Nogo-A, a membrane protein enriched in myelin of the adult CNS, inhibits neurite growth and regeneration; neutralizing antibodies or receptor blockers enhance regeneration and plasticity in the injured adult CNS and lead to improved functional outcome. Here we show that Nogo-A-specific knock-outs in backcrossed 129X1/SvJ and C57BL/6 mice display enhanced regeneration of the corticospinal tract after injury. Surprisingly, 129X1/SvJ Nogo-A knock-out mice had two to four times more regenerating fibers than C57BL/6 Nogo-A knock-out mice. Wild-type newborn 129X1/SvJ dorsal root ganglia in vitro grew a much higher number of processes in 3 d than C57BL/6 ganglia, confirming the stronger endogenous neurite growth potential of the 129X1/SvJ strain. cDNA microarrays of the intact and lesioned spinal cord of wild-type as well as Nogo-A knock-out animals showed a number of genes to be differentially expressed in the two mouse strains; many of them belong to functional categories associated with neurite growth, synapse formation, and inflammation/ immune responses. These results show that neurite regeneration in vivo, under the permissive condition of Nogo-A deletion, and neurite outgrowth in vitro differ significantly in two widely used mouse strains and that Nogo-A is an important endogenous inhibitor of axonal regeneration in the adult spinal cord.

SUMMARYWiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching. Nogo-A KO mice were generated by homologous recombination of exons 2 and 3 in the Nogo-A gene, as described previously (Dimou et al., 2006;Simonen et al., 2003). Antibodies11C7 antibody was raised against an 18-amino acid Nogo-A peptide corresponding to the rat sequence of amino acids 623-640 (Oertle et al., 2003). 7B12 antibody has an epitope in the C-terminal part of the Nogo-A-specific region (aa 763-820) (Oertle et al., 2003). Both antibodies are function-blocking antibodies (Liebscher et al., 2005) and are monospecific for Nogo-A (Dodd et al., 2005;Oertle et al., 2003). Polyclonal rabbit antibody Rb173A (Laura) recognizes the Nogo-A-specific region (aa 174-979) and the antibody Rb1 (Bianca) is specific for the N terminus of Nogo-A and Nogo-B (aa 1-172) (Dodd et al., 2005;Oertle et al., 2003). Nogo-A receptor complex components were blocked by anti-NgR (R&D Systems) or anti-Lingo1 (Abcam) antibodies. Compound Y27632 was used to inhibit ROCK (Sigma). Rabbit anti-neurofilament 160 antibody (Chemicon) was used for whole-mount staining and mouse anti--tubulin III (Abcam) was used for staining of dissociated DRG cultures. DRG cultures DRG explant culturesDRGs of newborn rats, wild-type and Nogo-A KO mice were plated on 20 g/ml poly-L-lysine (PLL)-coated four-well tissue culture plates. Cultures were incubated for 5-7 days at 37°C and 5% CO 2 atmosphere in F12 medium (Invitrogen) supplemented with 10% foetal bovine serum (Sigma), 100 g/ml nerve growth factor (NGF) and 10 g/ml gentamycin (Sigma). Cytosine arabinoside was added to inhibit mitosis of non-neuronal cells. Control, monoclonal mouse IgG antibody directed against wheat auxin, anti-Nogo-A antibodies 11C7 or 7B12, antibodies against NgR-1 or Lingo1 or the ROCK blocker Y27632 were added to the culture medium at a concentration of 10 g/ml at the beginnin...