Structural characterization of the human Nogo-A functional domains. Solution structure of Nogo-40, a Nogo-66 receptor antagonist enhancing injured spinal cord regeneration.

The recent discovery of the Nogo family of myelin inhibitors and the Nogo-66 receptor opens up a very promising avenue for the development of therapeutic agents for treating spinal cord injury. Nogo-A, the largest member of the Nogo family, is a multidomain protein containing at least two regions responsible for inhibiting central nervous system (CNS) regeneration. So far, no structural information is available for Nogo-A or any of its structural domains. We have subcloned and expressed two Nogo-A fragments, namely the 182 residue Nogo-A(567-748) and the 66 residue Nogo-66 in Escherichia coli. CD and NMR characterization indicated that Nogo-A(567-748) was only partially structured while Nogo-66 was highly insoluble. Nogo-40, a truncated form of Nogo-66, has been previously shown to be a Nogo-66 receptor antagonist that is able to enhance CNS neuronal regeneration. Detailed NMR examinations revealed that a Nogo-40 peptide had intrinsic helix-forming propensity, even in an aqueous environment. The NMR structure of Nogo-40 was therefore determined in the presence of the helix-stabilizing solvent trifluoroethanol. The solution structure of Nogo-40 revealed two well-defined helices linked by an unstructured loop, representing the first structure of Nogo-66 receptor binding ligands. Our results provide the first structural insights into Nogo-A functional domains and may have implications in further designs of peptide mimetics that would enhance CNS neuronal regeneration.     

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。     

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.     

Nogo与Nogo受体研究

nogo是新近发现的一种基因,编码3种蛋白质：Nogo-A、Nogo-B和Nogo-C.迄今为止,已证明它有抑制成熟中枢神经系统(CNS)神经元轴突再生及诱导细胞凋亡的作用.Nogo受体是一种糖基醇磷脂结合蛋白.对Nogo和Nogo受体的研究,对于CNS再生障碍及肿瘤的认识和治疗有重要意义.     

Nogo-B receptor possesses an intrinsically unstructured ectodomain and a partially folded cytoplasmic domain

RTN4/Nogo proteins containing three isoforms have been implicated in a large and diverse spectrum of biological functions. By contrast, only two functional receptors were known for them, namely NgR binding the 66-residue ectodomain shared by all three Nogos and NgBR specifically binding Nogo-B. The 297-residue NgBR was recently identified to be essential for stimulating chemotaxis and morphogenesis of endothelial cells but its structural property still remains completely unknown. In the present study, we expressed and subsequently conducted bioinformatics, CD and NMR characterization of NgBR and its two dissected domains. Very surprisingly, our results indicate that the NgBR ectodomain is intrinsically unstructured without both secondary and tertiary structures while the cytoplasmic domain is only partially folded with secondary structures but without a tight tertiary packing. Therefore, NgBR is a very rare example showing that the entire ectodomain of a transmembrane receptor could be predominantly disordered and the results presented here may bear important implications in understanding NgBR functions in the future.     

Rational design, solution conformation and identification of functional residues of the soluble and structured Nogo-54, which mimics Nogo-66 in inhibiting the CNS neurite outgrowth

The interaction between Nogo-66 and its receptor NgR represents a promising target for designing drugs to treat CNS axonal injury which often leads to permanent disability. Unfortunately, the isolated Nogo-66 is highly insoluble while its truncated form Nogo-40 is soluble but unstructured, thus retarding further characterization and application. Here, we rationally design another soluble form Nogo-54. CD and NMR characterization reveals that Nogo-54 is structured, and importantly, is able to mimic Nogo-66 in inhibiting neurite outgrowth. Strikingly, mutating its C-terminal four residues (Lys50, Glu51, Arg53, and Arg54) leads to a mutant Nogo-54m which has no dramatic structural change but whose inhibitory activity is completely abolished. This strongly suggests that the four charged residues contribute significantly to the inhibitory action of Nogo-66. Furthermore, our study also provides a soluble and structured mimic as well as a possible antagonist for Nogo-66 which may hold promising potential for various medical applications.     

Nogo goes in the pure water: solution structure of Nogo-60 and design of the structured and buffer-soluble Nogo-54 for enhancing CNS regeneration

The inability to determine the structure of the buffer-insoluble Nogo extracellular domain retarded further design of Nogo receptor (NgR) antagonists to treat CNS axonal injuries. Very surprisingly, we recently discovered that Nogo-60 was soluble and structured in salt-free water, thus allowing the determination of the first Nogo structure by heteronuclear NMR spectroscopy. Nogo-60 adopts an unusual helical structure with the N- and C-terminal helices connected by a long middle helix. While the N-helix has no contact with the rest of the molecule, the C-helix flips back to pack against the 20-residue middle helix. This packing appears to trigger the formation of the stable Nogo-60 structure because Nogo-40 with the last helix truncated is unstructured. The Nogo-60 structure offered us rationales for further design of the structured and buffer-soluble Nogo-54, which may be used as a novel NgR antagonist. Furthermore, our discovery may imply a general solution to solubilizing a category of buffer-insoluble proteins for urgent structural investigations.     

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.     

A multi-domain fragment of Nogo-A protein is a potent inhibitor of cortical axon regeneration via Nogo receptor 1

Nogo-A limits axon regeneration and functional recovery after central nervous system injury in adult mammals. Three regions of Nogo-A (Nogo-A-24, Nogo-66, and Nogo-C39) interact with the neuronal Nogo-66 receptor 1 (NgR1). Nogo-66 also interacts with a structurally unrelated cell surface receptor, paired immunoglobulin-like receptor (PirB). We show here that the other two NgR1-interacting domains, Nogo-A-24 and Nogo-C39, also bind to PirB with high affinity. A purified 22-kDa protein containing all three NgR1- and PirB-interacting domains (Nogo-22) is a substantially more potent growth cone-collapsing molecule than Nogo-66 for chick dorsal root ganglion neurons and mature cortical neurons. Moreover, Nogo-22 inhibits axon regeneration of mature cortical neurons in vitro more potently than does Nogo-66. Although all three NgR1-interacting domains of Nogo-A also interact with PirB, expression of PirB in mature cortical cultures is nearly undetectable. Consistent with a relatively minor role for PirB in mature cortical neurons, Nogo-22 inhibition of axon regeneration is abolished by genetic deletion of NgR1. Thus, NgR1 is the predominant receptor for Nogo-22 in regenerating cortical neurons.     

Immunity to the extracellular domain of Nogo-A modulates experimental autoimmune encephalomyelitis

Nogo-66, the extracellular 66 aa loop of the Nogo-A protein found in CNS myelin, interacts with the Nogo receptor and has been proposed to mediate inhibition of axonal regrowth. It has been shown that immunization with Nogo-A promotes recovery in animal models of spinal cord injury through induction of Ab production. In this report, studies were performed to characterize the immune response to Nogo-66 and to determine the role of Nogo in experimental autoimmune encephalomyelitis (EAE). Immunization of EAE-susceptible mouse strains with peptides derived from Nogo-66 induced a CNS immune response with clinical and pathological similarities to EAE. The Nogo-66 peptides elicited strong T cell responses that were not cross-reactive to other encephalitogenic myelin Ags. Using a large scale spotted microarray containing proteins and peptides derived from a wide spectrum of myelin components, we demonstrated that Nogo-66 peptides also generated a specific Ab response that spreads to several other encephalitogenic myelin Ags following immunization. Nogo-66-specific T cell lines ameliorated established EAE, via Nogo-66-specific Th2 cells that entered the CNS. These results indicate that some T cell and B cell immune responses to Nogo-66 are associated with suppression of ongoing EAE, whereas other Nogo-66 epitopes can be encephalitogenic.     

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.     

Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration

Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury.     

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.     

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.     

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。     

A new role for Nogo as a regulator of vascular remodeling

Although Nogo-A has been identified in the central nervous system as an inhibitor of axonal regeneration, the peripheral roles of Nogo isoforms remain virtually unknown. Here, using a proteomic analysis to identify proteins enriched in caveolae and/or lipid rafts (CEM/LR), we show that Nogo-B is highly expressed in cultured endothelial and smooth muscle cells, as well as in intact blood vessels. The N terminus of Nogo-B promotes the migration of endothelial cells but inhibits the migration of vascular smooth muscle (VSM) cells, processes necessary for vascular remodeling. Vascular injury in Nogo-A/B-deficient mice promotes exaggerated neointimal proliferation, and adenoviral-mediated gene transfer of Nogo-B rescues the abnormal vascular expansion in those knockout mice. Our discovery that Nogo-B is a regulator of vascular homeostasis and remodeling broadens the functional scope of this family of proteins.     

Functions of Nogo proteins and their receptors in the nervous system

The membrane protein Nogo-A was initially characterized as a CNS-specific inhibitor of axonal regeneration. Recent studies have uncovered regulatory roles of Nogo proteins and their receptors--in precursor migration, neurite growth and branching in the developing nervous system--as well as a growth-restricting function during CNS maturation. The function of Nogo in the adult CNS is now understood to be that of a negative regulator of neuronal growth, leading to stabilization of the CNS wiring at the expense of extensive plastic rearrangements and regeneration after injury. In addition, Nogo proteins interact with various intracellular components and may have roles in the regulation of endoplasmic reticulum (ER) structure, processing of amyloid precursor protein and cell survival.     

Nogo-A, -B, and -C are found on the cell surface and interact together in many different cell types

Nogo-A, -B, and -C are generated from the Nogo/RTN-4 gene and share a highly conserved C-terminal domain. They lack an N-terminal signal sequence and are predominantly localized to the endoplasmic reticulum (ER). We found the N terminus of endogenous Nogo-A exposed on the surface of fibroblasts, DRG neurons, and myoblasts. Surface-expressed Nogo-A was also present on presynaptic terminals of the neuromuscular junction and on DRG neurons in vivo. Surface biotinylations confirmed the presence of all Nogo isoforms on the surface. To search for proteins that interact with Nogo-A and suggest a function for the large intracellular pool of Nogo-A, immunoprecipitations were performed. Surprisingly, the most predominant proteins that interact with Nogo-A are Nogo-B and Nogo-C as seen with radiolabeled lysates and as confirmed by Western blotting in multiple cell lines. Nogo-A, -B, and -C share a 180-amino acid C-terminal domain with two highly conserved hydrophobic stretches that could form a channel or transporter in the ER and/or on the cell surface.     

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。