MicroRNAs are Necessary for BMP-7-induced Dendritic Growth in Cultured Rat Sympathetic Neurons

Abstract

Neuronal connectivity is dependent on size and shape of the dendritic arbor. However, mechanisms controlling dendritic arborization, especially in the peripheral nervous system, are not completely understood. Previous studies have shown that bone morphogenetic proteins (BMPs) are important initiators of dendritic growth in peripheral neurons. In this study, we examined the hypothesis that post-transcriptional regulation mediated by microRNAs (miRNAs) is necessary for BMP-7-induced dendritic growth in these neurons. To examine the role of miRNAs in BMP-7-induced dendritic growth, microarray analyses was used to profile miRNA expression in cultured sympathetic neurons from the superior cervical ganglia of embryonic day 21 rat pups at 6 and 24 h after treatment with BMP-7 (50 ng/mL). Our data showed that BMP-7 significantly regulated the expression of 43 of the 762 miRNAs. Of the 43 miRNAs, 22 showed robust gene expression; 14 were upregulated by BMP-7 and 8 were downregulated by BMP-7. The expression profile for miR-335, miR-664-1*, miR-21, and miR-23b was confirmed using qPCR analyses. Functional studies using morphometric analyses of dendritic growth in cultured sympathetic neurons transfected with miRNA mimics and inhibitors indicated that miR-664-1*, miR-23b, and miR-21 regulated early stages of BMP-7-induced dendritic growth. In summary, our data provide evidence for miRNA-mediated post-transcriptional regulation as important downstream component of BMP-7 signaling during early stages of dendritic growth in sympathetic neurons.

Keywords: Bone morphogenetic proteins; Dendrite; MicroRNA; Sympathetic neurons.

Fig. 1

Cluster diagram showing miRNAs differentially regulated by BMP-7 in cultured sympathetic neurons. The columns are clustered based on experimental conditions, with rows showing the individual miRNA transcripts. The six columns in each panel represent two control samples, two samples from cultures exposed to BMP-7 for 6 h, and two samples from cultures exposed to BMP-7 at 24 h. The relative expression of the miRNAs is shown on a color scale where red represents the highest level of upregulation and green represents the lowest level of downregulation at different time points. Statistical significance was assessed by one-way ANOVA, followed by Tukey’s test. a–c show the miRNAs altered at p < 0.01, p < 0.05, and p < 0.1, respectively

Fig. 2

Microarray analyses of high abundance miRNAs differentially regulated by BMP-7. Relative microarray signal for 23 miRNAs in total RNA samples obtained from neurons exposed to BMP-7 at 50 ng/mL for 6 h or 24 h. a miRNAs upregulated by BMP-7. b miRNAs downregulated by BMP-7. Statistically significant differences were determined using two-way ANOVA (p < 0.05). * Significant difference between control and BMP-7-exposed group at p < 0.05; #Significant differences following BMP-7 treatment over time at p < 0.05

Fig. 3

qPCR analysis of BMP-7 effects on miR-335, miR-664-1*, miR-21, miR-23b, and miR-26a expression. Total RNA extracted from cultured sympathetic neurons at 6 and 24 h after treatment with control medium or BMP-7 at 50 ng/mL was analyzed by qPCR to quantify expression of 5 miRNAs identified as differentially regulated by BMP-7 in the microarray screen. Expression of the target miRNA was normalized against snoRNA and U6 RNA for miR-335, miR-664-1*, snoRNA for miR-23b, and U6 RNA for miR-21. The figure shows data for the two biological replicates, each of which was run as 3 technical replicates. *Significantly different from control at p < 0.05 (N = 2)

Fig. 4

Optimizing small RNA transfection efficiency in cultured embryonic rat sympathetic neurons. a, b Representative photomicrographs of GAPDH protein in cultured sympathetic neurons transfected with either control siRNA (a) or 75 pmol of FAM-labeled siRNA for GAPDH (b). c Transfection efficiency in cultures transfected with 30 versus 75 pmol of FAM-labeled siRNA for GAPDH. N = 100 cells counted for each condition

Fig. 5

Overexpression of miR-23b and miR-21 or inhibition of miR-664-1* altering BMP-7-induced dendritic growth in sympathetic neurons. Representative fluorescence micrographs of cultured sympathetic neurons immunostained for the dendrite-selective cytoskeletal protein, microtubule associated protein-2 (MAP-2). Neurons were grown in the absence (a) or presence (b–g) of BMP-7 at 50 ng/ml from DIV 5-10. BMP-7-induced dendritic growth was examined in untransfected neurons (b) or in neurons transfected with negative controls for miRNA mimics (c), negative controls for miRNA inhibitors (d), specific miRNA mimics (e, f), or specific mRNA inhibitors (g). Bar = 50 μM

Fig. 6

Effect of miRNA modulation on number of primary dendrites in sympathetic neurons. Sympathetic neurons were transfected with mimics for miR-21, miR-23b, or miR-26a or inhibitors for miR-335 or miR-664-1* using Lipofectamine RNAimax. Controls were untransfected or transfected with negative controls (NC) for the miRNA mimics and inhibitors. Following transfection, cultures were treated with control medium or BMP-7 (50 ng/mL) for 5 days. The number of dendrites per neuron was quantified in cultures immunostained for MAP-2 to identify dendritic processes. Data are expressed as the mean ± SEM. a–c show the graphical representation of the data and d–f show the numerical data including the N for each condition, mean, and SEM. Statistical significance was assessed using one-way ANOVA, followed by Tukey’s post hoc test. *Significantly different from cultures transfected with respective negative controls in the presence of BMP-7 at p ≤ 0.05

Fig. 7

Effect of miRNA modulation on total dendritic arbor of BMP-7-treated neurons. Sympathetic neurons were transfected with mimics for miR-21 or miR-23b or inhibitors for miR-664-1* using Lipofectamine RNAimax. Controls were untransfected or transfected with negative controls (NC) for the miRNA mimics and inhibitors. Following transfection, cultures were treated with control medium or BMP-7 (50 ng/mL) for 5 days. The total length of the dendritic arbor per neuron was quantified in cultures immunostained for MAP-2 to identify dendritic processes. Data are expressed as the mean ± SEM. a–c show the graphical representation of the data and d–f show the numerical values including N for each condition, mean, and SEM. Statistical significance was assessed using one-way ANOVA, followed by Tukey’s post hoc test. *Significantly different from cultures transfected with respective negative controls in the presence of BMP-7 at p ≤ 0.05