The Nogo-66 receptor: focusing myelin inhibition of axon regeneration Aaron W. McGee and Stephen M. Strittmatter Departments of Neurology and Neurobiology, Yale University School of Medicine, PO Box 208018, New Haven, CT 06520, USA CNS myelin inhibits axonal outgrowth in vitro and is one of several obstacles to functional recovery following spinal cord injury. Central to our current understanding of myelin-mediated inhibition are the membrane protein Nogo and the Nogo-66 receptor (NgR). New findings implicate NgR as a point of convergence in signal transduction for several myelin-associated inhibitors. Additional studies have identified a potential corecep- tor for NgR as p75 NTR , and a second-messenger path- way involving RhoA that inhibits neurite elongation. Although these findings expand our understanding of the molecular determinants of adult CNS axonal regrowth, the physiological roles of myelin-associated inhibitors in the intact adult CNS remain ill-defined. Classic peripheral nerve graft studies illustrate that damaged axons have the capacity for regenerative out- growth when provided with a permissive substrate [1,2]. It is not the absence of growth-promoting molecules in the CNS but, rather, the presence of axon outgrowth inhibitors in CNS astroglial ‘scars’ [3] and CNS myelin [4,5] that suppresses the regrowth of damaged axons. Inhibition by CNS myelin can be alleviated using antibodies directed against inhibitory components of myelin [6,7] or by trophic-factor supplements [8]. Understanding how the various factors associated with myelin contribute to repressing axon extension could provide opportunities for positive intervention to improve the regenerative response of neurons after injury. Accordingly, much recent work has focused upon the identification and character- ization of factors within CNS myelin that inhibit neurite outgrowth and promote the collapse of growth cones in vitro and in vivo. Nogo The IN-1 monoclonal antibody was generated against a fraction of myelin enriched for inhibitory activity [7], and it improves axon outgrowth and functional recovery follow- ing injury when infused into the lesion site in several injury models [6,9,10]. Three groups identified the Nogo gene and the protein that corresponds to the IN-1 antigen, and demonstrated inhibition of axon growth in vitro with recombinant Nogo protein [11–13]. Nogo is differentially spliced to generate three proteins with alternative N termini. The longest isoform is termed Nogo-A and contains a unique sequence (‘amino-Nogo’) with a large percentage of acidic residues. The C terminus of Nogo has homology to the reticulon family of proteins and contains two predicted transmembrane domains and a short extracellular loop. Northern analysis has shown the three Nogo isoforms to have overlapping distributions: Nogo-A is predominantly expressed in the CNS, Nogo-B is a minor isoform and Nogo-C is enriched in the periphery, especially skeletal muscle [14]. Nogo-A is expressed by CNS myelin-forming oligodendrocytes but not by periph- eral Schwann cells [14–16], and can be observed in immunoelectron micrographs at the innermost adaxonal and outermost myelin membranes [14,15]. In addition, Nogo-A is expressed in a range of central and peripheral neurons [14–16]. Interestingly, both amino-Nogo and a 66 amino acid segment within the extracellular loop (Nogo-66) have been reported to inhibit neurite outgrowth in vitro. The topology of Nogo, predicted from amino acid composition, immuno- histochemistry [13] and homology to the reticulons, is such that the N terminus (containing amino-Nogo) and the C terminus are cytosolic, whereas the short 66 amino acid axon-inhibitory loop between the transmembrane domains protrudes into the lumenal or extracellular space. Selective blockade of Nogo-66 with a 40 amino acid peptide derived from the same region (the Nogo extracellular peptide, NEP1 – 40), partially blocks the inhibitory activity of CNS myelin, and improves locomotor activity when infused into the intrathecal space following a dorsal hemisection injury [17]. However, administration of antibodies directed against the N-terminal domain can induce axon sprouting from uninjured neurons [18] and also might promote functional recovery. One interpret- ation of the observation that both amino-Nogo and Nogo-66 are inhibitory is that Nogo-66 might serve to inhibit axon sprouting and outgrowth subsequent to myelination, whereas amino-Nogo is an additional inhibi- tory factor presented by ruptured myelin membranes after injury. Alternatively, a proportion of Nogo-A might adopt a second topology, in which amino-Nogo is extracellular, and then both of these regions could inhibit regeneration. Nogo receptor A protein that interacts with Nogo-66 was identified by an alkaline-phosphatase (AP)-fusion-protein expression screening strategy [19]. This protein binds with high (nanomolar) affinity to both AP and glutathione-S-trans- ferase (GST) proteins fused to Nogo-66. Transfection of the cDNA encoding this putative receptor into retinal ganglion Corresponding author: Stephen M. Strittmatter (stephen.strittmatter@yale.edu). Review TRENDS in Neurosciences Vol.26 No.4 April 2003 193 http://tins.trends.com 0166-2236/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0166-2236(03)00062-6