The Nogo receptor,its ligands and axonal regeneration in the spinal cord; a review

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.An erratum to this article can be found at     

Nogo and Nogo-66 receptor in human and chick: implications for development and regeneration.

Antibodies to the myelin protein Nogo increase axonal regrowth after central nervous system injury. We have investigated whether Nogo expression contributes to loss of regenerative potential during development by using chick embryos, which regenerate their spinal cord until embryonic day (E) 13, when myelination begins. We show that Nogo-A and the Nogo receptor (NgR) are developmentally regulated both in chick and human embryos, are first detected at developmental stages when the chick spinal cord regenerates, and are not down-regulated after injury at permissive stages for regeneration. Therefore, expression of Nogo-A and NgR in pre-E13 chick spinal cords is not sufficient to inhibit regeneration. Nogo-A expression in the chick early embryo is primarily observed in axons, whereas NgR is mainly located on neuronal cell bodies, both in spinal cord and eye, and in striated muscle including the heart. With the onset of myelination, there is down-regulation of Nogo-A expression in neurons. Therefore, loss of regenerative potential might be linked to changes in its cellular localization. The possibility that only Nogo expressed in mature oligodendrocytes can exercise inhibitory effects would reconcile the lack of inhibition we observe in developing chick spinal cords before the onset of myelination with evidence from other laboratories on the inhibitory effects of Nogo in mature central nervous system. The distinctive and complementary patterns of Nogo-A and NgR expression and their conservation throughout evolution support the view that Nogo signaling represents a key pathway in nervous system and striated muscle development. Its putative role in target innervation and establishment of neural circuitry is discussed.     

Nogo与Nogo受体在正常小鼠大脑的分布

NOgo与NOgo受体对抑制中枢神经系统的再生有着重要的作用。本研究用本室制备的抗Nogo和抗Nogo受体的多克隆抗体进行免疫组织化学染色,在低倍镜下计数阳性细胞、观察Nogo与Nogo受体在正常小鼠脑内的分布。结果显示:Nogo样和Nogo受体样免疫阳性结构在大脑皮层、海马、下丘脑、苍白球、尾壳核和黑质等处的神经元细胞核中有较强的表达,而在胞浆和突起内表达较弱。由此我们得出结论,Nogo和Nogo受体蛋白在成年小鼠脑内分布广泛。     

Nogo受体(N-20)在大鼠视神经损伤后视网膜的表达

观察Nogo受体(NgR)在Wistar大鼠视神经(ON)损伤后视网膜各层的表达变化及分布规律,为Nogo蛋白抑制中枢神经再生的理论提供形态学依据。本实验各组动物均采用眶内眼球后2 mm ON切断术,术后动物分别存活3 d和7 d后取视网膜固定、冰冻后做水平切片,用免疫组织化学的方法观察NgR的表达情况。结果显示:NgR在正常对照组视网膜内3层有表达;在切断ON后实验各组有强烈表达;各组在损伤ON和移植神经组织后存活3 d时也有强烈表达,至7 d时表达有所下降。以上结果表明:NgR在大鼠ON损伤后视网膜的表达位于节细胞层、神经纤维层和内网层;其表达程度与移植物的种类有关,并可随存活时间的延长而下降。     

Association study of Nogo‐related genes with schizophrenia in a Japanese case–control sample

Many studies have suggested that myelin dysfunction may be causally involved in the pathogenesis of schizophrenia. Nogo (RTN4), myelin‐associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMG) all bind to the common receptor, Nogo‐66 receptor 1 (RTN4R). We examined 68 single nucleotide polymorphisms (SNPs) (51 with genotyping and 17 with imputation analysis) from these four genes for genetic association with schizophrenia, using a 2,120 case–control sample from the Japanese population. Allelic tests showed nominally significant association of two RTN4 SNPs (P = 0.047 and 0.037 for rs11894868 and rs2968804, respectively) and two MAG SNPs (P = 0.034 and 0.029 for rs7249617 and rs16970218, respectively) with schizophrenia. The MAG SNP rs7249617 also showed nominal significance in a genotypic test (P = 0.017). In haplotype analysis, the MAG haplotype block including rs7249617 and rs16970218 showed nominal significance (P = 0.008). These associations did not remain significant after correction for multiple testing, possibly due to their small genetic effect. In the imputation analysis of RTN4, the untyped SNP rs2972090 showed nominally significant association (P = 0.032) and several imputed SNPs showed marginal associations. Moreover, in silico analysis (PolyPhen) of a missense variant (rs11677099: Asp357Val), which is in strong linkage disequilibrium with rs11894868, predicted a deleterious effect on Nogo protein function. Despite a failure to detect robust associations in this Japanese cohort, our nominally positive signals, taken together with previously reported biological and genetic findings, add further support to the “disturbed myelin system theory of schizophrenia” across different populations. © 2011 Wiley‐Liss, Inc.     

髓鞘相关抑制因子与中枢神经系统损伤

轴突生长的抑制因素是中枢神经系统受损后再生困难的主要原因之一。髓鞘相关糖蛋白(MAG),Nogo蛋白和少突胶质细胞-髓鞘糖蛋白(OMgp)是3种主要的髓鞘相关抑制因子（MAIFs）。Ephrin-B3是另外一种髓鞘相关抑制因子。Nogo受体,p75受体和LINGO-1组成Nogo受体复合体。Rho-A和蛋白激酶C是MAIFs发挥轴突生长抑制作用的重要胞内分子。拮抗MAIFs或是阻断MAIFs的信号通路,可促进中枢神经损伤后的轴突再生。     

Neuronal Nogo‐A in New‐born Retinal Ganglion Cells: Implication for the Formation of the Age‐related Fiber Order in the Optic Tract

Nogo‐A is highly expressed in oligodendrocytes in the adult central nervous system (CNS). Recently it was found that Nogo‐A is also expressed in some neuronal types during development. Here, we examined the expression pattern of Nogo‐A in both the retina and optic tract (OT) of mouse embryos from E12 to E15. After perturbation of its function in the OT for 5 hr in the brain slice culture system using a Nogo‐A specific antibody or antagonist of its receptor (NEP1‐40), the optic nerve fibers and growth cones were traced with DiI. We showed that most Tuj‐1 positive new‐born neurons at E12 were Nogo‐A positive. At E15, retinal neurons reduced the Nogo‐A expression. It was worth noting that some projecting axons expressed Nogo‐A along the retinofugal pathway. On the basis of their specific locations within the superficial half of the OT and the colocalization with GAP‐43 (a marker for the newly born growth cones and axons), we concluded that those Nogo‐A positive axons were the newly arrived retinal fibers. Blocking the function of Nogo‐A with Nogo‐A antibody or NEP1‐40 resulted in the shift of DiI labeled axons and growth cones from the superficial half to the whole depth of the OT. These results indicate that Nogo‐A in the newly born retinal ganglion cells (RGCs) and their axons are involved in sorting out the newly arrived axons to the subpial region of the OT. Anat Rec, 299:1027–1036, 2016. © 2016 Wiley Periodicals, Inc.     

The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI

The neurotrophin receptor p75(NTR) is involved in the regulation of axonal elongation by neurotrophins as well as several myelin components, including Nogo, myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (OMgp). Neurotrophins stimulate neurite outgrowth by inhibiting Rho activity, whereas myelin-derived proteins activate RhoA and thereby inhibit growth. Here we show that direct interaction of the Rho GDP dissociation inhibitor (Rho-GDI) with p75(NTR) initiates the activation of RhoA, and this interaction between p75(NTR) and Rho-GDI is strengthened by MAG or Nogo. We also found that p75(NTR) facilitates the release of prenylated RhoA from Rho-GDI. The peptide ligand that is associated with the fifth alpha helix of p75(NTR) inhibits the interaction between Rho-GDI and p75(NTR), thus silencing the action mediated by p75(NTR). This peptide has potential as a therapeutic agent against the inhibitory cues that block regeneration in the central nervous system.     

A p75(NTR) and Nogo receptor complex mediates repulsive signaling by myelin-associated glycoprotein

Myelin-associated glycoprotein (MAG), an inhibitor of axon regeneration, binds with high affinity to the Nogo-66 receptor (NgR). Here we report that the p75 neurotrophin receptor (p75(NTR)) is a co-receptor of NgR for MAG signaling. In cultured human embryonic kidney (HEK) cells expressing NgR, p75(NTR) was required for MAG-induced intracellular Ca2+ elevation. Co-immunoprecipitation showed an association of NgR with p75(NTR) that can be disrupted by an antibody against p75(NTR) (NGFR5), and extensive coexpression was observed in the developing rat nervous system. Furthermore, NGFR5 abolished MAG-induced repulsive turning of Xenopus axonal growth cones and Ca2+ elevation, both in neurons and in NgR/p75(NTR)-expressing HEK cells. Thus we conclude that p75(NTR) is a co-receptor of NgR for MAG signaling and a potential therapeutic target for promoting nerve regeneration.     

The use of a gold nanoparticle-based adjuvant to improve the therapeutic efficacy of hNgR-Fc protein immunization in spinal cord-injured rats

As a common receptor for three myelin associated inhibitors, Nogo-66 receptor (NgR) mediates their inhibitory activities on neurite outgrowth in the adult mammalian central nervous system (CNS). Therapeutic vaccination protocol targeting NgR emulsified with Freund's adjuvant (FA) has been used in spinal cord injury (SCI) models. However, the vaccine emulsified with FA may induce some side effects, which are not suitable for further clinical application. As an adjuvant, gold nanoparticles (GNPs) could stimulate a stronger immune response without producing detectable toxicity and physiological damage than FA. There is, however, uncertainty regarding the efficacy of axon regeneration and neuroprotection in vaccines with GNPs as an adjuvant. In this investigation, a recombinant protein vaccine targeting NgR, human NgR-Fc (hNgR-Fc) fusion protein conjugated with 15?nm GNPs was prepared and its effects on axonal regeneration and functional recovery in spinal cord-injured rats were investigated. The results showed that adult rats immunized with the protein vaccine produced higher titers of anti-NgR antibody than that with FA, and the antisera promoted neurite outgrowth in presence of MAG in?vitro. In a spinal cord dorsal hemisection model, vaccine immunized with GNPs promoted axonal regeneration more effectively than FA, resulted in significant protection from neuronal loss, and improved functional recovery. Thus, as an adjuvant, 15?nm GNPs can effectively boost the immunogenicity of hNgR-Fc protein vaccine, and promote the repair of spinal cord-injured rats. The utilization of GNPs, for clinical considerations, may be a more beneficial supplement than FA to the promising therapeutic vaccination strategy for promoting SCI repair.     

Structural features of the Nogo receptor signaling complexes at the neuron/myelin interface

Upon spinal cord injury, the central nervous system axons are unable to regenerate, partially due to the repulsive action of myelin inhibitors, such as the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp). These inhibitors bind and signal through a single receptor/co-receptor complex that comprises of NgR1/LINGO-1 and either p75 or TROY, triggering intracellular downstream signaling that impedes the re-growth of axons. Structure–function analysis of myelin inhibitors and their neuronal receptors, particularly the NgRs, have provided novel information regarding the molecular details of the inhibitor/receptor/co-receptor interactions. Structural and biochemical studies have revealed the architecture of many of these proteins and identified the molecular regions important for assembly of the inhibitory signaling complexes. It was also recently shown that gangliosides, such as GT1b, mediate receptor/co-receptor binding. In this review, we highlight these studies and summarize our current understanding of the multi-protein cell-surface complexes mediating inhibitory signaling events at the neuron/myelin interface.     

Novel contributors to B cell activation during inflammatory CNS demyelination; An oNGOing process

Over the past two decades, the development of targeted immunotherapeutics for relapsing-remitting multiple sclerosis has been successfully orchestrated through the efficacious modulation of neuroinflammatory outcomes demonstrated in the experimental autoimmune encephalomyelitis (EAE) model. In this model, the focus of developing immunomodulatory therapeutics has been demonstrated through their effectiveness in modifying the pro-inflammatory Th1 and Th17-dependent neuropathological outcomes of demyelination, oligodendrocytopathy and axonal dystrophy. However, recent successful preclinical and clinical trials have advocated for the significance of B cell-dependent immunopathogenic responses and has led to the development of novel biologicals that target specific B cell phenotypes. In this context, a new molecule, B-cell activating factor (BAFF), has emerged as a positive regulator of B cell survival and differentiation functioning through various signaling pathways and potentiating the activity of various receptor complexes through pleiotropic means. One possible cognate receptor for BAFF includes the Nogo receptor (NgR) and its homologs, previously established as potent inhibitors of axonal regeneration during central nervous system (CNS) injury and disease. In this review we provide current evidence for BAFF-dependent signaling through the NgR multimeric complex, elucidating their association within the CNS compartment and underlying the importance of these potential pathogenic molecular regulators as possible therapeutic targets to limit relapse rates and potentially MS progression.     

Nogo(N-18)在大鼠脑干及小脑分布的免疫组织化学研究

采用免疫组织化学方法(SP法),研究了Nogo(N-18)在大鼠脑干及小脑的分布并探讨其存在的意义。结果表明,Nogo(N-18)在正常大鼠脑干以及小脑的神经核团有广泛的表达,阳性物质很强地表达于神经元的胞核内,神经元胞体、突起内表达较弱。结论:Nogo(N-18)在脑干及小脑的神经核团广泛表达提示其作为一种髓鞘源性神经突生长抑制因子在中枢神经系统中的存在,可能在正常的神经活动中起重要作用。     

原代培养大鼠嗅球成鞘细胞Nogo(N-18)的共聚焦激光扫描显微镜观察

目的 观察原代培养的成年大鼠嗅球成鞘细胞(OECs)Nogo(N-18)蛋白的表达，探讨Nogo与成鞘细胞促进神经再生作用的关系。方法 原代培养嗅球OECs，采用免疫组织化学和双标免疫荧光细胞化学技术，结合激光共聚焦扫描显微镜观察。结果 原代培养的嗅球OECs的Nogo(N-18)免疫细胞化学反应呈阳性，Nogo(N-18)蛋白主要分布于胞浆，而在胞膜及突起分布较少。结论 嗅球(OECs)含有Nogo—A蛋白，提示Nogo-18蛋白在嗅神经系统可能没有决定抑制轴突再生的作用。     

Axonal regeneration of optic nerve after crush in Nogo66 receptor knockout mice

Mature retinal ganglion cells (RGCs) cannot regenerate injured axons because some neurite growth inhibitors, including the C-terminal of Nogo-A (Nogo66), myelin-associated glycoprotein (MAG) and Omgp, exert their effects on neuron regeneration through the Nogo receptor (NgR). In this study, the axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush was investigated both in vivo and in vitro in NgR knockout mice. We used NgR knockout mice as the experimental group, and C57BL/6 mice as the control group. Partial ON injury was induced by using a specially designed ON clip to pinch the ON 1 mm behind the mouse eyeball with 40 g pressure for 9 s. NgR mRNA was studied by in situ hybridization (ISH). NgR protein was studied by Western blot. Growth Associated Protein 43 (GAP-43), a plasticity protein expressed highly during axon regeneration, was studied by immunofluorescence staining on the frozen sections. RGCs were cultured and purified. The axonal growth of RGCs was calculated by a computerized image analyzer. We found that compared with the control group, the GAP-43 expression was significantly higher and the axonal growth was significantly more active at every observation time point in the experimental group. These results indicate that NgR genes play an important role in the axonal regeneration after ON injury, while knockout of NgR is effective for eliminating this inhibition and enhancing axonal regeneration.     

Effects of Nogo-A and its receptor on the repair of sciatic nerve injury in rats

Regeneration of injured peripheral nerves is an extremely complex process. Nogo-A (neurite outgrowth inhibitor-A) inhibits axonal regeneration by interacting with Nogo receptor in the myelin sheath of the central nervous system (CNS). The aim of this study was to investigate the effects of Nogo-A and its receptor on the repair of sciatic nerve injury in rats. Sprague-Dawley rats (n=96) were randomly divided into 4 groups: control group (control), sciatic nerve transection group (model), immediate repair group (immediate repair), and delayed repair group (delayed repair). The rats were euthanized 1 week and 6 weeks after operation. The injured end tissues of the spinal cord and sciatic nerve were obtained. The protein expressions of Nogo-A and Nogo-66 receptor (NgR) were detected by immunohistochemistry. The protein expressions of Nogo-A, NgR, and Ras homolog family member A (RhoA) were detected by western blot. At 1 week after operation, the pathological changes in the immediate repaired group were less, and the protein expressions of Nogo-A, NgR, and RhoA in the spinal cord and sciatic nerve tissues were decreased (P<0.05) compared with the model group. After 6 weeks, the pathological changes in the immediate repair group and the delayed repair group were alleviated and the protein expressions decreased (P<0.05). The situation of the immediate repair group was better than that of the delayed repair group. Our data suggest that the expression of Nogo-A and its receptor increased after sciatic nerve injury, indicating that Nogo-A and its receptor play an inhibitory role in the repair process of sciatic nerve injury in rats.     

维甲酸在轴突再生过程中的作用与机制

背景：维甲酸信号通路在神经系统形成、神经元的特化以及轴突生长过程中极为重要,近年的研究结果显示维甲酸在轴突再生过程中具有重要作用,但是却鲜有关于其确切作用分子机制的研究报道。 目的：对近年来维甲酸信号通路在轴突再生过程中的作用机制进行总结分析。 方法：以“维甲酸,中枢神经系统,神经损伤,轴突再生,作用机制”为中文捡索词,以“Retinoic acid, the central nervous system, nerve damage, axon regeneration, signaling pathway,mechanism”为英文检索词,检索维普和中国知网(CNKI)期刊全文数据库、PubMed网络数据库、BioMed Centeral 、Springer 、The Free Medical Journals、EBSCO和外文生物医学期刊全文数据库(Foreign Journals Integration System)2000年1月至2013年12月有关维甲酸在轴突再生中作用机制的研究报道,排除重复性研究和不典型报道。 结果与结论：急性中枢神经系统损伤后,机体轴突再生和功能恢复的能力极为有限。为了保持机体的某些特有功能,神经元轴突必须再生并再支配它的作用靶点,以实现机体结构和功能的恢复。中枢神经系统损伤后,维甲酸信号通路通过表达转录因子RAβ2 受体,可诱导轴突的再生;同时在背根神经节神经元中,经慢病毒转染表达RARβ2后可以引起胞内cAMP水平升高,从而促进神经轴突生长;在脊髓损伤后以及体外轴突生长抑制环境中,RA-RARβ途径可以直接抑制中枢神经再生抑制因子Nogo受体(NgR)复合体-Lingo-1的转录,从而促进轴突的再生。维甲酸信号通路正是通过以上一系列的分子机制在轴突再生过程中其重要的作用 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Nogo‐B expression,in arterial intima,is impeded in the early stages of atherosclerosis in humans

Nogo‐B (Reticulon 4B) is considered to be a novel vascular marker, which may have a protective role in injury‐induced neointima formation and atherosclerosis. Nogo A/B is found to be crucial for monocyte/macrophage recruitment in acute inflammation and it is expressed in CD68 + macrophages. We hypothesize that macrophage infiltration in atherosclerosis is not dependent on Nogo‐B expression in arterial wall. We have assessed Nogo‐B expression and macrophage accumulation in the iliac arteries of healthy organ donors and organ donors with cardiovascular risk factors. Paraffin sections of 66 iliac arteries, from 44 deceased organ donors (17 women and 27 men), were studied. The healthy and cardiovascular risk (CVR) subgroups were created. With regard to staging of the atherosclerotic process, the thickness of arterial intima was measured in digitalized images of H+E stained tissue sections. Immunohistochemical reactions (Nogo‐B and CD68) were carried out in all arteries (66 samples). Western blotting (WB‐19 samples) and real‐time PCR (27 samples) were performed on selected arteries. Significantly higher Nogo‐B expression was demonstrated in the intima of the healthy subjects' subgroup, using immunohistochemistry. WB and real‐time PCR revealed a trend toward lower Nogo‐B expression in the adventitia of the CVR subgroup. Furthermore, the thickness of the intima was found to negatively correlate with the expression of Nogo‐B in the intima and media (r = ?0.32; p < 0.05; r = ?0.32; p < 0.05). Macrophage infiltrates were more prominent in intima of CVR subjects (0.65 vs 3.52 a.u.; p < 0.01). Macrophage density in intima increased with atherosclerosis progression (r = 0.37; p < 0.01). CD68 macrophages density in adventitia was lower in CVR arteries than in healthy arteries. The expression of Nogo‐B, in arterial intima, is impeded in the early stages of atherosclerosis. Accumulation of arterial intimal CD68 macrophages has been shown to progress; however, the overall macrophage density in the adventitia is reduced in arteries shown to have intimal thickening. Macrophage infiltration is not accompanied by Nogo‐B expression in atherosclerotic arteries.     

NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1(-/-); Ngr2(-/-); Ngr3(-/-); which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1(-/-); Ngr3(-/-)), but not Ngr1 and Ngr2 (Ngr1(-/-); Ngr2(-/-)), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1(-/-); Ngr3(-/-) mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.