Abstract
In the injured nervous system, myelin-associated glycoprotein (MAG) on residual myelin binds to receptors on axons, inhibits axon outgrowth, and limits functional recovery. Conflicting reports identify gangliosides (GD1a and GT1b) and glycosylphosphatidylinositol-anchored Nogo receptors (NgRs) as exclusive axonal receptors for MAG. We used enzymes and pharmacological agents to distinguish the relative roles of gangliosides and NgRs in MAG-mediated inhibition of neurite outgrowth from three nerve cell types, dorsal root ganglion neurons (DRGNs), cerebellar granule neurons (CGNs), and hippocampal neurons. Primary rat neurons were cultured on control substrata and substrata adsorbed with full-length native MAG extracted from purified myelin. The receptors responsible for MAG inhibition of neurite outgrowth varied with nerve cell type. In DRGNs, most of the MAG inhibition was via NgRs, evidenced by reversal of inhibition by phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves glycosylphosphatidylinositol anchors, or by NEP1-40, a peptide inhibitor of NgR. A smaller percentage of MAG inhibition of DRGN outgrowth was via gangliosides, evidenced by partial reversal by addition of sialidase to cleave GD1a and GT1b or by P4, an inhibitor of ganglioside biosynthesis. Combining either PI-PLC and sialidase or NEP1-40 and P4 was additive. In contrast to DRGNs, in CGNs MAG inhibition was exclusively via gangliosides, whereas inhibition of hippocampal neuron outgrowth was mostly reversed by sialidase or P4 and only modestly reversed by PI-PLC or NEP1-40 in a non-additive fashion. A soluble proteolytic fragment of native MAG, dMAG, also inhibited neurite outgrowth. In DRGNs, dMAG inhibition was exclusively NgR-dependent, whereas in CGNs it was exclusively ganglioside-dependent. An inhibitor of Rho kinase reversed MAG-mediated inhibition in all nerve cells, whereas a peptide inhibitor of the transducer p75(NTR) had cell-specific effects quantitatively similar to NgR blockers. Our data indicate that MAG inhibits axon outgrowth via two independent receptors, gangliosides and NgRs.
Highlights
The identity of the axonal receptors for myelin-associated glycoprotein (MAG) has been a matter of controversy
MAG Inhibition of dorsal root ganglion neurons (DRGNs) Neurite Outgrowth Is Primarily via Nogo receptors (NgRs) and Secondarily via Gangliosides—Postnatal DRGNs extended long neurites within 24 h when plated on control surfaces (Fig. 1)
Our results indicate that MAG inhibition of neurite outgrowth from Hippocampal Neurons (HNs) is predominantly ganglioside-dependent, a portion of the myelin-mediated inhibition appears to involve NgR and p75NTR
Summary
Materials—Phosphatidylinositol-specific phospholipase C (PI-PLC; Bacillus cereus) and Y-27632 (Rho kinase inhibitor) were from Sigma-Aldrich. Cells were collected by centrifugation (500 ϫ g for 7 min) and resuspended in growth medium (Neurobasal medium (Invitrogen) containing 0.13 mM L-glutamine, 0.25% (v/v) heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 0.5% B-27 supplement (Invitrogen), 100 units/ml penicillin, and 100 g/ml streptomycin). Cells were collected by centrifugation, resuspended in growth medium (minimal essential medium (Invitrogen) containing 25 mM Hepes, 25 mM KCl, 10% (v/v) heat-inactivated horse serum, 5% (v/v) fetal bovine serum (Hyclone), 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine), plated (55,000 cells in 100 l/well) on 96-well plates (Nunc, Roskilde, Denmark) precoated with 125 g/ml poly-D-lysine (molecular weight, Ͼ300,000), and cultured at 37 °C in a 5% CO2 incubator for 48 h. Inhibitory Substrata—MAG was extracted from purified myelin membranes using mild detergent and adsorbed to culture surfaces as described previously [12]. Statistical p values were obtained using Student’s t test
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