Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth - PubMed (original) (raw)

Comparative Study

. 2005 Jul 26;102(30):10694-9.

doi: 10.1073/pnas.0504021102. Epub 2005 Jul 14.

Affiliations

Comparative Study

Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth

M Douglas Benson et al. Proc Natl Acad Sci U S A. 2005.

Abstract

The inability of CNS axons to regenerate after traumatic spinal cord injury is due, in part, to the inhibitory effects of myelin. The three major previously identified constituents of this activity (Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein) were isolated based on their potent inhibition of axon outgrowth in vitro. All three myelin components transduce their inhibitory signals through the same Nogo receptor/p75 neurotrophin receptor/LINGO-1 (NgR1/p75/LINGO-1) complex. In this study, we considered that molecules known to act as repellants in vertebrate embryonic axonal pathfinding may also inhibit regeneration. In mice, ephrin-B3 functions during development as a midline repellant for axons of the corticospinal tract. We therefore investigated whether this repellant was expressed in the adult spinal cord and retained inhibitory activity. We demonstrate that ephrin-B3 is expressed in postnatal myelinating oligodendrocytes and, by using primary CNS neurons, show that ephrin-B3 accounts for an inhibitory activity equivalent to that of the other three myelin-based inhibitors, acting through p75, combined. Our data describe a known vertebrate axon guidance molecule as a myelin-based inhibitor of neurite outgrowth.

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Figures

Fig. 1.

Fig. 1.

Expression of ephrin-B3 in the spinal cord. (A) X-Gal staining of transverse spinal cord sections from mice containing the ephrin-B3/lacZ knockin allele (_a_-h). Ephrin/β-gal expression is reduced at the midline after postnatal day 10 and appears in the white matter; b, d, f, and h show the dorsal columns in a, c, e, and g at higher magnification. In situ hybridization confirms the lacZ expression pattern (_i_-l). Note the absence of expression in the dorsal root ganglion and Schwann cells (g Inset) and concentrated expression around the CST (g and h, arrows). (Scale bars, 100 μm.) (B) Immunofluorescent detection of β-gal (red) in the EB3/LacZ mouse spinal cord colocalizes with the oligodendrocyte marker APC (green). (Insets) High magnification confocal image of β-gal/APC colocalization. (Scale bar, 100 μm; Inset scale bar, 50 μm.) (C) Aliquots of purified myelin from EB3/LacZ mice and control mice incubated with X-Gal and spotted on filter paper demonstrate the presence of ephrin-B3/β-gal in myelin membranes. (D) Sections of adult Brain (Brn) and spinal cord (cord) from WT and olig1 knockout mice were hybridized with an ephrin-B3 antisense probe. No ephrin expression was observed in corpus collosum or dorsal white matter (arrows)of the mutant tissue. (Scale bar, 200 μm.)

Fig. 2.

Fig. 2.

EphA4 activation and axon repulsion/inhibition in ephrin-B3-treated postnatal cortical neurons. (A) Immunofluorescent localization of EphA4 receptor (red) and phosphotyrosine (green) in cortical neurons plated on laminin (_a_-c) and treated with control IgG/Fc (_d_-f), preclustered ephrin-B3/Fc (_g_-i) chimeric protein, or plated on WT myelin (_j_-l), or EB3 knockout myelin (_m_-o). DAPI-stained nuclei are shown in blue. Arrowheads indicate phosphorylated clusters of EphA4. (Scale bar, 10 μm.) (B) Total neurite outgrowth of 1,317 EphA4-positive cortical neurons plated on polyornithine with the indicated concentrations of ephrin B3/Fc (EB3/Fc), MAG/Fc, or Fc alone (IgG/Fc). ANOVA analysis with a post hoc Fisher's test shows a significant effect of EB3/Fc and MAG/Fc, but not IgG/Fc, treatment (F = 13.16, P < 0.001). All EB3/Fc and MAG/Fc concentrations, except 63 nM MAG/Fc, were significantly different from untreated control and from IgG/Fc-treated groups (P < 0.001 for EB3/Fc, and P < 0.01 for MAG/Fc), as well as from each other at each concentration (P < 0.01 at 63 nM and P < 0.001 at 125 nM). IgG/Fc controls were not significantly different from untreated controls at the concentrations shown.

Fig. 3.

Fig. 3.

Comparison of Nogo/MAG/OMgp and ephrin-B3 components of myelin inhibition of neurite outgrowth. (A) Total neurite outgrowth of 2,063 cortical neurons from p75+/+ or p75-/- mice from four independent experiments grown on poly(DL)-ornithine, WT myelin, or ephrin-B3 knockout myelin with control growth on poly(DL)-ornithine set at 100% for each genotype, and each group expressed as a percentage of the control, ±SEM, for its appropriate genotype. Statistical analysis was performed by ANOVA (F = 40.173, P < 0.0001 for myelin substrate; F = 23.993, P < 0.0001 for p75 genotype). All groups except p75-/- on ephrin-B3 knockout myelin are statistically different from controls (P < 0.0001), and differences in neurite outgrowth due to ephrin-B3 knockout from myelin and p75 knockout from neurons are all significant (P < 0.0001 for columns 2 vs. 3, 5 vs. 6, 3 vs. 6, and 2 vs. 5) as measured by a post hoc Fisher's protected test (WT, WT mouse myelin; KO, ephrin-B3 knockout myelin). (B) Representative micrographs of immunofluorescent labeling of cortical neurons from one of the experiments quantitated in A. Green labeling is acetylated tubulin, red is EphA4, blue is DAPI. (Scale bar, 10 μm.) (C) Total neurite outgrowth of 1,902 CGNs from p75+/+ or p75-/- mice from three independent experiments grown as in A. ANOVA analysis (F = 78.705, P < 0.0001 for substrate; F = 7.322, P < 0.01 for p75 genotype) shows that all groups are statistically different from controls (P < 0.0001). Differences in neurite outgrowth due to ephrin-B3 knockout from myelin and p75 knockout from neurons are all significant (P < 0.0001 for columns 2 vs. 3, 5 vs. 6, and 3 vs. 6; P < 0.03 for columns 2 vs. 5) as measured by a post hoc Fisher's protected test. (D) β-gal activity of purified myelin and of detergent soluble and insoluble myelin fractions from mice containing the ephrin-B3/LacZ fusion allele is represented as absorbance at 420 nm normalized to total protein ±SD for triplicate samples. pellet, Insoluble fraction; sup., solubilized fraction.

Fig. 4.

Fig. 4.

Relief of myelin inhibition of neurite outgrowth by recombinant EphA4 and p75. Total neurite outgrowth (as a percentage of control), ±SEM, of 1,679 cortical (A) and 1,658 cerebellar granule (B) neurons grown on WT myelin in the presence of 250 nM of the recombinant Fc protein indicated for each group. ANOVA was performed (F = 5.410, P < 0.01 for cortical neurons; F = 26.938, P < 0.0001 for CGNs) with a post hoc Fisher's protected test. Columns 3, 4, and 5 (EphA4/Fc, p75/Fc, and combined Fcs, respectively) are all different from column 2 (IgG/Fc) (P < 0.001, 0.01, and 0.05 respectively for cortical neurons in A, and P < 0.0001 in all cases for CGNs in B). The CGN EphA4/Fc+p75/Fc group (column 5 in B) is statistically indistinguishable from polyornithine controls (column 1 in B).

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