Behavioral characterization of mice lacking the neurite outgrowth inhibitor Nogo-A

The membrane protein Nogo-A inhibits neurite outgrowth and regeneration in the injured central nervous system, primarily because of its expression in oligodendrocytes. Hence, deletion of Nogo-A enhances regeneration following spinal cord injury. Yet, the effects of Nogo-A deletion on general behavior and cognition have not been explored. The possibility of potential novel functions of Nogo-A beyond growth inhibition is strongly suggested by the presence of subpopulations of neurons also expressing Nogo-A – not only during development but also in adulthood. We evaluated here Nogo-A ^−/− mice in a series of general basic behavioral assays as well as functional analyses related to brain regions with notable expression levels of Nogo-A. The SHIRPA protocol did not show any major basic behavioral changes in Nogo-A ^−/− mice. Anxiety-related behavior, pain sensitivity, startle reactivity, spatial learning, and associative learning also appeared indistinguishable between Nogo-A ^−/− and control Nogo-A^+/+ mice. However, motor co-ordination and balance were enhanced in Nogo-A ^−/− mice. Spontaneous locomotor activity was also elevated in Nogo-A ^−/− mice, but this was specifically observed in the dark (active) phase of the circadian cycle. Enhanced locomotor reaction to systemic amphetamine in Nogo-A ^−/− mice further pointed to an altered dopaminergic tone in these mice. The present study is the first behavioral characterization of mice lacking Nogo-A and provides significant insights into the potential behavioral relevance of Nogo-A in the modulation of dopaminergic and motor functions.     

Nogo-A expression in the brain of mice with cerebral malaria

Cerebral malaria (CM) is associated with a high rate of transient or persistent neurological sequelae. Nogo-A, a protein that is highly expressed in the endoplasmic reticulum (ER) of the mammalian central nervous system (CNS), is involved in neuronal regeneration and synaptic plasticity in the injured CNS. The current study investigates the role of Nogo-A in the course of experimental CM. C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. Brain homogenates of mice with different clinical severity levels of CM, infected animals without CM and control animals were analyzed for Nogo-A up-regulation by Western blotting and immunohistochemistry. Brain regions with Nogo-A upregulation were evaluated by transmission electron microscopy. Densitometric analysis of Western blots yielded a statistically significant upregulation of Nogo-A in mice showing moderate to severe CM. The number of neurons and oligodendrocytes positive for Nogo-A did not differ significantly between the studied groups. However, mice with severe CM showed a significantly higher number of cells with intense Nogo-A staining in the brain stem. In this region ultrastructural alterations of the ER were regularly observed. Nogo-A is upregulated during the early course of experimental CM. In the brain stem of severely affected animals increased Nogo-A expression and ultrastructural changes of the ER were observed. These data indicate a role of Nogo-A in neuronal stress response during experimental CM.     

Nogo A和srGAP蛋白在NIH 3T3细胞中的共表达

为检测Nogo A和srGAPs蛋白在NIH 3T3细胞上的表达,应用Western印迹的方法检测Nogo A蛋白的表达. 从NIH 3T3细胞抽提物中检测到约230 kD特异性的Nogo A反应条带;利用双重免疫细胞荧光化学标记法和激光共聚焦显微镜成像技术分别检测Nogo A与srGAPs或Rho蛋白在NIH 3T3细胞上的共表达状况,可观察到Nogo A与srGAPs共存于3T3细胞的细胞浆、突起和生长锥样结构上,亦可观察到Nogo A与Rho蛋白的共存.结果表明,NIH 3T3细胞中共表达Nogo A、srGAPs和Rho分子. 这为研究Nogo A与Rho信号转导途径间的关系奠定了基础.     

Phosphorylation at Tyr-694 of Nogo-A by Src-family kinases

Nogo-A is a neurite outgrowth inhibitor protein associated with myelin in the central nervous system. Unexpectedly, targeted disruption of Nogo-A in mice results in little or no improvement of axonal regeneration, suggesting that Nogo-A has other functions and/or receives complex regulations to exert its inhibitory functions. Here, we have found that Nogo-A becomes phosphorylated at Tyr-694 in the N-terminal region. The phosphorylation is mediated co-operatively by Src-family tyrosine kinases, which play many important roles in the nervous system. Levels of tyrosine phosphorylation of Nogo-A seem to be irrelevant to developmental stages of oligodendrocytes, and might be regulated by specific extracellular stimuli. Identification of tyrosine phosphorylation of Nogo-A will introduce an additional level of complexity into Nogo-A functions.     

Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.     

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.     

Molecular regulation of Nogo-A in neural cells: Novel insights into structure and function

Nogo-A is part of the reticulon family of proteins localized to the myelin and oligodendroglial plasma membranes. Nogo-A specifically initiates signal transduction cascades limiting axonal regrowth following injury and disease in the adult mammalian central nervous system (CNS). Recent novel data support the contention that neuronal Nogo-A plays an important role in regulating cytoskeletal re-organization without the requirement of signaling through its cognate receptor (Nogo receptor). These data, along with the recent findings that the N-terminus of Nogo-A can interact with integrins and that NgR1 interacts with the amyloid precursor protein extracellularly, as well as novel findings showing ubiquitin ligases binding with Nogo-A intracellularly add a layer of complexity to its functional role in the CNS.     

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.     

Nogo-A interacts with TrkA to alter nerve growth factor signaling in Nogo-A-overexpressing PC12 cells

The Nogo-A protein, originally discovered as a potent myelin-associated inhibitor of neurite outgrowth, is also expressed by certain neurons, especially during development and after injury, but its role in neuronal function is not completely known. In this report, we overexpressed Nogo-A in PC12 cells to use as a model to identify potential neuronal signaling pathways affected by endogenously expressed Nogo-A. Unexpectedly, our results show that viability of Nogo-A-overexpressing cells was reduced progressively due to apoptotic cell death following NGF treatment, but only after 24 h. Inhibitors of neutral sphingomyelinase prevented this loss of viability, suggesting that NGF induced the activation of a ceramide-dependent cell death pathway. Nogo-A over-expression also changed NGF-induced phosphorylation of TrkA at tyrosines 490 and 674/675 from sustained to transient, and prevented the regulated intramembrane proteolysis of p75^NTR, indicating that Nogo-A was altering the function of the two neurotrophin receptors. Co-immunoprecipitation studies revealed that there was a physical association between TrkA and Nogo-A which appeared to be dependent on interactions in the Nogo-A-specific region of the protein. Taken together, our results indicate that Nogo-A influences NGF-mediated mechanisms involving the activation of TrkA and its interaction with p75^NTR.     

HBO suppresses Nogo-A,Ng-R,or RhoA expression in the cerebral cortex after global ischemia

Nogo-A, a myelin-associated neurite outgrowth inhibitory protein, binds with the Ng-R receptor to activate RhoA intracellular signals and inhibit the plasticity after CNS injury. We evaluated the effect of hyperbaric oxygen (HBO) on the expression of Nogo-A, Ng-R, and RhoA after transient global ischemia in a rat 2 vessel occlusion global ischemic model. Male SD rats (n=78) were randomly divided into 13 groups: 1 sham group, 6 groups of global ischemia, and 6 groups of HBO treatment after global ischemia. HBO (3ATA) was applied for 2 hr at 1 hr after global ischemia. Rats were sacrificed at 6, 12, 24, 48, and 96 hr and 7 days. Global ischemia (10 min) produced a marked increase of Nogo-A/B, Nogo-A, Ng-R, and RhoA expression. Immunohistochemistry showed increased Nogo-A/B and Nogo-A located in the myelin sheath of ischemic brain cortex. Ng-R expressed on the surface of neurons and their processes, and RhoA expressed inside the cytoplasm of neurons in ischemic brain. HBO significantly reduced neurological injury, decreased the levels of Nogo-A, Ng-R, and RhoA in ischemic injured cortex (p<0.05).     

Serum Nogo-A levels are not elevated in amyotrophic lateral sclerosis patients

Improved biomarkers would facilitate the diagnosis and treatment of amyotrophic lateral sclerosis (ALS). Muscle content of the neuritic outgrowth inhibitor Nogo-A is increased in patients with ALS and other denervating conditions. Seeking a less invasive diagnostic method, we sought to determine whether or not Nogo increases in the serum of ALS patients. We developed a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) protocol to screen serum samples from 172 ALS patients and 172 healthy controls for Nogo-A immunoreactivity. Unexpectedly, there was a trend toward decreased levels of serum Nogo-A in ALS. Mean serum Nogo-A level in ALS patients was 0.71?nM (95% confidence interval (CI) 0.42–1.00), as opposed to 1.15?nM (95% CI 0.72–1.59) in healthy controls. A significantly larger percentage of healthy control sera (11.0% vs 4.7%) displayed markedly elevated levels of Nogo-A. Additional study is required to determine the factors that lead to elevated Nogo-A levels in a subset of both ALS patients and healthy controls.     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

Lack of enhanced spinal regeneration in Nogo-deficient mice

The failure of regeneration of severed axons in the adult mammalian central nervous system is thought to be due partly to the presence of endogenous inhibitors of axon regeneration. The nogo gene encodes three proteins (Nogo-A, -B, and -C) that have been proposed to contribute to this inhibition. To determine whether deletion of nogo enhances regenerative ability, we generated two lines of mutant mice, one lacking Nogo-A and -B but not -C (Nogo-A/B mutant), and one deficient in all three isoforms (Nogo-A/B/C mutant). Although Nogo-A/B-deficient myelin has reduced inhibitory activity in a neurite outgrowth assay in vitro, tracing of corticospinal tract fibers after dorsal hemisection of the spinal cord did not reveal an obvious increase in regeneration or sprouting of these fibers in either mouse line, suggesting that elimination of Nogo alone is not sufficient to induce extensive axon regeneration.     

Inhibition of Retinal Ganglion Cell Axonal Outgrowth Through the Amino-Nogo-A Signaling Pathway

Nogo-A is a myelin-derived inhibitor playing a pivotal role in the prevention of axonal regeneration. A functional domain of Nogo-A, Amino-Nogo, exerts an inhibitory effect on axonal regeneration, although the mechanism is unclear. The present study investigated the role of the Amino-Nogo–integrin signaling pathway in primary retinal ganglion cells (RGCs) with respect to axonal outgrowth, which is required for axonal regeneration. Immunohistochemistry showed that integrin αv, integrin α5 and FAK were widely expressed in the visual system. Thy-1 and GAP-43 immunofluorescence showed that axonal outgrowth of RGCs was promoted by Nogo-A siRNA and a peptide antagonist of the Nogo-66 functional domain of Nogo-A (Nep1–40), and inhibited by a recombinant rat Nogo-A-Fc chimeric protein (△20). Western blotting revealed increased integrin αv and p-FAK expression in Nogo-A siRNA group, decreased integrin αv expression in △20 group and decreased p-FAK expression in Nep1–40 group. Integrin α5 expression was not changed in any group. RhoA G-LISA showed that RhoA activation was inhibited by Nogo-A siRNA and △20, but increased by Nep1–40 treatment. These results suggest that Amino-Nogo inhibits RGC axonal outgrowth primarily through the integrin αv signaling pathway.     

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.     

An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity

The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system. The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect. However, this murine IgM/kappa antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure-function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis. We describe here the affinity maturation of a bacterially produced functional IN-1 F(ab) fragment via protein engineering. A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments. After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 F(ab) fragment was obtained, carrying five side chain substitutions within CDR-L3. Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 microM. The affinity of the unmutated IN-1 F(ab) fragment was 8-fold lower. The engineered F(ab) fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections. Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture. This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered F(ab) fragment as a reagent for promoting axonal regeneration in vivo.     

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition

Background  

Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS) and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition.     

Expression of Nogo-66 receptor in human astrocytoma is correlated with tumor malignancy

Nogo-A is a myelin-associated neuronal growth inhibitory molecule in central nervous system after trauma. However, the physiological functions of Nogo-A in neural development and in healthy oligodendrocytes are largely unknown. In this study, we investigated the expression of Nogo-66 receptor (NgR) protein in 60 cases of human astrocytoma by Western blot RT-PCR and immunohistochemistry. The correlation between the expression of NgR and pathologic grades of astrocyoma was further analyzed. The results showed that the expression of NgR protein and NgR mRNA immunoreactivity score, were decreased markedly with the increasing pathological grades. Double immunostaining results showed that Nogo-A and NgR were colocalized at the interface of astrocytoma cells and extracellular matrix. Our results indicated that NgR may have inhibitory effects on tumor activity and Nogo-A may restrict migration of tumor cells via NgR.     

Nogo-A蛋白在一氧化碳中毒大鼠海马组织中的动态表达

目的:观察一氧化碳中毒后大鼠海马组织中Nogo-A蛋白动态表达情况,探讨Nogo-A蛋白在一氧化碳中毒后神经系统损伤中的作用与影响。方法:随机数字法将雄性SD大鼠30只分为正常对照组(NC组)、CO中毒组(CO组)、CO中毒后24小时组(CO-24h)、CO中毒后48小时组(CO-48h)、CO中毒后7天组(CO-7d),每组各6只。CO气体腹腔注射染毒法建立一氧化碳中毒模型。建模后不同时间点利用免疫组织化学染色(IHC)、蛋白质免疫印迹技术检测(WB)Nogo-A蛋白在海马组织中的表达情况并分析其变化规律。结果:IHC结果表明NC、CO组、CO-24h组、CO-48h组、CO-7d组中Nogo-A蛋白平均光密度值分别为0.0928±0.0038、0.01172±0.0042、0.1452±0.0056、0.1271±0.0057、0.1088±0.0055;WB结果提示一氧化碳中毒后海马组织中Nogo-A蛋白的表达较NC组增高,在24h时表达达到高峰(P0.05);24h后表达开始逐步下降,至染毒第7天时,海马组织中Nogo-A蛋白的表达明显下降,但仍高于NC组(P0.05)。结论:在本研究中,大鼠海马组织中Nogo-A蛋白表达的增高与一氧化碳中毒相关;Nogo-A蛋白表达在24h时表达达到高峰;24h后表达开始逐步下降。