Phosphorylation-regulated cleavage of the reticulon protein Nogo-B by caspase-7 at a noncanonical recognition site.

Reticulons (RTNs) are a large family of transmembrane proteins present throughout the eukaryotic domain in virtually every cell type. Despite their wide distribution, their function is still mostly unknown. RTN4, also termed Nogo, comes in three isoforms, Nogo-A, -B, and -C. While Nogo-A has been described as potent inhibitor of nerve growth, Nogo-B has been implicated in vascular remodeling and regulation of apoptosis. We show here that Nogo-B gets cleaved by caspase-7, but not caspase-3, during apoptosis at a caspase nonconsensus site. By a combination of MS and site-directed mutagenesis we demonstrate that proteolytic processing of Nogo-B is regulated by phosphorylation of Ser(16) within the cleavage site. We present cyclin-dependent kinase (Cdk)1 and Cdk2 as kinases that phosphorylate Nogo-B at Ser(16) in vitro. In vivo, cleavage of Nogo-B is markedly increased in Schwann cells in a lesion model of the rat sciatic nerve. Taken together, we identified an RTN protein as one out of a selected number of caspase targets during apoptosis and as a novel substrate for Cdk1 and 2. Furthermore, our data support a functionality of caspase-7 that is distinct from closely related caspase-3.

Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish.

Reticulons (RTNs) are a family of evolutionary conserved proteins with four RTN paralogs (RTN1, RTN2, RTN3, and RTN4) present in land vertebrates. While the exact functions of RTN1 to RTN3 are unknown, mammalian RTN4-A/Nogo-A was shown to inhibit the regeneration of severed axons in the mammalian central nervous system (CNS). This inhibitory function is exerted via two distinct regions, one within the Nogo-A-specific N-terminus and the other in the conserved reticulon homology domain (RHD). In contrast to mammals, fish are capable of CNS axon regeneration. We performed detailed analyses of the fish rtn gene family to determine whether this regeneration ability correlates with the absence of the neurite growth inhibitory protein Nogo-A. A total of 7 rtn genes were identified in zebrafish, 6 in pufferfish, and 30 in eight additional fish species. Phylogenetic and syntenic relationships indicate that the identified fish rtn genes are orthologs of mammalian RTN1, RTN2, RTN3, and RTN4 and that several paralogous fish genes (e.g., rtn4 and rtn6) resulted from genome duplication events early in actinopterygian evolution. Accordingly, sequences homologous to the conserved RTN4/Nogo RHD are present in two fish genes, rtn4 and rtn6. However, sequences comparable to the first approximately 1,000 amino acids of mammalian Nogo-A including a major neurite growth inhibitory region are absent in zebrafish. This result is in accordance with functional data showing that axon growth inhibitory molecules are less prominent in fish oligodendrocytes and CNS myelin compared to mammals.

Postnatal ontogeny of hippocampal expression of the mineralocorticoid and glucocorticoid receptors in the common marmoset monkey.

The mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are nuclear transcription factors that mediate many of the basal and stress functions and effects of the corticosteroid hormones, including those related to brain development. Despite this, relatively little is known about the postnatal ontogeny of MR and GR gene and protein expression in the central nervous system, and this is particularly true of the primates, including humans. Here we describe the postnatal ontogeny of central MR and GR gene and protein expression in the common marmoset monkey. By developing marmoset-specific riboprobes and using in situ hybridization, it was demonstrated that MR mRNA expression in the dentate gyrus and Ammon's horn was significantly greater in marmoset infants (aged 4-6 weeks) than in neonates (1-2 days), juveniles (4-5 months) and adults (3-6 years), with expression in the latter three ontogenetic stages being broadly similar. In the same subjects and ontogenetic stages, GR mRNA expression was developmentally consistent in the marmoset dentate gyrus and Ammon's horn, as well as in the paraventricular nucleus of the hypothalamus. Qualitative immunohistochemical comparison of infants and adults demonstrated that MR protein expression in the hippocampus was, as for mRNA, also greater in infants than adults, and that hippocampal GR protein was, as for mRNA, also similar in infants and adults. The increase in MR mRNA expression between the stages of neonate and infant co-occurred with a reduction in basal plasma ACTH and cortisol titres. The ontogenetic profiles of MR and GR gene expression in the marmoset monkey are therefore fundamentally different from those described for the rat and the mouse. This evidence for the postnatal ontogeny of central corticosteroid nuclear receptor expression in a primate is important for understanding both the developmental stage-specific significance of stress exposure and its potential long-term effects on health and disease.

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

Identification of two NOGO/RTN4 genes and analysis of Nogo-A expression in Xenopus laevis.

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.

Identification of Nogo-66 receptor (NgR) and homologous genes in fish.

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.

Serum and cerebrospinal fluid antibodies to Nogo-A in patients with multiple sclerosis and acute neurological disorders.

Nogo-A is a protein associated with central nervous system (CNS) myelin thought to impair regenerative responses and to suppress sprouting and plastic changes of synaptic terminals. In this study, we report that serum IgM autoantibodies to the recombinant large N-terminal inhibitory domain of Nogo-A are a frequent finding in multiple sclerosis (MS) and acute inflammatory (IND) and non-inflammatory neurological diseases (OND), but not in neurodegenerative diseases (ND), systemic inflammatory disease and healthy controls. Furthermore, we demonstrate intrathecal production of anti-Nogo-A antibodies measured by increased IgG indices. Intrathecal anti-Nogo antibodies were significantly more frequent in patients with relapsing-remitting as compared to chronic progressive (CP) MS. We also found a highly significant negative correlation of these antibody responses with age indicating that they are more frequent in younger patients. We finally demonstrate that human anti-Nogo-A antibodies recognize native Nogo-A in brain extracts, oligodendrocytes and cells expressing human Nogo-A.

Rapid induction of autoantibodies against Nogo-A and MOG in the absence of an encephalitogenic T cell response: implication for immunotherapeutic approaches in neurological diseases.

Vaccinations against various antigens of the central nervous system (CNS) are gaining increasing interest as a therapeutic approach in a variety of neurological diseases such as spinal cord injury, ischemic stroke, Alzheimer disease, or spongiform encephalopathy. In the present work, the time window after spinal cord injury allowing potentially therapeutic antibody to penetrate the damaged blood-brain barrier (BBB) was measured by intravenous injection of a monoclonal anti-Nogo-A antibody. Although an influx of Nogo antibodies at the lesion site was detectable up to 2 wk after injury, a significant decrease in BBB permeability was noticed within the first week. Clearly, therefore, a vaccination protocol with a rapid antibody response is required for acute therapeutic interventions after CNS trauma. We designed a conjugate vaccine paradigm with particular focus on the safety and the kinetics of the antibody response. As antigen targets, we used Nogo-A and the strongly encephalitogenic myelin-oligodendrocyte glycoprotein (MOG). Intrasplenic autoimmunization of rats with a Nogo-A-specific region fused to the Tetanus toxin C-fragment (TTC) resulted in a fast IgM response against Nogo-A. A specific switch to IgG was observed as soon as 4-7 days after intrasplenic immunization in TTC-primed animals. In spite of the induction of a specific IgG response after intrasplenic immunization, no signs of experimental autoimmune disease (EAE) or inflammatory infiltrates on histological examinations were observable. In contrast to subcutaneous immunization with MOG, in vitro cytokine secretion assays (IL-2, IL-10, and IFN-gamma) did not reveal activation of MOG-specific T cells after intrasplenic immunization. Our findings have critical implications for future strategies in the development of safe and efficient therapeutic vaccines for neurological diseases.

Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions.

Nogo-A is a potent neurite growth inhibitor in vitro and 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.

A reticular rhapsody: phylogenic evolution and nomenclature of the RTN/Nogo gene family.

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 and its paRTNers.

Reticulons (RTNs) are a relatively new eukaryotic gene family with unknown functions but broad expression and peculiar topological features. RTNs are widely distributed in plants, yeast and animals and are characterized by a approximately 200-amino-acid C-terminal domain, including two long hydrophobic sequences. Nogo/RTN4 can inhibit neurite growth from the cell surface via specific receptors, whereas more general, 'ancestral', RTN functions might relate to those of the endoplasmic reticulum - for example, intracellular trafficking, cell division and apoptosis. Here, we review the taxonomic distribution and tissue expression of RTNs, summarize recent discoveries about RTN localization and membrane topology, and discuss the possible functions of RTNs.

Do cancer cells die because of Nogo-B?

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.

Anti-Nogo-A antibody infusion 24 hours after experimental stroke improved behavioral outcome and corticospinal plasticity in normotensive and spontaneously hypertensive rats.

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.

Genomic structure and functional characterisation of the promoters of human and mouse nogo/rtn4.

The reticulon-family member Nogo-A is a potent neurite growth inhibitory protein in vitro and may play a role in the restriction of axonal regeneration after injury and of structural plasticity in the CNS of higher vertebrates. Of the three major isoforms of Nogo, Nogo-A is mostly expressed in the brain, Nogo-B is found in a ubiquitous pattern, and Nogo-C is most highly expressed in muscle. Seven additional splice-variants derived both from differential splicing and differential promoter usage have been identified. Analysis of the TATA-less Nogo-A/B promoter (P1) shows that conserved GC-boxes and a CCAAT-box within the first 500bp upstream of the transcription start are responsible for its regulation. No major differences in the methylation status of the P1 CpG-island in tissues expressing or not expressing Nogo-A/B could be detected, suggesting that silencer elements are involved in the regulation. The specific expression pattern of Nogo-A/B is due to differential splicing. The basal Nogo-C promoter (P2) is regulated by a proximal and a distal element. The 5'UTR of Nogo-C harbours a negative control element. These data may help to identify factors that can modulate Nogo transcription, thus offering an alternative approach for Nogo neutralisation.

Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1.

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, 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.

Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions.

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.