Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo

doi:10.1073/pnas.0409026102

Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo

^*Department of Biological Sciences, ^§Program in Neurosciences, ^**Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305; ^¶Departments of Microbiology and Immunology and Structural Biology, Stanford University School of Medicine, Stanford, CA 94305; and ^∥Reeve–Irvine Research Center, University of California, Irvine, CA 92697

Communicated by Corey S. Goodman, Renovis, South San Francisco, CA, December 4, 2004 (received for review October 5, 2004)

Abstract

Axon regeneration failure in the adult mammalian CNS is attributed in part to the inhibitory nature of CNS myelin. Three myelin-associated, structurally distinct proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, have been implicated in this inhibition. Neuronal Nogo receptor (NgR) binds to each of the three inhibitors and has been proposed to mediate their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p75^NTR. To assess the contribution of NgR to mediating myelin inhibitory signals and regeneration failure in vivo, we generated and characterized NgR-deficient mice. Nogo transcripts are up-regulated in NgR mutants, indicating that NgR regulates Nogo in vivo. However, neurite outgrowth from NgR-deficient postnatal dorsal root ganglion or cerebellar granule neurons is inhibited by myelin and by a Nogo-66 substrate to the same extent as is from wild-type neurons, whereas p75^NTR-deficient neurons are less inhibited. The NgR ligand-binding domain promotes neurite outgrowth on Nogo-66, regardless of the genotype of the neurons, indicating that the NgR ligand-binding domain can act independent of NgR. Thus, NgR is not essential for mediating inhibitory signals from CNS myelin, at least in the neurons tested, whereas p75^NTR plays a central role in this response. Neither NgR-nor p75^NTR-deficient mice showed enhanced regeneration of corticospinal tract axons in comparison with wild-type controls after spinal dorsal hemisection. Our results thus fail to support a central role for NgR in axonal growth inhibition in vitro or in corticospinal tract regeneration block in vivo.

Footnotes

↵ †† To whom correspondence should be sent at the present address: Research Drug Discovery, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. E-mail: marctl{at}gene.com.
↵ † Present address: Research Drug Discovery, Genentech, Inc., South San Francisco, CA 94080.
↵ ‡ B.Z. and J.A. contributed equally to this work.
Author contributions: B.Z., J.A., and M.T.-L. designed research; B.Z., J.A., C.H., L.C., and O.S. performed research; B.Z., J.A., X.-L.H., and K.C.G. contributed new reagents/analytic tools; B.Z., J.A., C.H., L.C., O.S., and M.T.-L. analyzed data; and B.Z., J.A., and M.T.-L. wrote the paper.
Abbreviations: NgR, Nogo receptor; MAG, myelin-associated glycoprotein; OMgp, oligodendrocyte myelin glycoprotein; GPI, glycosylphosphatidylinositol; DRG, dorsal root ganglion; PDL, poly-d-lysine; LBD, ligand-binding domain.
Note. While this work was under review, Strittmatter and colleagues (22) published results on the analysis of a NgR mutant mouse they generated. Consistent with our results, they did not detect any enhanced regeneration of the CST in their mutants (22). They did report, however, enhanced regeneration of other tracts, most notably, the raphe spinal fibers; whether this result reflects a reduction in inhibition of such fibers by myelin inhibitors, or some other change in these mice, remains to be determined. Considering that NgR is thought to mediate the inhibitory signals not only from Nogo-66 but also MAG and OMgp, it is not clear why CST axons fail to regenerate in NgR mutants, although they appear to do so in at least some Nogo-deficient mutant lines (10). Kim et al. (22) also reported that NgR-deficient DRG growth cones do not collapse in response to Nogo-66 and other myelin inhibitors. In these experiments, the inhibitors caused a modest increase in the collapse of wild-type growth cones from ≈40% (seen in control cultures) to ≈60%, an increase that was not seen with their NgR knockout neurons. However, it is unclear whether this collapse was sustained, that is, whether these neurons are inhibited in longer-term cultures by an immobilized myelin or Nogo-66 substrate, which is the assay used previously by the same group (2, 5) to suggest a role for NgR in mediating inhibition, and also the assay used here that showed no change in inhibition after NgR deletion.
Freely available online through the PNAS open access option.