Gangliosides and Nogo Receptors Independently Mediate Myelin-associated Glycoprotein Inhibition of Neurite Outgrowth in Different Nerve Cells

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 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 ...

Sialidase enhances recovery from spinal cord contusion injury

“…It is reasonable to propose that the therapeutic benefit of sialidase was a result of conversion of MAG-binding gangliosides (GD1a and GT1b) to GM1, which does not bind MAG (35). Supporting this hypothesis are data demonstrating that sialidase reverses the inhibitory effects of MAG on axon outgrowth from some types of neurons in vitro (13,14,17). The increase in serotonergic fibers caudal to the lesion in the current study and our prior observation that sialidase enhanced axon outgrowth from motor neurons into a peripheral nerve graft (19) provide evidence that sialidase treatment enhances axon outgrowth in vivo.…”

“…Therapies that reverse ARIs may increase axon sprouting and outgrowth generally, or may selectively affect nerve subpopulations (13,14). In the current study, anterograde labeling of corticospinal tract (CST) axons, for example, did not reveal a significant treatmentrelated increase in labeled axons within 7 mm caudal (P = 0.7) or rostral (P = 0.8) to the lesion.…”

“…Gangliosides are the most abundant sialoglycans on nerve cells (15). In some nerve cells, MAG-mediated axon outgrowth inhibition in vitro is reversed by blocking ganglioside biosynthesis, by competitive inhibition using MAG-binding sialoglycans (16), and by sialidase, an enzyme that cleaves the key MAG-binding terminal sialic acid from GD1a and GT1b (14,17).…”

“…Some neurons respond to MAG primarily via sialoglycans, whereas others use NgRs and other receptors (13,14). Gangliosides are the most abundant sialoglycans on nerve cells (15).…”

Ibuprofen Enhances Recovery from Spinal Cord Injury by Limiting Tissue Loss and Stimulating Axonal Growth

“…Among the myelin inhibitors, MAG utilizes both NgRdependent and NgR-independent pathways, so its mechanism of action is the most diverse of these inhibitors Mehta et al, 2007;Venkatesh et al, 2007). Acutely, MAG induces growth cone collapse in cultured dorsal root ganglion neurons (Liu et al, 2002) (Fig.…”