Progressive axonal degeneration follows demyelination in many neurological diseases, including multiple sclerosis and inherited demyelinating neuropathies, such as Charcot-Marie-Tooth disease. One glial molecule, the myelin-associated glycoprotein (MAG), located in the adaxonal plasmalemma of myelin-producing cells, is known to signal to the axon and to modulate axonal caliber through phosphorylation of axonal neurofilament proteins. This report establishes for the first time that MAG also promotes resistance to axonal injury and prevents axonal degeneration both in cell culture and in vivo. This effect on axonal stability depends on the RGD domain around arginine 118 in the extracellular portion of MAG, but it is independent of Nogo signaling in the axon. Exploiting this pathway may lead to therapeutic strategies for neurological diseases characterized by axonal loss.

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 nonadditive 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.The injured adult mammalian central nervous system is a highly inhibitory environment for axon regeneration due in part to endogenous axon regeneration inhibitors (ARIs) 4 at least three of which are expressed on residual myelin that persists at sites of central nervous system injury (1, 2). Knowledge of myelin-derived ARIs, their receptors on axons, and the downstream signaling pathways that limit axon outgrowth may provide new opportunities to reverse inhibition and enhance recovery after traumatic central nervous system injury (3, 4). One well established inhibitor of axon regeneration is myelinassociated glycoprotein (MAG), a transmembrane protein of the immunoglobulin superfamily that is expressed on the innermost wrap of myelin directly apposed to the axon surface. MAG is essential to the long term stability of myelinated axons and positively regulates axon cytoarchitecture (5). However, in the injured nervous system, MAG on residual ...

Myelin-associated glycoprotein (MAG), a protein expressed on the innermost wrap of myelin, contributes to long-term axon stability as evidenced by progressive axon degeneration in Mag-null mice. Recently, MAG was also found to protect axons from acute toxic insults. In the current study, rat dorsal root ganglion neurons were cultured on control substrata and substrata adsorbed with myelin proteins. Neurons on myelin-adsorbed surfaces were resistant to acute degeneration of neurites induced by vincristine, a cancer chemotherapeutic agent with neuropathic side effects. Myelin-mediated protection was reversed by anti-MAG antibody and was absent when cells were cultured on extracts from Mag-null mouse myelin, confirming the protective role of MAG. Gangliosides (sialylated glycosphingolipids) are one functional class of axonal receptors for MAG. In the current studies, a direct role for gangliosides in mediating the acute protective effects of MAG was established. Treatment of neurons with sialidase, an enzyme that cleaves the terminal sialic acids required for MAG binding, reversed MAG's protective effect, as did treatment with (1R,2R)-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, an inhibitor of glycosphingolipid biosynthesis. In contrast, treatment with phosphatidylinositol-specific phospholipase C, an enzyme that cleaves Nogo receptors (NgR, another class of MAG receptor), or with a peptide inhibitor of an NgR-associated signaling molecule p75NTR, failed to diminish MAG-mediated protection. Inhibiting the Rho-associated protein kinase ROCK reversed protection. We conclude that MAG protects neurites from acute toxic insult via a ganglioside-mediated signaling pathway that involves activation of RhoA. Understanding MAG-mediated protection may provide opportunities to reduce axonal damage and loss.

J. Neurochem. (2011) 116, 900–908. Abstract In addition to supporting rapid nerve conduction, myelination nurtures and stabilizes axons and protects them from acute toxic insults. One myelin molecule that protects and sustains axons is myelin‐associated glycoprotein (MAG). MAG is expressed on the innermost wrap of myelin, apposed to the axon surface, where it interacts with axonal receptors that reside in lateral membrane domains including gangliosides, the glycosylphosphatidylinositol‐anchored Nogo receptors, and β1‐integrin. We report here that MAG protection extends beyond the axon to the neurons from which those axons emanate, protecting them from excitotoxicity. Compared to wild type mice, Mag‐null mice displayed markedly increased seizure activity in response to intraperitoneal injection of kainic acid, an excitotoxic glutamate receptor agonist. Mag‐null mice also had larger lesion volumes in response to intrastriatal injection of the excitotoxin NMDA. Prior injection of a soluble form of MAG partially protected Mag‐null mice from NMDA‐induced lesions. Hippocampal neurons plated on proteins extracted from wild‐type rat or mouse myelin were resistant to kainic acid‐induced excitotoxicity, whereas neurons plated on proteins from Mag‐null myelin were not. Protection was reversed by anti‐MAG antibody and replicated by addition of soluble MAG. MAG‐mediated protection from excitotoxicity was dependent on Nogo receptors and β1‐integrin. We conclude that MAG engages membrane‐domain resident neuronal receptors to protect neurons from excitotoxicity, and that soluble MAG mitigates excitotoxic damage in vivo.