A novel Rieske-type protein derived from an apoptosis-inducing factor-like (AIFL) transcript with a retained intron 4 induces change in mitochondrial morphology and growth arrest

Upon spinal cord injury, the myelin inhibitors, including the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp), bind to and signal via a single neuronal receptor/co-receptor complex comprising of Nogo receptor 1(NgR1)/LINGO-1 and p75 or TROY, impeding regeneration of injured axons. We employed a cell-free system to study the binding of NgR1 to its co-receptors and the myelin inhibitor Nogo-A, and show that gangliosides mediate the interaction of NgR1 with LINGO-1. Solid phase binding assays demonstrate that the sialic acid moieties of gangliosides and the stalk of NgR1 are the principal determinants of these molecular interactions. Moreover, the tripartite complex comprising of NgR1, LINGO-1 and ganglioside exhibits stronger binding to Nogo-A (Nogo-54) in the presence of p75, suggesting the gangliosides modulate the myelin inhibitor-receptor signaling.     

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.     

Why do Nogo/Nogo‐66 receptor gene knockouts result in inferior regeneration compared to treatment with neutralizing agents?

IN-1, the monoclonal antibody against the exon 3-encoded N-terminal domain of Nogo-A, and the Nogo-66 receptor (NgR) antagonist NEP1-40 have both shown efficacy in promoting regeneration in animal spinal cord injury models, the latter even when administered subcutaneously 1 week after injury. These results are supportive of the hypothesis that the Nogo-NgR axis is a major path for inhibition of spinal cord axonal regeneration and uphold the promises of these neutralizing agents in clinical applications. However, mice with targeted disruption of Nogo and NgR have, surprisingly, only modest regenerative capacity (if any) compared with treatment with IN-1 or NEP1-40. Disruption of the Nogo gene by various groups yielded results ranging from significant regenerative improvement in young mice to no improvement. Likewise, knockout of NgR produced some improvement in raphespinal and rubrospinal axonal regeneration, but not that of corticospinal neurons. Other than invoking possible differences in genetic background, we suggest here some possible and testable explanations for the above phenomena. These possibilities include effects of IN-1 and NEP1-40 on the CNS beyond neutralization of Nogo and NgR functions, and the latter's possible role in the CNS beyond that of neuronal growth inhibition.     

Nogo and axon regeneration

Nogo-A is one of several neurite growth inhibitory components present in oligodendrocytes and CNS myelin membranes. Nogo has a crucial role in restricting axonal regeneration and compensatory fibre growth in the injured adult mammalian CNS. Recent studies have shown that in vivo applications of Nogo neutralizing antibodies, peptides blocking the Nogo receptor subunit NgR, or blockers of the postreceptor components Rho-A and ROCK induce long-distance axonal regeneration and compensatory sprouting, accompanied by an impressive enhancement of functional recovery, in the rat and mouse spinal cord.     

The role of Nogo-A in axonal plasticity, regrowth and repair

Axonal damage leads to permanent deficits in the adult central nervous system (CNS) not only because of the weak intrinsic ability of adult neurons to activate their growth program but importantly also because of the presence of specific growth inhibitors in the CNS tissue and the environment of the damaged axons. The well-studied myelin-derived protein Nogo-A is involved in various cellular and molecular events contributing to the failure of CNS axons to regrow and reconnect after transection. Recent studies have shown that, by acting in a negative way on the cytoskeleton and on the growth program of axotomized neurons, Nogo-A exerts fast and chronic inhibitory effects on neurite outgrowth. On the other hand, the blockade of Nogo-A results in a marked enhancement of compensatory and regenerative axonal extension in vivo; this enhancement is often paralleled by significant functional recovery, for example, of locomotion or skilled forelimb reaching after spinal cord or stroke lesions in rats and monkeys. Surprisingly, the blockade of Nogo-A or its receptor NgR in the hippocampus has recently been demonstrated to enhance long-term potentiation. A role of Nogo-A in synaptic plasticity/stability might therefore represent an additional, new and important aspect of CNS circuit remodeling. Function-blocking anti-Nogo-A antibodies are currently being tested in a clinical trial for improved outcome after spinal cord injury.     

Epitope mapping of the neuronal growth inhibitor Nogo-A for the Nogo receptor and the cognate monoclonal antibody IN-1 by means of the SPOT technique

Nogo-A is a potent inhibitor of axonal outgrowth in the central nervous system of adult mammals, where it is expressed as a membrane protein on oligodendrocytes and in myelin. Here we describe an attempt to identify linear peptide epitopes in its sequence that are responsible for the interaction either with the Nogo receptor (NgR) or with the neutralizing monoclonal antibody IN-1. Analysis of an array of immobilized overlapping 15 mer peptides covering the entire amino acid sequence of human Nogo-A (1192 residues) revealed a single epitope with prominent binding activity both towards the recombinant NgR and the IN-1 F(ab) fragment. Further truncation and substitution analysis yielded the minimal epitope sequence 'IKxLRRL' (x not equal to P), which occurs within the so-called Nogo66 region (residues 1054-1120) of Nogo-A. The bacterially produced Nogo66 fragment exhibited binding activity both for the recombinant NgR and for the IN-1 F(ab) fragment on the Western blot as well as in ELISA. Unexpectedly, the synthetic epitope peptide and the recombinant Nogo66 showed cross-reactivity with the 8-18C5 F(ab) fragment, which is directed against myelin oligodendrocyte glycoprotein (MOG) as a structurally unrelated target. On the other hand, the recombinant N-terminal domain of Nogo-A (residues 334-966) was shown to specifically interact on the Western blot and in an ELISA with the IN-1 F(ab) fragment but not with the recombinant NgR, which is in agreement with previous results. Hence, our data suggest that there is a distinct binding site for the Nogo receptor in the Nogo66 region of Nogo-A, whereas its interaction with NgR is less specific than anticipated before. Although there probably exists a non-linear epitope for the neutralizing antibody IN-1 in the N-terminal region of Nogo-A, which is likely to be accessible from outside the cell, a previously postulated second binding site for NgR in this region (called Nogo-A-24) remains elusive.     

Characterization of myelin ligand complexes with neuronal Nogo-66 receptor family members

Nogo, MAG, and OMgp are myelin-associated proteins that bind to a neuronal Nogo-66 receptor (NgR/NgR1) to limit axonal regeneration after central nervous system injury. Within Nogo-A, two separate domains are known interact with NgR1. NgR1 is the founding member of the three-member NgR family, whereas Nogo-A (RTN4A) belongs to a four-member reticulon family. Here, we systematically mapped the interactions between these superfamilies, demonstrating novel nanomolar interactions of RTN2 and RTN3 with NgR1. Because RTN3 is expressed in spinal cord white matter, it may have a role in myelin inhibition of axonal growth. Further analysis of the Nogo-A and NgR1 interactions revealed a novel third interaction site between the proteins, suggesting a trivalent Nogo-A interaction with NgR1. We also confirmed here that MAG binds to NgR2, but not to NgR3. Unexpectedly, we found that OMgp interacts with MAG with a higher affinity compared with NgR1. To better define how these multiple structurally distinct ligands bind to NgR1, we examined a series of Ala-substituted NgR1 mutants for ligand binding activity. We found that the core of the binding domain is centered in the middle of the concave surface of the NgR1 leucine-rich repeat domain and surrounded by differentially utilized residues. This detailed knowledge of the molecular interactions between NgR1 and its ligands is imperative when assessing options for development of NgR1-based therapeutics for central nervous system injuries.     

Effect of ganglioside combined with Chip Jiaji electro-acupuncture on Nogo-NgR signal pathway in SCI rats

At present, the effect of ganglioside combined with Jiaji electroacupuncture (Jiaji EA) on SCI still remains unclear. This study explores the effect of ganglioside combined with electroacupuncture on Nogo/NgR signal pathway in spinal cord tissue of spinal cord injury (SCI) rats. Basso Beattie Bresnahan (BBB) score was used to evaluate spinal cord function after modeling and 14 days post ganglioside and electroacupuncture treatment. RT-qPCR and western blot were performed to evaluate the expression levels of targets in spinal cord tissue. After 14 days of treatment, the BBB scores of Jiaji EA group, ganglioside group and combination group were all improved. The expression levels of IL-1β, IL-6 and TNF-α in Jiaji EA group, ganglioside group and combination group were significantly lower than those in model group. Both of mRNA and protein expression levels of Nogo-A, NgR and LINGO-1 in the model group were significantly higher than those in the Jiaji EA group, ganglioside group and combination group. Ganglioside combined with Jiaji EA has a stronger effect on promoting the recovery of nerve function. Its mechanism of action may be related to its inhibition of the expression of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α and Nogo-NgR signal pathway to promote neuronal growth. Our results will provide fundamental information for further SCI studies.     

The Nogo-66 receptor family in the intact and diseased CNS

The Nogo-66 receptor family (NgR) consists in three glycophosphatidylinositol (GPI)-anchored receptors (NgR1, NgR2 and NgR3), which are primarily expressed by neurons in the central and peripheral mammalian nervous system. NgR1 was identified as serving as a high affinity binding protein for the three classical myelin-associated inhibitors (MAIs) Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), which limit axon regeneration and sprouting in the injured brain. Recent studies suggest that NgR signaling may also play an essential role in the intact adult CNS in restricting axonal and synaptic plasticity and are involved in neurodegenerative diseases, particularly in Alzheimer's disease pathology through modulation of β-secretase cleavage. Here, we outline the biochemical properties of NgRs and their functional roles in the intact and diseased CNS.     

Nogo-A and Nogo-66 receptor in amyotrophic lateral sclerosis

Nogo/reticulon (RTN)-4 has been strongly implicated as a disease marker for the motor neuron disease amyotrophic lateral sclerosis (ALS). Nogo isoforms, including Nogo-A, are ectopically expressed in the skeletal muscle of ALS mouse models and patients and their levels correlate with the disease severity. The notion of a direct involvement of Nogo-A in ALS aetiology is supported by the findings that Nogo-A deletion in mice reduces muscle denervation and prolongs survival, whereas overexpression of Nogo-A destabilizes motor nerve terminals and promotes denervation. Another intriguing, and somewhat paradoxical, recent finding revealed that binding of the Nogo-66 receptor (NgR) by either agonistic or antagonistic Nogo-66-derived peptides protects against p75 neurotrophin receptor (p75(NTR))-dependent motor neuron death. Ligand binding by NgR could result in subsequent engagement of p75(NTR), and this association could preclude pro-apoptotic signalling by the latter. Understanding the intricate interplay among Nogo isoforms, NgR and p75(NTR) in ALS disease progression may provide important, therapeutically exploitable information.     

A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.     

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.     

Nogo receptor 3, a paralog of Nogo-66 receptor 1 （NgR1）, may function as a NgR1 co-receptor for Nogo-66

Nogo-A is a major myelin associated inhibitor that blocks regeneration of injured axons in the central nervous system (CNS).Nogo-66 (a 66-residue domain of Nogo-A) expressed on the surface of oligodendrocytes has been shown to directly interact with Nogo-66 receptor 1 (NgR1).A number of additional components of NgR1 receptor complex essential for its signaling have been uncovered.However,detailed composition of the complex and its signaling mechanisms remain to be fully elucidated.In this study,we show that Nogo receptor 3 (NgR3),a paralog of NgR1,is a binding protein for NgR1.The interaction is highly specific because other members of the reticulin family,to which Nogo-A belongs,do not bind to NgR3.Neither does NgR3 show any binding activity with Nogo receptor 2 (NgR2),another NgR1 paralog.Majority of NgR3 domains are required for its binding to NgR1.Moreover,a truncated NgR3 with the membrane anchoring domain deleted can function as a decoy receptor to reverse neurite outgrowth inhibition caused by Nogo-66 in culture.These in vitro results,together with previously reported overlapping expression profile between NgR1 and NgR3,suggest that NgR3 may be associated with NgR1 in vivo and that their binding interface may be targeted for treating neuronal injuries.     

神经轴突生长抑制因子Nogo 家族的研究进展*

Nogo家族是一类神经轴突生长抑制因子家族,目前成员包括Nogo-A,Nogo-B,Nogo-C三个亚型。Nogo家族成员因C末端具有保守的RHD结构域而归属于RTNs家族,表明它们的分布和功能与内质网密切相关。Nogo家族C末端还具有一个进化保守的66氨基酸的功能段称为Nogo-66,体外表达的Nogo-66片段具有抑制神经突生长的作用。Nogo家族成员结构上的区别主要表现在不同剪切长短的N末端序列。Nogo-A主要在中枢和外周神经系统中广泛分布,Nogo-C主要分布在骨骼肌,而Nogo-B则几乎遍布于各种组织与细胞之中。目前,发现可介导Nogo胞内信号转导通路的受体主要是膜外糖蛋白偶联的NgR和跨膜受体p75NTR组成的共受体,但NgR与Nogo-A在胚胎发育中时空表达并不同步提示可能还有其它受体存在。虽然Nogo家族作为神经轴突生长抑制因子被发现,但越来越多的研究表明其可能在胚胎发育、细胞凋亡或神经退行性变等重大事件中扮演重要角色。本文拟就Nogo家族迄今为止突出的研究进展作一综述,旨在为下一步的功能研究工作提供理论参考和依据。     

Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.     

New Insights on Neuronal Alterations in Jimpy Mutant Brain

In spite of abundant data on oligodendrocyte abnormalities in dysmyelinated jimpy brain, little is known about the axonal damage and the expression of neuronal genes. Recent findings indicate that Nogo-A, oligodendrocyte-myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) inhibit axonal growth by binding a common receptor, the Nogo-A receptor (NgR)-p75 complex. In order to evaluate neuronal modifications in the absence of myelin and in the presence of abnormal oligodendrocytes at different developmental stages, the expression of these inhibitory proteins and their receptors was investigated in jimpy mutant brain. Despite the decrease in oligodendrocyte number at P15 and P25 in jimpy, Nogo-A and OMgp mRNA levels are not significantly different compared with control, suggesting an overexpression of neuronal Nogo-A and OMgp in mutant. Double immunolabeling for Nogo-A and neurofilaments shows strong axonal staining of Nogo-A in jimpy and its down-regulation in oligodendrocytes. The current data raise questions about functions of Nogo-A other than neurite growth inhibition in the CNS. No significant changes in NgR mRNA levels were observed in jimpy, where the increase in p75 level can be correlated with the cell death of oligodendrocytes. In the paranodal region, the cell adhesion molecule neurofascin glial isoform NFN155 mRNA level is reduced by 40% whereas neuronal form NFN186 is up-regulated. These results may explain the failure of paranodal region organization, even with normal level of CASPR (paranodin) mRNA detected in jimpy brain.     

Nogo在中枢神经损伤再生中的作用机制

Nogo是中枢神经系统（CNS）少突胶质细胞分泌的一种髓磷脂蛋白,它的主要功能是抑制损伤后轴突的再生,它含有两个完全独立的具有抑制活性的结构域：位于细胞内的amino—Nogo和位于细胞表面的Nogo-66。Nogo-66是通过与受体复合体NgR／p75／Lingo—1结合,触发Rho信号通路来发挥作用。Nogo及其信号转导机制日益成为CNS损伤再生的研究热点,就Nogo在CNS损伤再生中的作用机制作一综述。     

中枢神经再生抑制因子及免疫治疗研究

成体哺乳动物中枢神经损伤后早期轴突再生失败的一个主要原因是由于髓磷脂抑制分子的存在。Nogo、髓磷脂相关糖蛋白以及少突胶质细胞髓磷脂糖蛋白等神经再生抑制因子的发现,大大促进了中枢神经再生分子机制的研究。它们均能独立通过Nogo-66受体产生对轴突再生的抑制效应,髓磷脂抑制分子及其信号转导机制的研究日益成为中枢神经再生的研究热点,髓磷脂及其信号转导分子特别是Nogo-66受体、p75神经营养素受体成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制上述抑制因子及相关受体NgR或p75NTR可能有助于中枢神经损伤的修复,围绕这些抑制因子及其相关受体介导的信号转导途径,人们提出了多种治疗中枢神经损伤的新思路,其中免疫学方法尤其受到关注。