Identification of a functional epitope of the Nogo receptor by a combinatorial approach using ribosome display

The Nogo receptor (NgR) plays a central role in mediating growth-inhibitory activities of myelin-derived proteins, thereby severely limiting axonal regeneration after injury of the adult mammalian central nervous system (CNS). The inhibitory proteins Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) all bind to the extracellular leucine-rich repeat (LRR) domain of NgR, which provides a large molecular surface for protein-protein interactions. However, epitopes within the LRR domain of NgR for binding Nogo, MAG and OMgp have not yet been revealed. Here, we report an evolutionary approach based on the ribosome display technology for detecting regions involved in ligand binding. By applying this method of "affinity fingerprinting" to the NgR ligand binding domain we were able to detect a distinct region important for binding to Nogo. Several residues defining the structural epitope of NgR involved in interaction with Nogo were subsequently confirmed by alanine scanning mutagenesis.     

Crystal structure of the Nogo-receptor-2

The inhibition of axon regeneration upon mechanical injury is dependent on interactions between Nogo receptors (NgRs) and their myelin-derived ligands. NgRs are composed of a leucine-rich repeat (LRR) region, thought to be structurally similar among the different isoforms of the receptor, and a divergent "stalk" region. It has been shown by others that the LRR and stalk regions of NgR1 and NgR2 have distinct roles in conferring binding affinity to the myelin associated glycoprotein (MAG) in vivo. Here, we show that purified recombinant full length NgR1 and NgR2 maintain significantly higher binding affinity for purified MAG as compared to the isolated LRR region of either NgR1 or NgR2. We also present the crystal structure of the LRR and part of the stalk regions of NgR2 and compare it to the previously reported NgR1 structure with respect to the distinct signaling properties of the two receptor isoforms.     

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。     

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Structure and axon outgrowth inhibitor binding of the Nogo-66 receptor and related proteins

The myelin-derived proteins Nogo, MAG and OMgp limit axonal regeneration after injury of the spinal cord and brain. These cell-surface proteins signal through multi-subunit neuronal receptors that contain a common ligand-binding glycosylphosphatidylinositol-anchored subunit termed the Nogo-66 receptor (NgR). By deletion analysis, we show that the binding of soluble fragments of Nogo, MAG and NgR to cell-surface NgR requires the entire leucine-rich repeat (LRR) region of NgR, but not other portions of the protein. Despite sharing extensive sequence similarity with NgR, two related proteins, NgR2 and NgR3, which we have identified, do not bind Nogo, MAG, OMgp or NgR. To investigate NgR specificity and multi-ligand binding, we determined the crystal structure of the biologically active ligand-binding soluble ectodomain of NgR. The molecule is banana shaped with elongation and curvature arising from eight LRRs flanked by an N-terminal cap and a small C-terminal subdomain. The NgR structure analysis, as well as a comparison of NgR surface residues not conserved in NgR2 and NgR3, identifies potential protein interaction sites important in the assembly of a functional signaling complex.     

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.     

Nogo受体相关研究进展

中枢神经系统损伤后其再生能力较弱已被人们所熟知,原因在于髓磷脂抑制物如Nogo、MAG、Omgp等抑制因子的作用,这些抑制因子通过与神经元上的Nogo受体(NgR)特异性结合,发挥对神经轴突再生的抑制作用。Nogo是一种存在于中枢神经系统少突胶质细胞上的髓磷脂蛋白,其作用主要在于神经细胞损伤后抑制其突触再生,这同时也是对损伤部位其他细胞免于进一步损伤的保护作用。存在于细胞表面的Nogo-66结构是与NgR特异性结合的功能域。NgR是一种存在于神经元表面,传递抑制轴突生长信号的复合共受体。近年来随着对NgR、Nogo及其下游信号通路其他相关蛋白研究的深入,提示多种神经系统疾病与之相关。我们简要综述近年来关于NgR的研究进展。     

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.     

Nogo on the go

Growth inhibition in the central nervous system (CNS) is a major barrier to axon regeneration. Recent findings indicate that three distinct myelin proteins, myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp), inhibit axon growth by binding a common receptor, the Nogo66 receptor (NgR), and likely converge on a common signaling cascade.     

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.     

NgR RNA interference, combined with zymosan intravitreal injection, enhances optic nerve regeneration

Mature retinal ganglion cells like other CNS neurons are unable to regenerate their axons after injury. Regenerative failure has been attributed, in part, to two factors: the existence of myelin-derived inhibitors that bind to the Nogo receptor (NgR) and a deficiency of trophic support factors. We investigated the regrowth of injured axons both by inhibiting NgR by RNA interference and by recruiting exogenous trophic support by zymosan intravitreal injection. Our results showed that either approach can stimulate optic nerve axon regrowth but regenerated axons can grow longer and extend further when both methods are combined. We conclude that endogenous NgR inhibition and exogenous trophic support both play independent, important roles in enhancing optic nerve axon regrowth and that the regenerative effect can be augmented when the two are combined. This may provide a therapeutic strategy for promoting axon regeneration in the CNS as well.     

TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration

Myelin-associated inhibitory factors (MAIFs) are inhibitors of CNS axonal regeneration following injury. The Nogo receptor complex, composed of the Nogo-66 receptor 1 (NgR1), neurotrophin p75 receptor (p75), and LINGO-1, represses axon regeneration upon binding to these myelin components. The limited expression of p75 to certain types of neurons and its temporal expression during development prompted speculation that other receptors are involved in the NgR1 complex. Here, we show that an orphan receptor in the TNF family called TAJ, broadly expressed in postnatal and adult neurons, binds to NgR1 and can replace p75 in the p75/NgR1/LINGO-1 complex to activate RhoA in the presence of myelin inhibitors. In vitro exogenously added TAJ reversed neurite outgrowth caused by MAIFs. Neurons from Taj-deficient mice were more resistant to the suppressive action of the myelin inhibitors. Given the limited expression of p75, the discovery of TAJ function is an important step for understanding the regulation of axonal regeneration.     

中枢神经系统轴突再生抑制蛋白

中枢神经系统 (CNS)轴突再生的主要障碍之一是存在抑制再生的蛋白 ,迄今 ,已在少突胶质细胞 /髓鞘中相继发现至少三个重要的轴突再生抑制蛋白 ,即髓鞘相关糖蛋白 (MAG)、Nogo A和少突胶质细胞 /髓鞘糖蛋白 (OMgp)。最近的研究又证实 ,这三个不同的抑制成分可能主要通过与一个共同的受体Nogo6 6受体 (NgR)结合而发挥作用。这些研究成果扩充了对CNS损伤后轴突再生障碍的理解 ,也为探讨CNS损伤的治疗新策略提供了新的思路。     

Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth

Myelin inhibitors of axonal regeneration, like Nogo and MAG, block regrowth after injury to the adult CNS. While a GPI-linked receptor for Nogo (NgR) has been identified, MAG's receptor is unknown. We show that MAG inhibits regeneration by interaction with NgR. Binding of and inhibition by MAG are lost if neuronal GPI-linked proteins are cleaved. Binding of MAG to NgR-expressing cells is GPI dependent and sialic acid independent. Conversely, NgR binds to MAG-expressing cells. MAG, but not a truncated MAG that binds neurons but does not inhibit regeneration, precipitates NgR from NgR-expressing cells, DRG, and cerebellar neurons. Importantly, NgR antibody, soluble NgR, or dominant-negative NgR each prevent inhibition of neurite outgrowth by MAG. Also, MAG and Nogo66 compete for binding to NgR. These results suggest redundancy in myelin inhibitors and indicate therapies for CNS injuries.     

The Expression Patterns of Nogo-A and NgR in the Neonatal Rat Visual Nervous System

Previous studies have shown that Nogo-NgR system plays multiple roles in controlling axonal regeneration and neuronal plasticity. However, the investigation on the distributions of Nogo and NgR was limited to restricted areas or special ages, and their expression patterns in the neonatal visual nervous system is still unknown. Frozen sections were made from eyeballs and brains of three neonatal rats, and expression of Nogo and NgR proteins were examined by immunofluorescence under a laser confocal microscopy. Nogo and NgR were observed to express in the cell bodies of retina, lateral geniculate bodies and visual cortex of the neonatal rats. Our results provide evidences that the Nogo-NgR system may play an active role in the plasticity of neonatal visual system. Y. Xiaolei, Y. Rongdi and J. Shuxing contributed equally to this work.     

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.     

The carboxyl-terminal region of Crtac1B/LOTUS acts as a functional domain in endogenous antagonism to Nogo receptor-1

Myelin-derived axon growth inhibitors, such as Nogo, bind to Nogo receptor-1 (NgR1) and thereby limit the action of axonal regeneration after injury in the adult central nervous system. Recently, we have found that cartilage acidic protein-1B (Crtac1B)/lateral olfactory tract usher substance (LOTUS) binds to NgR1 and functions as an endogenous NgR1 antagonist. To examine the functional domain of LOTUS in the antagonism to NgR1, analysis using the deletion mutants of LOTUS was performed and revealed that the carboxyl-terminal region (UA/EC domain) of LOTUS bound to NgR1. The UA/EC fragment of LOTUS overexpressed together with NgR1 in COS7 cells abolished the binding of Nogo66 to NgR1. Overexpression of the UA/EC fragment in cultured chick dorsal root ganglion neurons suppressed Nogo66-induced growth cone collapse. These findings suggest that the UA/EC region is a functional domain of LOTUS serving for an antagonistic action to NgR1.     

Segregation of Nogo66 receptors into lipid rafts in rat brain and inhibition of Nogo66 signaling by cholesterol depletion

NogoA, a myelin-associated component, inhibits neurite outgrowth. Nogo66, a portion of NogoA, binds to Nogo66 receptor (NgR) and induces the inhibitory signaling. LINGO-1 and p75 neurotrophin receptor (p75), the low-affinity nerve growth factor receptor, are also required for NogoA signaling. However, signaling mechanisms downstream to Nogo receptor remain poorly understood. Here, we observed that NgR and p75 were colocalized in low-density membrane raft fractions derived from forebrains and cerebella as well as from cerebellar granule cells. NgR interacted with p75 in lipid rafts. In addition, disruption of lipid rafts by beta-methylcyclodextrin, a cholesterol-binding reagent, reduced the Nogo66 signaling. Our results suggest an important role of lipid rafts in facilitating the interaction between NgRs and provide insight into mechanisms underlying the inhibition of neurite outgrowth by NogoA.     

Multiple signals regulate axon regeneration through the nogo receptor complex

Several myelin-derived proteins have been identified as components of central nervous system (CNS) myelin, which prevents axonal regeneration in the adult vertebrate CNS. The discovery of the receptor for these proteins was a major step toward understanding the failure of axon regeneration. The receptor complex consists of at least three elements: the p75 receptor (p75^NTR), the Nogo receptor and LINGO-1. Downstream from the receptor complex, RhoA activation has been shown to be a key element of the signaling mechanism of these proteins. Rho activation arrests axon growth, and blocking Rho activation promotes axon regeneration in vivo. Recent studies have identified conventional protein kinase C as an additional necessary component for axon growth inhibition. Possible crosstalk downstream of these signals should be explored to clarify all the inhibitory signals and may provide an efficient molecular target against injuries to the CNS.