The CS-E motif is a potent inhibitor of axon growth. (A) Structures of synthetic glycopolymers displaying pure CS-A, CS-C, and CS-E disaccharides. (B) The synthetic CS-E glycopolymer inhibits neurite outgrowth of chick E7 DRGs, whereas the CS-A glycopolymer, CS-C glycopolymer, and monovalent CS-E disaccharide have little effect. (C) The synthetic CS-E glycopolymer induces DRG growth cone collapse. (D) CSPGs from CS-E-deficient mice show significant loss of inhibitory activity on DRG neurite outgrowth. Mouse P8 DRGs were cultured on CSPGs purified from Chst15 knockout or wild-type mice. Statistical analyses were performed using the one-way ANOVA test (*P < 0.0001, relative to control).

A monoclonal antibody binds specifically to CS-E and blocks CSPG-mediated neurite inhibition. (A) Binding of the CS-E antibody to carbohydrate microarrays. Little binding to other sulfated CS polysaccharides or glycosaminoglycan classes was detected. Experiments were performed in triplicate (n = 10 per condition). (B) Dose-dependent binding of the anti-CS-E antibody to CSPGs, as shown by an enzyme-linked immunosorbent assay. The experiment was performed in triplicate, and average values (± SD, error bars) are shown for one representative experiment. (C) The CS-E antibody blocks CSPG-mediated inhibition of neurite outgrowth. Dissociated chick E7 DRGs were cultured on a substratum of P-Orn (control) or CSPGs (0.5 μg/mL) in the presence of the indicated antibodies (0.1 mg/mL) for 12 h. Quantitation from three experiments is shown (One-way ANOVA, *P < 0.0001, relative to CSPG without antibody treatment control; n = 50–200 cells per experiment).

CS-E-enriched polysaccharides inhibit DRG neurite outgrowth and induce growth cone collapse. (A) Dissociated chick E7 DRGs were cultured on a substratum of poly-DL-ornithine (P-Orn control), CSPGs, chondroitinase ABC-treated CSPGs, or CS polysaccharides enriched in the CS-A, CS-C, or CS-E sulfation motifs. Representative images and quantitation of average neurite length (± SEM, error bars) from three experiments (n = 50–200 cells per experiment). (B) Polysaccharides enriched in the CS-E sulfation motif, but not the CS-A or CS-C motifs, inhibit DRG neurite outgrowth in a dose-dependent manner. (C) CS-E-enriched polysaccharides repel axon crossing in a boundary assay. Polysaccharides (1 mg/mL) or PBS control were mixed with Texas Red and spotted on P-Orn coated coverslips. Dissociated rat P5-9 CGN neurons were immunostained with an anti-βIII-tubulin antibody. Representative images and quantitation of percentage of axon crossing (± SEM, error bars) from two experiments (n = 30–50 axons per experiment). (D) CS-E-enriched polysaccharides induce growth cone collapse. DRG explants from chick E7-9 embryos were grown on a P-Orn/laminin substratum, treated with medium (control) or the indicated polysaccharides, and stained with rhodamine-phalloidin. Representative images and quantitation of growth cone collapse (± SEM, error bars) from five experiments (n = 50–100 growth cones per experiment). Arrows indicate collapsed growth cones. All statistical analyses were performed using the one-way ANOVA test (*P < 0.0001, relative to control).

The CS-E sulfation motif inhibits axon growth via PTPσ. (A) Inhibitors against EGFR (AG1478, 15 nM) and ROCK (Y27632, 5 μM) rescued CS-E-and CSPG-mediated inhibition of neurite outgrowth in dissociated rat P5-9 CGN cultures, whereas JNK inhibitor II (10 μM) had no effect. Quantitation of neurite outgrowth from three experiments is reported. (One-way ANOVA, *P < 0.0001, relative to CS-E control without inhibitors, **P < 0.0001, relative to CSPG control without inhibitors; n = 50–200 cells per experiment). (B) PTPσ binds selectively to CS-E-enriched polysaccharides on glycosaminoglycan microarrays. Microarrays were incubated with PTPσ-Fc, followed by a Cy3-conjugated antihuman IgG secondary antibody, and analyzed using a GenePix 5000a scanner. Graphs show quantification from three experiments (n = 10 per condition). (C) Coprecipitation of CS-E and PTPσ. Full-length PTPσ-mycHis was expressed in COS-7 cells and incubated with biotinylated CS-E or CS-C polysaccharides bound to streptavidin beads. PTPσ binding was detected by immunoblotting with an anti-myc antibody. (D) Specific, high affinity binding of CS-E polysaccharides to PTPσ. (E) PTPσ -/- neurons show significantly less inhibition by CS-E than wild-type control neurons. For each genotype, the percentage inhibition of neurite outgrowth is plotted relative to neurons treated with only P-Orn. Quantification from three experiments is shown. (One-way ANOVA, *P < 0.005, relative to control; n =  > 200 cells per experiment).

CS-E neutralizing antibodies promote optic nerve regeneration. (A) a–c: Immunofluorescence labeling of CS-E expression in optic nerve sections at day 1 sham-operation (a), optic nerve crush injury (b), or optic nerve crush injury plus ChABC treatment (c). Note upregulation of CS-E around the injury site (b) that was removed by ChABC treatment (c). d–g: Representative epifluorescence photomicrographs of optic nerve sections taken from mice treated with control IgG (d), CS-E antibody (e), ChABC (f) or ChABC plus CS-E antibody (g). Asterisk indicates the crush site. Retinal ganglion cell axons (red) are labeled by an anterograde axon tracer, CTB, which was injected into the vitreous 3 d prior to scarify, followed by immunostaining with goat-anti-CTB antibody. In control antibody-treated mice (d), few regenerating axons are evident. In contrast, numerous regenerating axons were seen extending pass the crush site in CS-E antibody, ChABC or the combine-treated groups (e–g). Scale bars: 75 μm (a–g); 25 μm (d′–f′). Arrowheads indicate regenerating RGC axons. (B) Quantification of the numbers of regenerating axons at different distances from the injury site. Nerve fibers were counted at 125-μm intervals from the crush site from three nonconsecutive sections, and the number of fibers at a given distance was calculated (± SEM, error bars). Both the anti-CS-E-and ChABC-treated groups showed significantly more regenerating axons as compared with the control IgG antibody-treated group (ANOVA with Bonferroni posttests at each distance, *P < 0.001 as compared to controls; n = 6 for each group). (C) Quantification of the distances of axon regeneration. Longest distance of axon regeneration was measured from at least four nonconsecutive optic nerve sections from each mouse (± SEM, error bars). Combined treatment of CS-E mAb and CPT-cAMP more than tripled the distance of axon regeneration but did not affect the number of regenerating axons compared to the anti-CS-E or CPT-cAMP treatment alone (Fig. S13).