All of our experiments were performed in accordance with the recommendations of the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong. The protocol was approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (Ref. No. 16-060-MIS).

Mouse NSCs were obtained from the SVZ of an adult mouse brain (Walker and Kempermann, 2014). Briefly, the lateral wall of lateral ventricle was dissected and dissociated into single cells by 0.05% trypsin-EDTA, and the cells were seeded into Petri dishes with KnockOut TM D-MEM/F-12 medium containing StemPro Neural Supplement (2%), bFGF (20 ng/ml), EGF (20 ng/ml), GlutaMAX-I Supplement (2 mM), heparin (6 units/ml) and ascorbic acid (200 μM). The SVZ-derived neurospheres were incubated for 6–7 days and replaced with fresh medium every 3 days. For neuron differentiation, NSCs were plated into dishes coated with poly-D-lysine (Sigma) and laminin (Invitrogen) in a 1:1 mix of Neurobasal Medium and DMEM/F12 supplemented with N2 (Gibco), B27 (Gibco), 10 ng/ml BDNF (Peprotech), and 200 mM ascorbic acid (Sigma). Half of the medium was replaced every other day. After 2 weeks of culture, the ratio of the mixture of Neurobasal Medium and DMEM/F12 was changed to 3:1; the N2 supplement was reduced to 0.5%, and the BDNF was increased to 20 ng/ml (Thier et al., 2012).

Primary cortical neuron cultures were prepared from embryonic day 18.5 (E18.5) mouse brain. Chamber slides (Nunc) were coated with 100 μg/ml poly-L-lysine (Sigma) and 5 μg/ml laminin (Invitrogen) at 37°C in an incubator for 3 h to overnight, then washed twice with distilled water, and air dried 20 min. Cortices were digested with 1× trypsin-EDTA for 15 min at 37°C, and then the reaction was stopped with trypsin inhibitor for 3 min at room temperature. After washing with dissection buffer containing 1× HBSS without Ca²⁺ and Mg²⁺ (Invitrogen), 10 mM HEPES buffer (Invitrogen), 0.5% glucose and 100 units/ml antibiotics (penicillin and streptomycin) (Invitrogen), the tissues were triturated by gently pipetting in plating medium containing MEM without glutamine (Life Technologies), 10% FBS (Gibco), 1 mM L-glutamine (Invitrogen), 10 mM Hepes (Invitrogen), and 50 units/ml antibiotics (penicillin and streptomycin) (Invitrogen) until fully dissociated. Cells were diluted to an appropriate concentration and plated in chamber slides (Nunc) pre-coated with poly-L-lysine (Sigma) and laminin (Invitrogen). Three hours later, cells were grown in culture medium containing neurobasal medium (Invitrogen), 2% B27 supplement (Invitrogen), 0.5 mM L-glutamine (Invitrogen), and 50 units/ml antibiotics (penicillin and streptomycin) (Invitrogen) (Kaech and Banker, 2006).

Proteins were extracted with RIPA buffer containing protease inhibitor cocktail, and the concentration of which was measured with a BCA kit. Proteins (10–20 μg) were loaded into 8–10% SDS-polyacrylamide gel. Following SDS-PAGE, proteins were transferred to nitroate cellulose membrane, blocked with 5% non-fat milk, incubated overnight with primary antibodies at 4°C, and washed three times with TBST for 10 min. After the proteins had been incubated with secondary antibodies for 2 h at room temperature, signals were detected by enhanced chemiluminescent (Thermo Fisher Scientific). The following primary antibodies were used: rabbit anti-PTEN (1:1000; Cell Signaling Technology #9188), rabbit anti-p^(Ser380) PTEN (1:1000; Cell Signaling Technology #9551), rabbit anti-AKT (1:1000; Cell Signaling Technology #4691), rabbit anti-p^(Ser473)-AKT (1:1000; Cell Signaling Technology #4060), rabbit anti-p^(Thr308)-AKT (1:1000; Cell Signaling Technology #13038), phospho-p44/42 MAPK (Erk1/2)^{(Thr202/Tyr204)} (1:1000; Cell Signaling Technology #9106), p44/42 MAPK (Erk1/2) (1:1000; Cell Signaling Technology #9102), mouse anti-β-actin (1:2000; ImmunoWay #YM3028), rabbit anti-Neurofilament-L (NF-L; 1:1000; Cell Signaling Technology #2837), mouse anti-Neurofilament-M (NF-M; 1:1000; Cell Signaling Technology #2838) and rabbit anti-BCAT2 (1:500; Abcam #ab95976). The secondary antibodies used were: anti-mouse HRP-linked antibody (1:2000; Cell Signal Technologies #7076) and anti-rabbit HRP-linked antibody (1:2000; Cell Signal Technologies #7074).

Cortical neurons were transfected with 60 nM scramble mimics as control, 60 nM miR-182-5p mimics, 60 nM negative control inhibitor mimics, 60 nM miR-182 inhibitor mimics, 60 nM negative control siRNA, 60 nM BCAT2 siRNA-1 mimics (TechDragon #siRNA no. 1330304) and 60 nM BCAT2 siRNA-2 mimics (TechDragon #siRNA no. 1330305) plus GFP-encoding plasmid at 1 day in vitro (DIV) by using Lipofectamine 3000 reagent (Invitrogen), incubated with lipofectamine 3000 and MicroRNA agomir or antagomir at 37°C, 5% CO₂ for 24 h, washed once with 1× PBS and replaced with fresh medium. 36 h later, analyzed for axon outgrowth; also transfected at 5 DIV, and 48 h later, analyzed for dendrite development. The GFP-encoding plasmid is promoted by the CAG promoter, and the plasmid was a generous gift from Professor Zhao Hui (School of Biomedical Sciences, The Chinese University of Hong Kong).

Lengths of axons and dendrites were quantified by NIH Image J. The statistical analysis of total dendritic branch tip number (TDBTN), average dendrite branching length (ADBL), and total dendrite branching length (TDBL) were done with GraphPad Prism 6. The projection images were traced with NIH Image J, and dendritic complexity was analyzed with Image J by using the Sholl analysis plugin (Anirvan Ghosh Lab). A series of concentric circles at 15 and 25 μm intervals were drawn around the soma, and crossings of dendrites with each circle were counted automatically. Images for axon and dendrite analysis were taken by using 20× objectives under Zeiss microscope with a CCD camera.

The total RNAs from mouse brain tissues were isolated with TRIzol (Ambion). For gene expression, 1 μg of total RNAs were used to do reverse transcription with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). For microRNA detection, 100 ng of total RNAs were reverse transcribed by using M-MuLV Reverse Transcriptase (New England Biolabs) and a RT primer for adding an anchored poly (T) tag to the miRNA sequence (Alvarez and Nourbakhsh, 2014). Then, the following PCR could be performed with a universal primer and a microRNA-specific primer. The PCR products were detected with SYBR Green dye, and SYBR assays were performed on an Applied Biosystems 7500 Real-Time PCR system. Relative expression was calculated by delta threshold cycles (Ct), using normalization to GAPDH for gene expression detection and to U6 for microRNA detection. The expression measures were recorded from triplicate samples, each of which was analyzed by triplicate qPCR assay. In our results, the average Cts of GAPDH, BCAT2, U6, and miR-182 are 23.64967, 25.017, 17.004, and 21.24467. The primer information for Real-Time PCR was described in Table 1.

Neural stem cells were cultured for 2 days in StemPro NSCs culture medium (Gibco). The NSCs and differentiated neurons were collected, washed three times with PBS, fixed in 4% PFA for 20 min at room temperature, and permeabilised with 0.3% Triton X-100 for 10 min. After blocked with 5% BSA for 10 min, cells were incubated with primary antibodies overnight at 4°C, and signals were detected by a fluorescence microscope. Cell nuclei were visualized with DAPI. Primary antibodies included: rabbit anti-Nestin (Santa Cruz Biotechnology #sc-20978) and rabbit anti-β III tubulin (Abcam #ab18207); secondary antibodies included a donkey anti-rabbit IgG secondary antibody (Life Technologies #A16036).

3′ UTR-containing fragments of BCAT2 were amplified by PCR using Platinum Pfx DNA Polymerase and inserted into the SacI and XbaI sites of pmir-GLO (Promega). The primer information was described in Table 1.

HEK-293T cells were plated in 96-well plates at 2.5 × 10⁴ cells per well and co-transfected with 60 nM scramble mimics or 60 nM miR-182 mimics plus 0.5 ng/μl plasmids of pmir-GLO-mBCAT2-3′ UTR using Lipofectamine 3000 (Life Technologies), and each group is triplicate. 293T cells were cultured for 2 days after transfection and lysed with Dual-Glo Luciferase reagent. Luciferase activity was detected with the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s protocol, and the results were determined by normalizing to the Renilla luciferase activities of three independent experiments.

We used PI (Sigma) /Hoechst (Life Technologies #H1399) and Trypan Blue to detect cell apoptosis, and used a kit (Promega, G3580) to detect cell proliferation ability. For the cell proliferation assay kit, cortical neurons were plated in 4 × 10⁴ cells per well in 96-well plate, and transfected with microRNAs at 1 DIV. Pipet 20 μl of one solution reagent into each well of 96-well plate containing 100 μl of culture medium. Incubate the plate at 37°C for 1–4 h, and then record the absorbance at 490 nm.

Datasets represented a gene list were submitted to the Ingenuity Pathway Analysis Tool (IPA tool; Ingenuity H Systems, Redwood City, CA, USA¹) for core analysis.

The results presented are the average of at least three experiments with standard errors of the mean. Statistical analyses were performed with an unpaired, two-tailed Student’s t-test and one-way ANOVA, and the data remain normally distributed. One-way ANOVA with post hoc test was performed for multiple comparisons. A p-value of <0.05 was considered to indicate statistical significance. Statistical analyses were performed using GraphPad Prism 6.

To identify microRNAs that may regulate neuronal development, NSCs were dissociated from the SVZ zone of adult mouse brain and differentiated into neurons (Gage, 2000). We stained the NSCs with primary antibody of anti-Nestin (Figure 1A) and stained the neurons differentiated from NSCs with β III tubulin (Figure 1B). Recent evidence shows that numbers of microRNAs are specifically expressed in neurons and promote axon extension (Jovicic et al., 2013; Hancock et al., 2014). In a companion paper, we designed primers and quantitatively identified microRNAs that were enriched in neurons relative to NSCs. Several microRNAs, particularly miR-182 and miR-138, showed enrichment in neurons but not in NSCs, whereas other microRNAs, such as miR-338, miR-21, and miR-219a were enriched in NSCs but not in neurons (Figure 1C). These results were consistent with previous results revealing that miR-182 is enriched in neurons compared with astrocytes (Smrt et al., 2010), and miRNAs specifically enriched in neurons were identified by microRNA array (Jovicic et al., 2013).

MiR-182 is known to be highly expressed in sensory cell types (Li et al., 2010) and involved in plasticity and memory (Griggs et al., 2013). We reasoned that if miR-182 were critical for neurodevelopment and function, it should be expressed in neurons of mouse brain. Hence, we detected miR-182 expression levels during different stages of mouse brain development and found that they increased significantly from E13.5 to adult (Figure 1D). We hypothesized whether miR-182 has functions in the later stage of neurite growth, and found that the expression of miR-182 was increased from 2 days after birth to adult (Supplementary Figure S1A). In vitro results showed that miR-182 expression levels were upregulated from 1 to 7 DIV in primary cultured neurons (Supplementary Figure S1B). Together, these data and other published literatures suggest that miR-182 plays functional roles in neurons.

MicroRNAs were found to play important roles in promoting neuronal differentiation and maturation. Here, we tried to investigate the function of miRNAs in neuronal maturation. As miR-182 is enriched in neurons, we reasoned that miR-182 might regulate neuron development during brain development. We co-transfected with miR-182, miR-138, and miR-31 each plus GFP-encoding plasmid into primary cultured cortical neurons at 1 DIV to investigate whether microRNAs overexpression in neurons could affect axon outgrowth. At 36 h after transfection, individual cortical neurons expressing GFP were imaged using fluorescence microscopy. The morphology of axons and soma, which was manually traced and measured by Image J software, showed that miR-182 promoted axon outgrowth by increasing axon length (Figures 2A,B). The statistical results are described in Figure 2C. In contrast, miR-138 showed no difference in axon outgrowth (data not shown). The overexpression of miR-182 was confirmed by qRT-PCR (Figure 2D). To verify the function of miR182 in regulating axon outgrowth, we blocked the endogenous miR-182 by transfecting with antisense oligonucleotides (Figures 2E,F). The results showed that miR-182 inhibitor could suppress axon outgrowth compared with the control group (Figure 2G), and miR-182 expression was detected by quantitative real-time PCR (Figure 2H).

As a neuron marker, neurofilaments (NFs), which consist of three subunits termed neurofilament-L (NF-L), neurofilament-M (NF-M) and neurofilament-H, are thought to provide structural support for mature axons (Nixon and Shea, 1992; Lee and Shea, 2014). We found that the expression of NF-L and NF-M was regulated by miR-182. Overexpression of miR-182 increased the expression of NF-L and NF-M in mRNA levels by RT-PCR, but β III tubulin and MAP2 as reference genes were not influenced by miR-182 (Figure 3A); in protein levels by western blot (Figure 3B). Western blot results showed that inhibition of miR-182 decreased the protein level of NF-L, but it had no influence on the protein levels of NF-M by western blot (Figure 3C). It indicated that miR-182 promoted axon outgrowth by regulating NF-L.

We tested the cell viability by PI/Hoechst staining and Trypan blue staining after transfection with microRNAs (Supplementary Figure S2A), and detected the cell dynamic viability after transfection for 3 days by cell proliferation assay (Supplementary Figure S2B). Supplementary Figure S2C showed the transfection efficiency of scramble mimics which conjugated with FAM fluorescence dye.

Dendrites have important functional implications in synapse formation and electroneurographic signal-passing in mature neurons (Jan and Jan, 2010). As miR-182 is known to be enriched in neurons, we further studied the functions of miR-182 on dendrite development and neuron maturation. To evaluate its influence on the complexity of dendrite tree, primary cortical neurons were co-transfected with GFP-encoding plasmid containing miR-182 mimics and scramble mimics at 5 DIV. Ectopic expression of miR-182 significantly increased the dendrite complexity (Figures 4A,B). We performed Sholl analysis to analyze the dendrite morphology by measuring the number of dendrites that intersected concentric circles at different distances from the soma (Sholl, 1953) (Figure 4C). The results showed that miR-182 significantly increased the complexity of the dendrite tree at a distance between 130 and 145 μm from the soma (Figure 4D). TDBTN and TDBL were significantly increased, but ADBL remained unchanged (Figures 4E–G).

In contrast, the inhibition of miR-182 expression resulted in a significant reduction of total dendritic length and branch numbers (Figures 4H,I); and the number of intersections was significantly reduced between the distance of 35–85 μm from the soma (Figures 4J,K); TDBTN and TDBL were significantly reduced (Figures 4L,M), but ADBL was not changed (Figure 4N). Together, these data suggested that miR-182 overexpression caused changes in dendrite branching and neuronal maturation; thus, it might play important roles during mouse brain development.

We investigated the mechanism of miR-182 in the regulation of neuronal development, and detected the activity of the PI3K/AKT, RAF/ERK, and JNK pathways. MicroRNAs regulate dendrite branching out via AKT pathway, which is known to play roles in the regulation of dendritic morphogenesis and to increase the complexity of the dendritic tree (Kumar et al., 2005; Xue et al., 2016). We found that overexpression of miR-182 increased the activity of AKT in T308 and S473 and blockage of the endogenous miR-182 inhibited AKT activity (Figures 5A,B), but there are no effects on Erk activity by miR-182 (Supplementary Figure S3). PTEN is an inhibitor of PI3K which promotes AKT activity, and the phosphorylation of PTEN S380 negatively regulates PTEN activity by preventing its recruitment into a protein complex (Vazquez et al., 2001). In our results, overexpression of miR-182 inhibited the activity of PTEN by increasing its phosphorylation (Figure 5A), and the phosphorylation of PTEN was decreased when the endogenous miR-182 was blocked (Figure 5B). Activation of the PI3K/AKT pathway increases dendritic complexity in neurons (Kumar et al., 2005), consistent with our findings of the induction of axon outgrowth and dendritic branching by miR-182.

Meanwhile, to investigate whether endogenous AKT activities are necessary for axon outgrowth, we inhibited the PI3K/AKT pathway by adding PI3K inhibitor LY294002 plus transfection with scramble mimics and miR-182 mimics. We found that the AKT activity (S473 and T308) and the phosphorylation of PTEN (S380) decreased compared with the non-drug group (Figure 5H). The statistic of axon length was consistent with the western blot result that LY294002 could rescue the influence on AKT activity regulated by miR-182 (Figures 5C–G). These data verified that miR-182 promoted neuronal maturation by activating the AKT pathway.

Taken together, these results showed that miR-182 inhibited the activity of PTEN and promoted the phosphorylation of AKT, indicating that miR-182 regulated axon outgrowth and dendrite branching out involving the PTEN/AKT pathway.

To explore the mechanism of miR-182 in regulation of neurite outgrowth, we attempted to identify the target gene of miR-182. First, we predicted the miR-182 target genes using three public databases, including miRanda, Target Scan, and miRDB; and identified nine candidate targets to be common (Figure 6A). We compared the expression of 14 target candidates of miR-182 by quantitative real-time PCR in primary cultured cortical neurons, which were transfected with scramble mimics and miR-182 mimics at 1 DIV and lysed at 3 DIV. We found that BCAT2 expression was significantly downregulated by miR-182 (Figure 6B). BCAT2 is known to be a target of miR-182 in PIGF mice and mouse embryonic fibroblasts (Li et al., 2016). Furthermore, a 3′ UTR-containing fragment of mouse BCAT2 was amplified and cloned into the luciferase reporter plasmid to detect the luciferase activity. The luciferase activity was significantly downregulated by miR-182 (Figure 6C), consistent with the results of a previous report (Li et al., 2016). Meanwhile, protein levels of BCAT2 were significantly decreased in cortical neurons overexpressing miR-182 (Figure 6D), but the expression of BCAT2 increased when blocking the endogenous miR-182 by the inhibitor (Figure 6E). Meanwhile, we detected the BCAT2 protein expression in different tissues of the E15.5 mouse; found that BCAT2 was highly expressed in cortex, lung, and liver (Figure 6F).

To verify the function of BCAT2, we transfected with two siRNA mimics into primary cultured neurons for downregulating the endogenous BCAT2, and deficiency of BCAT2 by siRNA-2 promoted axon outgrowth (Figures 7A–D). In western blot results, BCAT2 siRNA-2 increased the activity of AKT and decreased the activity of PTEN (Figure 7E). Meanwhile, we investigated the expression profile of BCAT2 in cultured neurons and brain tissue after birth, and found that the expression tendency was decreased (Figures 7F,G); it was consistent with the expression profile of miR-182 (Supplementary Figure S1). In cardiomyocytes, BCAT2 deletion promotes AKT activity by increasing the phosphorylation of Ser-473 (Li et al., 2016). MiR-182 may regulate neurite outgrowth by targeting BCAT2 and further increasing AKT activity and promoting neuronal maturation.