 of differentiation in AB.G.miR-451a and G-U6-451PT transduced cells (J) (n≥8 neurospheres per group) further confirmed the effect of miR-451a in neurite outgrowth from differentiating neurospheres. Statistical significance of the differences were tested by Kruskal-Wallis Test with Bonferroni correction for post-hoc analyses (I) or by t-test following confirmation of normal distribution with Wilk-Shapiro test (J). *p<0.05, ***p<0.001 in between compared groups; ^#p<0.05, ^###p<0.001 as compared to the respective control vectors (AB.G.ct or G-0). Error bars represent standard error of the mean (SEM). Scale bars: 100 μm.

Fig 4. NT2 cells show different morphology upon miR-451a overexpression.

MiR-451a overexpression resulted in a more compact cell shape with smaller soma size (A, B), while larger soma size was observed with miR-451 knock-down (C, D). Quantitative analysis of soma size in all groups of cells confirmed the significance of these observations (E). Mean fluorescence intensity of TNS4 fluorescent immunocytochemistry (ICC) in these cells showed significantly lower TNS4 expression in G-U6-451PT transduced group (miR-451a knock down) as compared to both AB.G.miR-451a (miR-451a overexpression) and G-0 (control vector) transduced groups (F). (G) shows a representative image of Western blot (WB) for TNS4 protein expression in all groups with β-Tubulin as internal control. ICC and WB pictures are representative of at least 3 different experiments. The statistical significance of the differences was assessed with Welch’s variance-weighted ANOVA followed by Games Howell test for post-hoc analysis (n≥6 neurospheres per group). Error bars show standard error of the mean (SEM). *p≤0.05, ***p≤0.001. Scale bars: 20 μm.

Fig 5. MRNA expression of miR-451a targets in AB.G.ct and AB.G.miR-451a transduced NT2 cells.

mRNA expression of predicted and validated target genes of miR-451a differ remarkably in AB.G.ct and AB.G.miR-451a transduced cells during neuronal differentiation. (A1-A6) show target genes that were significantly downregulated in response to miR-451a overexpression. (B1-B7) show a second group of target genes that exhibited a modified expression profile upon miR-451a overexpression. The statistical significance of the observed differences was assessed with the Mann-Whitney U-test, *p≤0.05. n = 3 biological replicates. Error bars show standard error of the mean (SEM).

Fig 6. MiRNA-451a expression in hippocampus of wild type mice.

miRNA-451a in situ hybridization was carried out on coronal brain sections of male C57BL/6N mice at postnatal day 5 (A), postnatal day 15 (B) and adult stage (C). (A´, B´, C´) show the respective regions at higher magnification. Intensively stained single cells were found predominantly in the subgranular zone and the hilus (arrows). (D) shows a hybridization control with a scrambled riboprobe. (E) shows quantitative analysis of miR-451a expression in hippocampal formation assessed by qPCR at postnatal day 5, day 15 and at the adult stage. In situ hybridizations were conducted with at least 15 coronal sections per animal with n≥5 animals per group. qPCR analysis was conducted with n = 3 animals per group with 8 qPCR replicates. ns: not significant. Scale bars: 100 μm.

Fig 7. MiR-451a^-/- mice show higher cell proliferation in adult neurogenic regions.

Proliferating cells were quantified in coronal brain sections of wild type and miR-451a^-/- mice at postnatal days 25, 30, 35, 40 and 50 by Ki67 immunostaining (A). Markedly higher numbers of Ki67⁺ cells were observed in miR-451a^-/- animals, particularly at days 25 and 40 in the subgranular zone (SGZ, demarcated with dashed lines) of the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ, demarcated with dashed lines) of lateral ventricles (LV). Representative microscopy images for day 25 and day 40 animals are shown in (B). Cell nuclei were counterstained with DAPI. Immunostaining and quantification were conducted on at least two coronal sections per animal with n≥4 animals per group per time point. The statistical significance of the observed differences was assessed with the Mann-Whitney U Test. The differences were statistically not significant. Circle: outliers, Star: extreme outliers. Scale bars: 50 μm.

Fig 8. Imbalanced expression of neuronal differentiation marker doublecortin in miR-451a^-/- mice.

Neuronal differentiation in the subgranular zone (SGZ) of dentate gyrus (DG) in hippocampus was evaluated by doublecortin (DCX) immunostaining of coronal brain sections of wild type and miR-451a^-/- animals. Qualitative evaluation revealed markedly lower expression of DCX in the miR-451a^-/- group at postnatal days 30 (A, B) and 50 (C, D). Immunostainings were conducted with at least two coronal sections per animal with n≥4 animals per group per time point. Scale bars: 50 μm.