MAG is a multifunctional myelin protein that enhances axon-myelin stability, regulates the axon cytoskeleton, and stabilizes the distribution of molecules at nodes of Ranvier (5
). In addition to its physiological functions, MAG is among a group of molecules that inhibit axon regeneration at sites of injury thereby limiting functional recovery (1
). These ARIs include MAG, Nogo, and OMgp on residual myelin and chondroitin sulfate proteoglycans on the astrocytic scar. Destruction of or blocking ARIs may enhance axon regeneration and functional recovery (3
). Identification of the molecular components of each ARI signaling system provides potential targets for therapeutic intervention.
Our data indicate that there are dual receptors that independently mediate MAG inhibition. Using native MAG extracted from rat or mouse myelin, or a soluble fragment of native MAG, and using a set of enzymatic and pharmacological agents to modulate potential MAG receptors, we found that different nerve cell types use different MAG receptors (). The clearest example of alternative receptors was in the response of DRGNs and CGNs to dMAG. dMAG acts exclusively via a PI-PLC- and NEP1–40-sensitive mechanism in DRGNs and exclusively via a sialidase- and P4-sensitive mechanism in CGNs ( and ). The near complete lack of overlap in receptor utility in these experiments implies the independent ability of NgRs and gangliosides to mediate MAG inhibition and argues against a requirement for functional association between the NgR pathway and the ganglioside pathway.
Enzymes and agents used in these studies
The dual independent receptor hypothesis is consistent with our other findings. The additive ability of PI-PLC and sialidase (or P4 and NEP1–40) to reverse MAG inhibition of DRGN neurite outgrowth to levels equivalent to either anti-MAG or MAG-null controls (, , and ) is consistent with independent pathways that act within cells or cell populations to restrict DRGN neurite outgrowth. In CGNs, sialidase or P4 reversal of axon outgrowth inhibition by MAG and the lack of significant reversal by PI-PLC or NEP-40 are consistent with a ganglioside-dependent pathway that is wholly independent of the NgR-dependent pathway.
The quantitatively similar reversal of MAG inhibition by sialidase and P4 in three different nerve cell types implicate gangliosides (sialoglycosphingolipids) rather than sialoglyco-proteins as functional MAG receptors (12
). Gangliosides are the major sialoglycans in the nervous system (44
); the major MAG-binding gangliosides, GD1a and GT1b, are among the most abundant and widely distributed (45
PI-PLC and the peptide inhibitor NEP1–40 led to similar quantitative reversal of MAG inhibition of neurite outgrowth from DRGNs and HNs. As GPI-anchored proteins, NgRs are sensitive to PI-PLC release (15
). NEP1–40, a sequence derived from Nogo-66, is a competitive antagonist of Nogo-NgR1 binding (17
). Although NEP1–40 had not been known to block MAG-NgR binding (13
), MAG and Nogo compete for a similar binding site on NgR (14
). We conclude that NEP1–40 and PI-PLC are acting at the same MAG receptor sites (NgR1 and/or NgR2) on DRGNs and HNs. An alternative hypothesis is that a portion of inhibition in our assays is due to extracted and adsorbed Nogo. Although this cannot be excluded in all cases, it is unlikely to account, for instance, for the nearly complete reversal of dMAG inhibition of DRGN neurite outgrowth by addition of either anti-MAG antibody or NEP1–40 (). Likewise addition of anti-MAG antibody or a combination of P4 and NEP1–40 reversed inhibition of DRGN neurite outgrowth on wild type mouse myelin extract to the same level as that on MAG-null mouse extract, implicating MAG as the primary inhibitor in our in vitro
assay system () and NEP1–40 as an effective blocker of MAG.
Both NgR1 and NgR2 bind MAG and mediate its inhibition (13
). Because the relative selectivity of NEP1–40 for NgR1 and NgR2 has not been detailed, further experiments will be needed to identify which is operative in DRGNs and HNs.
Prior reports identifying either gangliosides or NgRs as functional receptors for MAG used a variety of nerve cell types in vitro
, a variety of MAG sources (native and recombinant), and different measures of neurite outgrowth. Although our data reconcile some of these apparently conflicting findings, other discrepancies have yet to be resolved. In this light, it may be relevant that MAG is heavily glycosylated and that expression of recombinant forms in ectopic cells may result in sialylated glycoforms that bind to the glycan binding site of MAG and alter its binding. This hypothesis is supported by data showing that pretreatment of MAG-expressing CHO or COS cells with sialidase enhanced their ability to bind gangliosides (47
). Because some nerve cell types (or populations) display dual MAG receptor pathways (e.g.
DRGNs and HNs), one can envision that the nature of the MAG receptor used may depend on the glycosylation state of MAG or adjacent sialoglycans in experimental systems and perhaps in vivo
ARIs bind to axon or growth cone receptors, initiating a signal cascade that results in RhoA activation, engagement of Rho effectors (e.g.
Rho kinase), control of actin polymerization, and inhibition of axon outgrowth (1
). The molecules that link MAG-receptor binding to RhoA activation have not been fully elucidated. It has been proposed that MAG-NgR1 binds to the transmembrane neurotrophin receptor p75NTR
(or alternatively the related protein TROY) in complex with Lingo-1 to engage Rho GDP dissociation inhibitor and activate RhoA (34
). In each of the nerve cell systems tested, we found that Y-27632, an inhibitor of Rho kinase (37
), was effective in reversing MAG-mediated neurite outgrowth inhibition (, , and ). Because MAG accessed different receptors in the different nerve cell types, we conclude that RhoA is downstream of each receptor. In contrast, a p75NTR
-blocking peptide, TAT-Pep5 (16
), quantitatively tracked with PI-PLC and NEP1–40 in its ability to reverse MAG inhibition, having no effect on inhibited CGNs but partially reversing MAG inhibition of DRGNs and HNs (). We conclude that p75NTR
is not a required transducer for MAG-ganglioside-mediated inhibition. The quantitatively similar effects of the inhibitors of p75 and NgR are consistent with their functional association.
Previous studies reported a physical interaction between gangliosides and p75NTR
as part of an NgR-mediated signaling pathway in response to MAG-Fc-mediated inhibition of axon outgrowth from CGNs (35
). Our data, in contrast, indicate that ganglioside-mediated MAG inhibition of CGNs is independent of NgR or p75NTR
(–). The basis for this difference has yet to be determined, although different physical forms of MAG (native and MAG-Fc) and different axon outgrowth measures were used.
In our HN studies, PI-PLC modestly reversed MAG inhibition, sialidase robustly reversed inhibition, and the two were not additive ( and ). Similarly partial reversal by NEP1–40 was not additive with more robust reversal by P4. TAT-Pep5 reversal was equivalent to that of PI-PLC/NEP1–40. These data are consistent with a portion of the ganglioside-mediated MAG inhibition being associated with NgR/p75NTR in HNs. However, given the modest amount of reversal by PI-PLC/NEP1–40/TAT-Pep5 in HNs, we cannot rule out a minor inhibitory role of non-MAG inhibitors on our in vitro inhibitory substrate.
A model that fits our data is presented in . We propose that there are at least two independent pathways for MAG inhibition of neurite outgrowth, one via binding to gangliosides and a second via binding to NgRs. Both pathways link to RhoA activation. Published data support a role for p75NTR as a signal transducer for the NgR pathway. Our data using DRGNs and HNs are consistent with this pathway. MAG inhibition via gangliosides in CGNs did not appear to require NgR or p75NTR and therefore must signal via another transducer.
Dual receptor model of MAG-mediated inhibition of axon regeneration
A recent report further supports the model in (52
). Sialidase modestly but significantly attenuated axon outgrowth inhibition when CGNs were cultured on Chinese hamster ovary cells ectopically expressing MAG (MAG-CHO cells) but failed to attenuate inhibition of retinal ganglion neurons on the same MAG-expressing cells. Notably MAG inhibition of neurite outgrowth from retinal neurons obtained from mice lacking NgR1 was partially reversed by sialidase. Furthermore CGNs and retinal neurons from mice lacking p75NTR
or TROY remained sensitive to MAG-CHO inhibition. These data are fully consistent with at least two MAG receptors, one of which is sialidase-sensitive. The reason for more robust reversal of inhibition by sialidase and modifiers of NgRs in our study may be related to the physical nature of the inhibitor used (native MAG versus
MAG-CHO) or differences in neurite outgrowth measures.
The existence of dual (or multiple) independent receptors that mediate MAG inhibition of axon outgrowth from different neuronal cell types implies that a single receptor-targeted therapy may not be uniformly successful in enhancing therapeutic recovery from nerve injury or disease (53
). Quantifying the relative roles of the different MAG receptors on different axons may provide more accurate insights into the potential and limitations of different therapeutic interventions. Identifying the associated downstream transducing molecules and their points of convergence may also help in the development of therapeutics to enhance recovery from traumatic nerve injury and disease.