Figure 1.
ProBDNF treatment decreases neurite length on DRG or cortical neurons.
A, Time-lapse recordings show the collapsed neurite growth with proBDNF (left), and enhanced neurite growth with BDNF (right). Frames indicate 5, 10, 20, 30 min respectively. Scale bar, 20 µm. B, Treatment of DRG neurons with proBDNF decreased the rate of neurite extension within 3 min and remained decelerated through 30 min. n = 30 neurites/treatment. *p<0.05, **p<0.001, compared to BSA; Studens t test. C, Treated cortical or DRG neurons with proBDNF caused similar collapse in neurite length. n = 83–85 neurons/treatment. *p<0.05, **p<0.001, significantly different from untreated neurons; Studen
s t test. D, Expression of sortilin, p75NTR on the lysate of cultured cortical or DRG neurons processed for Western blot. Bands of 35 kDa of proBDNF, 75 kDa of p75NTR, and 110 kDa of sortilin were detected with their respective antibodies. ß-actin (42 kD) antibody was used as internal protein loading control. n = 3 independent experiments.
Figure 2.
ProBDNF decreases neurite length on DRG neurons in vitro.
Cultured DRG neurons were treated by different factors as shown, immunofluorescence stained by anti-MAP2 antibody. A, Normal sheep IgG. B, ProBDNF. C, Anti-proBDNF. D, BDNF. E, Anti-proBDNF. F, The neurite outgrowth assay were analyzed under the different culture conditions. Scale bar, 50 µm. n = 89∼92 neurons/treatment from three independent dishes. *p<0.05, **p<0.01, compared to IgG; one-way ANOVA with least-significant difference post hoc test.
Figure 3.
ProBDNF collapses neurite length in vitro through p75NTR.
A, Cultured DRG neurons were treated by proBDNF (3 ng/ml, 30 ng/ml) or myelin-associated glycoprotein (MAG 3 µg/ml, 30 µg/ml), respectively, immunofluorescence stained by anti-MAP2 antibody. C, proBDNF decreased neurite length on DRG neurons in a dose-dependent manner. **p<0.01, compared to untreated neurons; Studens t test. D, MAG decreased neurite length of DRG neurons in a dose-dependent manner. *p<0.05, **p<0.01, compared to untreated neurons; Student’s t test. E–G, Anti-MAP2 staining of p75NTR+/+ DRG neurons treated by control, proBDNF, MAG, respectively. H–J, Anti-MAP2 staining of p75NTR−/− DRG neurons treated by control, proBDNF, MAG, respectively. K, ProBDNF or MAG treated p75NTR+/+ DRG neurites displayed significant length decrease compared with control group. However, proBDNF or MAG treated p75NTR−/− DRG neurites had no effect compare with control group. Scale bar, 50 µm. n = 85∼90 neurons/treatment. *p<0.05, **p<0.01, compared to control; one-way ANOVA with least-significant difference post hoc test.
Figure 4.
ProBDNF increased RhoA activity in cultured cortical neurons through p75NTR.
A, Activated and total levels of RhoA or Cdc42 with proBDNF (0, 5, 10, 20 min) treatment which were processed for immunoblot (IB) assay. B, 10 and 20 min exposure to proBDNF caused significant production in activated RhoA normalized to total RhoA. **p<0.01, compared to untreated neurons; Studens t test. C, Exposure to proBDNF caused no differences in activated Cdc42 normalized to total Cdc42. D, RhoA activity increased on p75NTR+/+ cortical neurons after treated by proBDNF 20 min compared with p75NTR+/+ control neurons whereas p75NTR−/− cortical neurons exhibited no difference. n = 3∼4 independent experiments. **p<0.01, compared to control level; Studen
s t test.
Figure 5.
ProBDNF treatment increases RhoA activity and filopodial length on DRG growth cones.
A–H, Immunoreactivity of RhoA or Cdc42 activity in DRG growth cones after proBDNF (0, 5, 10, 20 min) treatment. Scale bar, 10 µm. I, Quantification of RhoA activity. There were significant inductions after exposure to proBDNF 10, 20 min. *p<0.05, **p<0.01, compared to untreated neurons; Studens t test. J, Quantification of Cdc42 activity. There was no change during the whole 20 min exposure of proBDNF. K–N, F-actin of DRG growth cones after proBDNF (0, 5, 10, 20 min) treatment. Scale bar, 5 µm. O, ProBDNF decreased filopodial length on DRG growth cones in a time-dependent manner. P, ProBDNF decreased filopodia number on DRG growth cones in a time-dependent manner. n = 35 neurites/treatment from at least three independent experiments. **p<0.01, compared to untreated neurons; Studen
s t test.
Figure 6.
ProBDNF inhibits neurite growth through activation of RhoA and ROCK.
Cultured DRG neurons were treated by different factors, followed by labelling of anti-MAP2 using immunocytochemistry. A, Treatment with normal sheep IgG. B, DRG neurons treated with proBDNF have shorter neurites. C, Pretreatment with C3-transferase (RhoA inhibitor) reverses proBDNF induced inhibition of neurite growth. D, Y27632 (ROCK inhibitor) completely reverses the proBDNF induced inhibition. Scale bar, 50 µm. E, Neurite outgrowth assay. ProBDNF significantly reduced the neurite length whereas pretreatment with C3 or Y27632 restored the reduction. n = 91∼95 neurons/treatment from three independent experiments. *p<0.01; Studens t test. F, GTP-RhoA was measured by pull-down assay. Activated CRMP-2 and total RhoA and ß-actin are also shown by immunoblot (IB). Lane 1, normal sheep IgG control group. Lane 2, proBDNF-induced RhoA activation. Lane 3, The RhoA inhibitor C3 abolishes proBDNF-induced RhoA activation. Lane 4, ROCK inhibition Y27632 blocks proBDNF-induced RhoA activation. Phosphorylation of the collapsin response mediator protein-2 (P-CRMP-2) was assessed with phospho-specific antisera. ß-actin was used as internal protein loading control. n = 3 independent experiments.
Figure 7.
ProBDNF decreases the nerve innervation in ventral planta skin in vivo through p75NTR.
A–C, Anti-neurofilament staining of footpad treated by normal sheep IgG (IgG), proBDNF, anti-proBDNF, respectively on p75NTR+/+ postnatal 5 days animals. D–F, Anti-neurofilament staining of footpad treated by IgG, proBDNF, anti-proBDNF, respectively on p75NTR−/− postnatal 5 days animals. G, Quantification of nerve fiber density. The maximal threshold of control IgG fiber density (area occupied/total area of field of view) was defined as 1, for the treatments, density of the fibers was divided by the IgG fiber density in the same optical field. ProBDNF reduced the nerve fibers density while anti-proBDNF increased the nerve fibers density on p75NTR+/+ animals. However, proBDNF or anti-proBDNF had no effect in p75NTR−/− mice. Scale bar, 100 µm. n = 40 sections/treatment from five independent animals. *p<0.01, compared to IgG; Studens t test.