| The lack of axonal growth after injury in the adult central nervous system (CNS) is due to the formation of a glial scar, the absence of neurotrophic factors, and the presence of growth-inhibitory molecules associated with myelin, rather than an intrinsic inability of neurons to regenerate. Three inhibitors have been identified in myelin: Myelin-Associated Glycoprotein (MAG), Nogo-A, and Oligodendrocyte-Myelin glycoprotein (OMgp). In this study we show that MAG inhibits regeneration by high affinity interaction (KD 8 nM) with the glycosylphosphatidylinositol (GPI)-linked Nogo66 receptor (NgR) and activation of a p75 neurotrophin receptor (p75NTR)-mediated signaling pathway. Treatment of neurons with PI-PLC to remove GPI-linked proteins results in the loss of both MAG binding and growth inhibition. NgR-AP binds MAG-expressing CHO cells and Nogo66 competes with MAG for binding to NgR-expressing cells. MAG precipitates NgR from cerebellar neurons (CN), dorsal root ganglia (DRG), and NgR-expressing CHO cells. Importantly, MAG-induced inhibition of neurite outgrowth in CN and DRG neurons is blocked by the addition of an anti-NgR antibody or soluble NgR-AP, and by expression of a dominant negative NgR construct.; As NgR is a GPI-linked protein it cannot itself transduce the signal for inhibition. Thus, it must be part of a larger receptor complex containing a transmembrane protein. We demonstrate that MAG-Fc, Nogo66-AP and NgR-AP precipitate p75NTR from cerebellar granule neurons (CN). Neurons that are sensitive to the activity of MAG express both NgR and p75NTR. Notably, MAG does not inhibit neurite outgrowth from the DRG subpopulation that binds isolectin-B4. This subpopulation expresses NgR but not p75NTR. Further, we find that p75NTR is required for the inhibition of axonal regeneration by both MAG and myelin. DRG from p75NTRexonIII −/− mice, which lack the p75NTR ligand binding domain, are not sensitive to either MAG or myelin inhibitory activity. Finally, neurite outgrowth inhibition by MAG, Nogo66 and myelin is abolished by expression of a truncated p75NTR construct lacking the intracellular domain. Because MAG and Nogo66 use a common receptor complex, these results point to a redundancy in myelin inhibitors of regeneration and suggest possible targets for the development of therapies to encourage regeneration after spinal cord and CNS injury. |
