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Myelin-associated axon growth inhibitors such as Nogo-A/RTN4-A impair axon regeneration in the adult mammalian central nervous system (CNS). Here, we describe the cloning and expression of two independent Xenopus laevis rtn4 orthologs. As in mammals, alternative transcripts are generated both through differential splicing and promoter usage, giving rise to Xenopus nogo-A, -B, -C and to a new isoform, nogo-N/rtn4-N. Xenopus is therefore the ‘lowest’ vertebrate where Nogo-A was identified. Xenopus Nogo-A/RTN4-A is predominantly expressed in the nervous system, whereas the other isoforms mainly occur in nonneuronal tissues. Nogo-A/RTN4-A specific antisera detect the protein in myelinated fiber tracts of the spinal cord, hindbrain, optic nerve, tectum opticum and in isolated oligodendrocytes. In addition, subpopulations of CNS neurons are Nogo-A/RTN4-A positive. This expression pattern is consistent with that observed for rat Nogo-A and suggests similar functions. Nogo-A in Xenopus myelin might therefore contribute to the failure of spinal cord regeneration in frogs—a feature that may have evolved during the transition from fish to land vertebrates.

Nogo-A is a potent neurite growth inhibitorin vitroand plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

Reticulon (RTN) genes code for a family of proteins relatively recently described in higher vertebrates. The four known mammalian paralogues (RTN1, -2, -3, and -4/Nogo) have homologous carboxyl termini with two characteristic large hydrophobic regions. Except for RTN4-A/Nogo-A, thought to be an inhibitor for neurite outgrowth, restricting the regenerative capabilities of the mammalian CNS after injury, the functions of other family members are largely unknown. The overall occurrence of RTNs in different phyla and the evolution of the RTN gene family have hitherto not been analyzed. Here we expound data showing that the RTN family has arisen during early eukaryotic evolution potentially concerted to the establishment of the endomembrane system. Over 250 reticulon-like (RTNL) genes were identified in deeply diverging eukaryotes, fungi, plants, and animals. A systematic nomenclature for all identified family members is introduced. The analysis of exon-intron arrangements and of protein homologies allowed us to isolate key steps in the history of these genes. Our data corroborate the hypothesis that present RTNs evolved from an intron-rich reticulon ancestor mainly by the loss of different introns in diverse phyla. We also present evidence that the exceptionally large RTN4-A-specific exon 3, which harbors a potent neurite growth inhibitory region, may have arisen de novo approximately 350 MYA during transition to land vertebrates. These data emphasize on the one hand the universal role of reticulons in the eukaryotic system and on the other hand the acquisition of putative new functions through acquirement of novel amino-terminal exons.

Nogo-A is a myelin-associated neurite outgrowth inhibitory protein limiting recovery and plasticity after central nervous system injury. In this study, a purified monoclonal anti—Nogo-A antibody (7B12) was evaluated in two rat stroke models with a time-to-treatment of 24 hours after injury. After photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks, long-term contralateral forepaw function was reduced to about 55% of prelesion performance until the latest time point investigated (9 weeks). Forepaw function was significantly better in the 7B12-treated group 6 to 9 weeks after PCI, and reached about 70% of prelesion levels. Cortical infarcts were also produced in spontaneously hypertensive rats (SHR) by permanent middle cerebral artery occlusion (MCAO). In the control group, forepaw function remained between 40% and 50% of prelesion levels 4 to 12 weeks after MCAO. In contrast, 7B12-treated groups showed significant improvement between 4 and 7 weeks after MCAO from around 40% of prelesion levels at week 4 to about 60% to 70% at 7 to 12 weeks after MCAO. Treatment in both models was efficacious without influencing infarct volume or brain atrophy. Neuroanatomically in the spinal cord, a significant increase of midline crossing corticospinal fibers originating in the unlesioned sensorimotor cortex was found in 7B12-treated groups, reaching 2.3 ± 1.5% after PCI (control group: 1.1 ± 0.5%) and 4.5 ± 2.2% after MCAO in SHR rats (control group: 1.8 ± 0.8%). Behavioral outcome and the presence of midline crossing fibers in the cervical spinal cord correlated significantly, suggesting a possible contribution of the crossing fibers for forepaw function after PCI and MCAO. The results suggest that specific anti—Nogo-A antibodies bear potential as a new rehabilitative treatment approach for ischemic stroke with a prolonged time-to-treatment window.

The adult mammalian CNS has a limited capacity for nerve regeneration and structural plasticity. The presence of glia-derived inhibitory factors myelin-associated glycoprotein (MAG) and Nogo-A have been suggested to provide a nonpermissive environment for elongating nerve fibers. In particular, Nogo-A, an integral membrane protein predominantly expressed by oligodendrocytes, has been demonstrated to impair neurite growth in vitro and in vivo . Structure function analysis revealed that Nogo-A protein contains at least two active domains, NiG and Nogo-66, with diverse effects on neurite outgrowth and cell spreading. We now provide evidence that these inhibitory domains mediate their effects via an antagonistic regulation of the small GTPases RhoA and Rac1, resulting in activation of RhoA and suppression of Rac1. By inactivating RhoA with C3 transferase or the downstream effector Rho-kinase ROCK with[Y27632][1], the inhibitory effects of both Nogo-A fragments and MAG on neurite outgrowth and oligodendrocyte-mediated growth cone collapse were abolished. Furthermore, we show that the recently cloned receptor for Nogo-66 and MAG, NgR, is not necessary for either NiG- or MAG-induced RhoA activation. [1]: /lookup/external-ref?link_type=GEN&access_num=Y27632&atom=%2Fjneuro%2F22%2F23%2F10368.atom

The Nogo-66 receptor NgR has been implicated in the mediation of inhibitory effects of central nervous system (CNS) myelin on axon growth in the adult mammalian CNS. NgR binds to several myelin-associated ligands (Nogo-66, myelin associated glycoprotein, and oligodendrocyte-myelin glycoprotein), which, among other inhibitory proteins, impair axonal regeneration in the CNS of adult mammals. In contrast to mammals, severed axons readily regenerate in the fish CNS. Nevertheless, fish axons are repelled by mammalian oligodendrocytes in vitro. Therefore, the identification of fish NgR homologs is a crucial step towards understanding NgR functions in vertebrate systems competent of CNS regeneration. Here, we report the discovery of four zebrafish (Danio rerio) and five fugu (Takifugu rubripes) NgR homologs. Synteny between fish and human, comparable intron-exon structures, and phylogenetic analyses provide convincing evidence that the true fish orthologs were identified. The topology of the phylogenetic trees shows that the extra fish genes were produced by duplication events that occurred in ray-finned fishes before the divergence of the zebrafish and pufferfish lineages. Expression of zebrafish NgR homologs was detected relatively early in development and prominently in the adult brain, suggesting functions in axon growth, guidance, or plasticity.

Nogo-A is a potent neurite outgrowth inhibitory protein in vitro and is suggested to play a role in the lack of regeneration in the central nervous system of adult higher vertebrates. A shorter splice isoform, ASY/Nogo-B, has recently been reported to act as a proapoptotic protein, the loss of which would be typical for cancer cells. Here, we show that the osteosarcoma cell line SaOS-2 and the cell line CHO do express high levels of endogenous Nogo-B and that stable transfectants overexpressing high levels of Nogo-B do not differ significantly from the respective parental wild-type or control cell lines both in respect to cell proliferation and to spontaneous apoptosis or cell death induced by staurosporine and tunicamycin. The deletion of the second transmembrane domain of Nogo-B, which has been claimed to abolish its proapoptotic activity, leads to a shift of the protein from the ER to a cytoplasmic localization, suggesting that ER stress of highly overexpressed Nogo-B may lead to aversive cellular reactions under particular conditions. Our data do not support a function of Nogo-B as a physiological pro-apoptotic protein in certain types of cancer.

Nogo-A is a neurite growth inhibitor involved in regenerative failure and restriction of structural plasticity in the adult CNS. Three major protein products (Nogo-A, -B, and -C) are derived from the nogo gene. Here we describe the embryonic and postnatal expression of the three Nogo isoforms in the rat by in situ hybridization and immunohistochemistry. Northern and Western blot analysis indicated that Nogo-A is predominantly expressed in the nervous system with lower levels also present in testis and heart. In CNS myelin, confocal and immunoelectron microscopy revealed that Nogo-A is expressed in oligodendrocyte cell bodies and processes and localized in the innermost adaxonal and outermost myelin membranes. Additionally, we find Nogo-A to be expressed by projection neurons, in particular during development, and by postmitotic cells in the developing cortex, spinal cord, and cerebellum. The expression levels of Nogo-A/B were not changed significantly after traumatic lesions to the cortex or spinal cord. Nogo-B showed widespread expression in the central and peripheral nervous systems and other peripheral tissues. Nogo-C was mainly found in skeletal muscle, but brain and heart were also found to express this isoform. The localization of Nogo-A in oligodendrocytes fits well with its role as a myelin-associated inhibitor of regenerative fiber growth and structural plasticity. However, expression of Nogo-A in other tissues and, in particular, in neurons and the widespread expression of the two shorter isoforms, Nogo-B and -C, suggest that the Nogo family of proteins might have function(s) additional to the neurite growth-inhibitory activity.