The OLN-93 oligodendroglia cell line was utilized in this study. OLN-93 cells are known to express several oligodendroglial markers; however, they do not exhibit characteristics of astrocytes (8). Cells were incubated at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM)/F12 with 10% fetal bovine serum (FBS; both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Growth medium was changed twice a week. When the cells reached 70% confluency, they were digested with 1% trypsin/EDTA (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 2 min. FBS was added to stop the digestion once the cells were round and floating. Cells were then seeded in plates or flasks and maintained in an incubator.

One subset of cells was directly fixed for fluorescent immunohistochemical staining using primary antibodies against proBDNF, p75NTR and sortilin, whereas another subset of cells was treated with serial concentrations of recombinant proBDNF (1, 3, 10, 30, 100 ng/ml); bovine serum albumin (BSA; 100 ng/ml) treatment was used as a control. Meanwhile, sheep anti-proBDNF antibody (5, 10 µg/ml), monoclonal proBDNF antibody (PB192E; 100 ng/ml), mouse anti-p75NTR antibody (10 µg/ml; 9,650 from Moses Chao), recombinant p75NTR extracellular domain-human FC fusion protein (p75NTRECD-fc; 3 µg/ml), and normal IgG (10 µg/ml) were also administrated in in vitro observations.

p75NTRECD-fc and the recombinant proBDNF with harbouring an RR-AA mutation on the cleavage site were produced as previously described and characterized by Fan et al (9). Sheep anti-proBDNF antibody is able to specifically recognise proBDNF; however, it cannot recognise mature BDNF and other NTs (7,8). Cell viability, proliferation, migration and apoptosis were determined using the MTT assay, BrdU staining, scratch assay and activated caspase-3 staining respectively.

First, cells were seeded at 10⁵ cells/well on a pre-treated glass coverslip in a 24-well plate. When they reached 70% confluency, 4% paraformaldehyde (PFA) was added to fix the cells at room temperature for 10 min. The fixed cells were subsequently subjected to a PBS wash three times before being immersed in blocking buffer (2% BSA, 0.5% Triton X-100, 0.1% Tween-20, 5% donkey serum) at room temperature for 60 min. Rabbit anti-p75 (1:1,000; Abcam, Cambridge, UK), sheep anti-proBDNF (5 µg/ml; University of South Australia, Adelaide, Australia) and rabbit anti-sortilin (1:1,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) antibodies were then introduced as primary antibodies and incubated with the cells at 4°C for overnight. Then, after washing the cells three times PBST, sheep anti-rabbit IgG CY3 (1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) and donkey anti-sheep IgG CY3 (1:1,000; EMD Millipore, Billerica, MA, USA) were applied as secondary antibodies. In addition, DAPI (1:1,000; Sigma-Aldrich; Merck KGaA) was diluted in the antibody solution to serve as a nuclear dye. Subsequently, cells were incubated with corresponding secondary antibody and DAPI at room temperature for 60 min. Finally, after washing the cells three times, fluorescent images of the cells were captured using an Olympus BX-50 (Olympus Corp., Tokyo, Japan) fluorescent microscope equipped with a CCD camera.

Cells were seeded at 10⁶ cells/well in a 6-well plate, incubated at 37°C and 5% CO₂ and subsequently lysed using RIPA buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mM phenylmethylsulphonyl fluoride, 1 µg/ml leupeptin] when they reached a confluency of 70%. After sonication and centrifugation to remove cell debris, the lysate was loaded onto a sodium dodecyl sulphate polyacrylamide gel, containing stacking (0.5%) and a separating gel (10%). On completion of electrophoresis, proteins were transferred onto a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) using the semi-dry method (Bio-Rad, Berkeley, CA, USA) at 20 V, 20 min. The membrane was then washed with PBS three times and blocked with a blocking buffer (5% skim milk in PBS) for 1 h at room temperature. Rabbit anti-p75 (1:1,000; Abcam), sheep anti-proBDNF (5 µg/ml; University of South Australia) and rabbit anti-sortilin (1:1,000; Sigma-Aldrich; Merck KGaA) antibodies were subsequently added as primary antibodies, followed by incubation at 4°C for overnight. Before and after triple washes with PBST, goat anti-rabbit IgG-HRP (1:3,000) and goat-anti-sheep IgG-HRP (1:3,000; both Sigma-Aldrich; Merck KGaA) were co-incubated with the membrane for 1 h at room temperature. ECL substrate solutions A & B (GE Healthcare) were mixed and added onto the membrane in order to observe any potential fluorescent protein bands. Finally, the film was developed in a dark room to obtain results.

Cells were seeded at 10⁴ cells/well in a 96-well plate, followed by incubation at 37°C and 5% CO₂ for overnight to allow the cells to attach to the bottom of the wells. After washing the cells with PBS three times, the cells were cultured in a serial concentration of proBDNF (1, 3, 10, 30, 100 ng/ml) or in a serial concentration of proBDNF and monoclonal proBDNF antibody PB192E (100 ng/ml) in an FBS-free medium. BSA (100 ng/ml) treatment was used as a control. After 20 h, 10 µl of MTT solution (5 mg/ml in PBS; Sigma-Aldrich-Aldrich; Merck KGaA) was added to each well, followed by incubation for an additional 4 h. Subsequently, 100 µl of filter-sterilized solubilisation solution (10% SDS in 0.01 M HCl) was added to each well and incubated overnight at 37°C to dissolve the insoluble purple formazan product in order to produce a coloured solution. Finally, the optical density (OD) of the solution in each well was measured at 595 nm using a multi-well scanning spectrophotometer (Bio-Rad Model 2550 EIA Reader); a wavelength of 620 nm was used as a reference.

Cells were at first seeded at 5×10⁵ cells per well on a pre-treated glass coverslip in a 24-well plate. Approximately 200 µl of BrdU (80 µM BrdU-medium solution; Sigma-Aldrich; Merck KGaA) was then added to each well, followed by incubation at 37°C and 5% CO₂ for 24 h. The cells were then fixed in 4% PFA and washed with PBS three times. After a 1 h incubation in HCl and neutralization in boric buffer (pH 8.0–8.5), the cells were blocked using a blocking buffer (2% BSA, 0.5% Triton X-100, 0.1% Tween-20, 5% donkey serum). An anti-BrdU antibody (1:1,000; DSHB; University of Iowa, Iowa City, USA) was incubated with the cells as a primary antibody. Then, after washing the cells with PBST three times, cells were subjected to secondary antibody of goat anti-mouse IgG CY3 (1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) and DAPI (1:1,000; Sigma-Aldrich; Merck KGaA). After washing the cells three times, the number of BrdU/DAPI-positive cells was counted at five random fields per slice at magnification, ×10 under a fluorescence microscope.

The scratch assay was performed as previously described (10). Briefly, OLN-93 cells cultured in a 6-well plate were wounded using a sterile P20 pipette tip, followed by washing with culture medium to remove cell debris. The cells were then treated with 1, 3, 10, 30, and 100 ng/ml proBDNF and 100 ng/ml BSA for 24 or 48 h, respectively. Subsequently, cells were fixed by 4% PFA for 10 min then stained by 0.05% crystal violet for 30 min. After washed by PBS, phase contrast images were captured in 6 different fields of every wound using an inverted microscope (Olympus IX-71; Olympus Corp.). Cells that migrated into the wound space were counted in the captured image. The experiments were performed in triplicates.

OLN-93 cells at 5×10⁴ cells per well were seeded in 24-well plates. When the cells reached 90% confluency, they were washed three times with PBS and treated with proBDNF, proBDNF antibody and BSA consecutively at 37°C and 5% CO₂ for 24 h. After treatment, cells were fixed in 4% PFA at room temperature for 10 min and then washed with PBS before being blocked using a blocking buffer (2% BSA, 0.5% Triton X-100, 0.1% Tween-20, 5% donkey serum). Next, rabbit anti-activated caspase-3 (1:1,000; EMD Millipore) was co-incubated with the cells at 4°C for overnight. Then, sheep anti-rabbit IgG FITC (1:1,000; Abcam) and DAPI (1:1,000; Sigma-Aldrich; Merck KGaA) were applied and co-incubated with the cells at room temperature for 1 h. After washing the cells 3 times, fluorescent images of the cells were captured using an Olympus BX-50 fluorescent microscope.

Five visual fields were randomly selected from superior, inferior and central parts as well as from left-hand and right-hand sides of each slide for fluorescence microscopy analysis. Images at magnifications of ×10, ×20 and ×60 were obtained using an Olympus BX-50 fluorescent microscope.

The total number of cells and positively-stained cells were counted using Image J. Quantitative analysis was performed for all experiments using SPSS for Windows v. 13.0. All data are presented as mean ± standard error of the mean (SEM). Data were analysed using one-way analysis of variance (ANOVA), followed by Tukey's post-hoc tests, where appropriate, or t-test for paired groups. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated at least 3 times.

OLN-93 cells were maintained in DMED/F12 1:1 medium. In the presence of 10% FBS, cell proliferation occurred at a high rate (8). All the cells expressed proBDNF, p75NTR and sortilin as shown by fluorescent immunocytochemistry (Fig. 1A-C). The typical oligodendroglia cell marker NG2 (Fig. 1D), which was also used to label oligodendrocytes in spinal cord was also expressed (11). Cell growth was observed to be density-dependent. Cells were mainly bipolar with long cellular extensions at lower confluency; they formed large clumps interconnected with long, thin cellular processes at higher confluency (Fig. 1E). Moreover, Western blot analysis using the cell lysate showed bands of proBDNF, p75NTR and sortilin at the corresponding molecular weights (Fig. 1F). These results indicated that OLN-93 cells expressed endogenous proBDNF and its receptors p75NTR and sortilin.

To examine the effect of proBDNF on OLN-93 cells, we used a monoclonal proBDNF antibody (PB192E), raised against the pro-domain of BDNF, to neutralise the effects of proBDNF. Cells treated with PB192E at 10 µg/ml were more active than those treated with normal IgG (Fig. 2A), indicating that endogenous proBDNF may have inhibited the cell growth.

MTT assay revealed that proBDNF imparts a toxic effect on cell viability. Treated with serial concentrations of proBDNF (1, 3, 10, 30, 100 ng/ml), the cell growth curve demonstrated a significant growth inhibition at 10 ng/ml, with the highest growth inhibition at 100 ng/ml (Fig. 2B) (P=0.000904), indicating a dose-dependent response.

Then, we treated the cells with serial concentrations of proBDNF (1, 3, 10, 30, 100 ng/ml) and PB192E antibody (100 ng/ml). Cells in this group had significantly higher bioactivity than those in the BSA and proBDNF-treated groups (Fig. 2B) (P=0.000194), indicating that PB192E antibody can neutralise exogenous proBDNF as well.

Quantitative assessment of the proliferative activities in OLN-93 cells were performed (Fig. 3). BrdU/DAPI double-labelling was applied to the OLN-93 cell proliferation assay (Fig. 3C). The proliferating cells were BrdU⁺/DAPI⁺, whereas the non-proliferating ones were only DAPI⁺. The percentage of BrdU⁺/DAPI⁺ cells in the proBDNF-treated groups was significantly lower than that in the BSA group, illustrating a dose-dependent inhibition (Fig. 3A) (P=0.0206 in 10; P=0.0420 in 100 ng/ml; P=0.000126 in 30 ng/ml).

To confirm our results, we used polyclonal sheep anti-proBDNF antibodies to neutralise endogenous proBDNF. We found that cells treated with polyclonal antibodies showed higher proliferative activities than those treated with normal sheep IgG (Fig. 3B) (P=0.000105). These experiments indicate that OLN-93 cells secrete endogenous proBDNF, which inhibits their growth.

To investigate the possible pathway by which proBDNF exerts an inhibitory on OLN-93 cells, recombinant fusion molecule of p75NTRECD-fc and p75NTR functional antibody were utilized. These have been used in our previous study as competitive inhibitors to block p75NTR signal transduction extracellularly (12). In this study, we treated OLN-93 cells with 3 µg/ml of p75NTRECD-fc protein and 10 µg/ml of anti-p75NTR for 24 h. Then, BrdU/DAPI double-labelling was conducted to check the proliferative activities after 24 h of incubation. Under both the treatments, the percentage of proliferating cells significantly increased in comparison with those in the normal sheep IgG control group (Fig. 3C) (P=0.00206 in p75NTRECD-fc; P=0.0151 in anti-p75NTR).

Moreover, we treated OLN-93 cells with proBDNF and p75NTR antibody. The cells were co-incubated with 100 ng/ml of proBDNF and 10 µg/ml of p75NTR antibody for 24 h. Treated cells had better proliferative activities than those in the proBDNF group (Fig. 3C) (P=0.000103) but had no significant differences in comparison with those in the BSA group (Fig. 3C) (P=0.264). This indicated that proBDNF can inhibit OLN-93 cell proliferation via the p75NTR pathway and that proBDNF can be blocked by disruption of p75NTR signal transduction with the soluble receptor body or antibody.

Both proliferation and migration are critical steps in the regeneration and repair after CNS injury. To investigate the function of proBDNF in OLN-93 cell migration, the scratch assay was used. After scratching, a cell-free wound region was generated in 6-well culture plates (Fig. 4Aa, d, g). Serial concentrations of proBDNF (10, 30, and 100 ng/ml) and proBDNF antibody (5 and 10 µg/ml) were used to treat the cells. After cell growth for 24 and 48 h, a number of cells were observed migrating from the edges into the cell-free scratch region (Fig. 4A). Cells treated with proBDNF showed lower migration activity. A higher concentration of proBDNF resulted in the migration of fewer cells, indicating a dose-dependent inhibition of cell migration by proBDNF (Fig. 4B and C) (P=0.0112 in 10 ng/ml; P=0.000104 in 30 ng/ml; P=0.000229 in 100 ng/ml).

In comparison with the proBDNF group, more cells migrated into the scratch wound region in the proBDNF antibody (5 or 10 µg/ml) treatment group; however, the population of migrated cells between different anti-proBDNF concentrations showed no statistical differences (Fig. 4B and C) (P=0.24 in 24 h; P=0.50 in 48 h).

As a member of the cysteine-aspartic acid protease family, caspase-3 is activated when cell apoptosis occurs (13). In this study, we applied the activated caspase-3 antibody to observe apoptosis of OLN-93 cells that were treated with proBDNF (10, 30, 100 ng/ml) and BSA (100 ng/ml) (Fig. 5A).

As expected, the percentage of apoptotic cells in the proBDNF group was significantly higher than that in the BSA group (Fig. 5B) (P=0.0000384). Moreover, with an increase in the dose of proBDNF, more cells were found to be caspase-3-positive (Fig. 5B) (P=0.024).