Myelin-associated glycoprotein in myelin and expressed by Schwann cells inhibits axonal regeneration and branching.

The mammalian CNS does not regenerate after injury due largely to myelin-specific inhibitors of axonal growth. The PNS, however, does regenerate once myelin is cleared and myelin proteins are down-regulated by Schwann cells. Myelin-associated glycoprotein (MAG), a sialic acid binding protein, is a potent inhibitor of neurite outgrowth when presented to neurons in culture. Here, we present additional evidence that strongly supports the suggestion that MAG contributes to the overall inhibitory properties of myelin. When myelin from MAG-/- mice is used as a substrate, axonal length is 100 and 60% longer for neonatal cerebellar and older DRG neurons, respectively, compared to MAG+/+ myelin. The converse is true for neurites from neonatal DRG neurons, which are twice as long on MAG+/+ relative to MAG-/- myelin, consistent with MAG's dual role of promoting axonal growth from neonatal DRG neurons but inhibiting growth in older DRG and all other postnatal neurons examined. Furthermore, desialylating neurons reverses inhibition by CNS myelin by 45%. Contrary to previous reports, under these conditions PNS myelin is also inhibitory for axonal regeneration. Importantly, results using PNS MAG-/- myelin as a substrate suggest that MAG contributes to this inhibition. Finally, when Schwann cells not expressing MAG and permissive for axonal growth are induced to express MAG by retroviral infection, not only is axonal outgrowth greatly inhibited by these cells but so also is neurite branching. This suggests for the first time that MAG not only affects axonal regeneration but may also play a role in the control of axonal sprouting.

Myelin-associated glycoprotein interacts with neurons via a sialic acid binding site at ARG118 and a distinct neurite inhibition site.

Inhibitory components in myelin are largely responsible for the lack of regeneration in the mammalian CNS. Myelin-associated glycoprotein (MAG), a sialic acid binding protein and a component of myelin, is a potent inhibitor of neurite outgrowth from a variety of neurons both in vitro and in vivo. Here, we show that MAG's sialic acid binding site is distinct from its neurite inhibitory activity. Alone, sialic acid-dependent binding of MAG to neurons is insufficient to effect inhibition of axonal growth. Thus, while soluble MAG-Fc (MAG extracellular domain fused to Fc), a truncated form of MAG-Fc missing Ig-domains 4 and 5, MAG(d1-3)-Fc, and another sialic acid binding protein, sialoadhesin, each bind to neurons in a sialic acid- dependent manner, only full-length MAG-Fc inhibits neurite outgrowth. These results suggest that a second site must exist on MAG which elicits this response. Consistent with this model, mutation of arginine 118 (R118) in MAG to either alanine or aspartate abolishes its sialic acid-dependent binding. However, when expressed at the surface of either CHO or Schwann cells, R118-mutated MAG retains the ability to inhibit axonal outgrowth. Hence, MAG has two recognition sites for neurons, the sialic acid binding site at R118 and a distinct inhibition site which is absent from the first three Ig domains.

Soluble myelin-associated glycoprotein (MAG) found in vivo inhibits axonal regeneration.

Myelin-associated glycoprotein (MAG) is a potent inhibitor of axonal regeneration when used as a substrate for growth. However, to be characterized definitively as inhibitory rather than nonpermissive, MAG must also inhibit axonal regeneration when presented in solution. Here, we show that soluble dMAG (extracellular domain only), released in abundance from myelin and found in vivo and chimeric MAG-Fc, can potently inhibit axonal regeneration. For both dMAG and MAG-Fc, inhibition is dose-dependent. If myelin-conditioned medium is immunodepleted of dMAG, or if a MAG antibody is included with MAG-Fc, inhibition is completely neutralized. Together with MAG's ability to induce growth cone collapse, these results demonstrate that MAG is an inhibitory molecule and not merely nonpermissive. The results also suggest that MAG binds to a specific receptor and initiates a signal transduction cascade to effect inhibition. Importantly, these results indicate that soluble dMAG detected in vivo could contribute to the lack of regeneration in the mammalian CNS after injury.

Myelin-associated glycoprotein inhibits axonal regeneration from a variety of neurons via interaction with a sialoglycoprotein.

Myelin-associated glycoprotein (MAG) is a potent inhibitor of axonal regeneration from both cerebellar neurons and adult dorsal root ganglion (DRG) neurons. In contrast, MAG promotes axonal growth from newborn DRG neurons. Here, we show that the switch in response to MAG from promotion to inhibition of neurite outgrowth by DRg neurons occurs sharply at Postnatal Day 3. To date, of all the neurons tested a postnatal switch in response is only observed for DRG neurons; MAG inhibits axonal growth from retinal, superior cervical ganglion, spinal, and hippocampal neurons of all postnatal ages. Furthermore, MAG binds to neurons from which it promotes and from which it inhibits outgrowth, in a sialic-acid-dependent manner. Now we show this binding is also trypsin-sensitive. Hence, the interaction is via a sialoglycoprotein. Binding of MAG to all the neurons tested here was also sialic-acid-dependent. Importantly, both inhibition and promotion of neurite outgrowth by MAG are reduced, or abolished completely, either by desialyation of the neurons prior to the outgrowth assay or by including small sialic-acid-bearing sugars in the cultures. These results suggest that MAG is likely to contribute to the lack of regeneration observed throughout the nervous system. Also, it is likely that MAG is exerting its effect, either directly or indirectly, on both promotion and inhibition of neurite outgrowth via a neuronal sialoglycoprotein.