Differential expression patterns of messenger RNAs encoding Nogo receptors and their ligands in the rat central nervous system

Nogo receptors (NgR1, -2, and -3) and their ligands, i.e., myelin-derived neurite outgrowth inhibitor (Nogo)-A, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp), have been considered to play pivotal roles in controlling axonal regeneration and neuronal plasticity. We show here that NgR1-3 mRNAs were differentially expressed exclusively in neurons situated in the telencephalon, diencephalons, and cerebellum, whereas we could not detect any NgR1-3 mRNA expression in the mesencephalon, pons, medulla oblongata, and spinal cord. On the other hand, Nogo-A mRNA was abundantly expressed in both neurons and oligodendrocytes throughout the central nervous system (CNS). MAG and OMgp mRNAs were also abundantly expressed in oligodendrocytes throughout the CNS. Interestingly, we did not detect NgR1-3 mRNAs in monoaminergic neurons in the substantia nigra, ventral tegmental area, locus caeruleus, and raphe nuclei, which are known to have high regenerative capacity. In addition, although neurons in the reticular thalamus and cerebellar nuclei are also known to show high capacity for regeneration, NgR1-3 mRNAs were not detected there. These data indicate that NgR1-3, Nogo-A, MAG, and OMgp mRNAs are differentially expressed in the rat CNS and suggest that the level of NgR1-3 expression in a neuron might determine its regenerative capacity.     

TROY and LINGO-1 expression in astrocytes and macrophages/microglia in multiple sclerosis lesions

Nogo constitutes a family of neurite outgrowth inhibitors contributing to a failure of axonal regeneration in the adult central nervous system (CNS). Nogo-A is expressed exclusively on oligodendrocytes where Nogo-66 segment binds to Nogo receptor (NgR) expressed on neuronal axons. NgR signalling requires a coreceptor p75(NTR) or TROY in combination with an adaptor LINGO-1. To characterize the cell types expressing the NgR complex in the human CNS, we studied demyelinating lesions of multiple sclerosis (MS) brains by immunohistochemistry. TROY and LINGO-1 were identified in subpopulations of reactive astrocytes, macrophages/microglia and neurones but not in oligodendrocytes. TROY was up-regulated, whereas LINGO-1 was reduced in MS brains by Western blot. These results suggest that the ternary complex of NgR/TROY/LINGO-1 expressed on astrocytes, macrophages/microglia and neurones, by interacting with Nogo-A on oligodendrocytes, might modulate glial-neuronal interactions in demyelinating lesions of MS.     

The role of Nogo-A in neuroregeneration: a review

Nogo, also known as Reticulon-4, is a protein that is specific to the central nervous system (CNS), and has been identified as an inhibitor of neurite outgrowth. Nogo-A is the largest member of the Nogo family and is responsible for inhibition of CNS regeneration. The structural information and biological functions of Nogo family members are reviewed in this study. The Nogo-66 receptor (NgR), a membrane protein which binds to Nogo, may play an important role in signal transduction for several myelin-associated inhibitors. The discovery of the Nogo family and the NgR provides an opportunity to develop interventions to promote axonal regeneration in the CNS after brain injury. Basic and clinical research of Nogo has increased our understanding of the mechanisms underlying spinal cord injury, multiple sclerosis, and neuroregenerative diseases. Understanding the biological functions of Nogo family members may open up a new avenue for the development of therapeutic agents. The anatomical and biological plastic changes are reviewed in animal models of injuries in the adult CNS. The role of Nogo A in neuroregeneration, and the mechanisms underlying functional recovery after CNS injury, are also detailed in this review.     

Myelin regulates immune cell adhesion and motility

The etiology of multiple sclerosis (MS) has not been fully elucidated, however evidence supports an autoimmune disease model notable for the infiltration of pro-inflammatory immune cells into sites of active demyelination and axonal injury. Previous findings demonstrate that neutralization of Nogo, a protein originally identified as a myelin-associated inhibitor (MAI) of axon regeneration, ameliorates experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS. More efficient axonal regeneration was suggested as a mechanism underlying the improved EAE outcome. However, neutralization of Nogo also led to an anti-inflammatory shift of T cell cytokines during EAE suggesting that another therapeutic mechanism may involve regulation of immune cell responses. Here we report that human immune cells from healthy individuals and MS patients express Nogo receptor1 (NgR1) indicating that they may be subject to regulation by MAIs. B cells, T cells and monocytes express NgR1 in a regulated fashion upon activation. While direct stimulation of human immune cells with an inhibitory fragment of Nogo does not impact their in vitro proliferation or cytokine production, the immune cells display reduced adhesion and enhanced motility in response to myelin, effects that are in part attenuated by antagonizing NgR1 signaling. We conclude that NgR1 alters the motility of immune cells exposed to myelin and may thus impact their behaviour within the CNS, particularly under conditions when immune cell activation is heightened.     

Small Nogo-66-binding peptide promotes neurite outgrowth through RhoA inhibition after spinal cord injury

Abortive regeneration in the adult mammalian central nervous system (CNS) is partially mediated through CNS myelin proteins, among which Nogo-A plays an important role. Nogo-66, which is located at the C-terminus of Nogo-A, inhibits axonal regrowth through the Nogo-66/NgR signalling pathway. In this study, two small peptides were tested in a neurite outgrowth assay and spinal cord injury (SCI) model to examine the effects of these molecules on the inhibition of Nogo-66/NgR signalling. PepIV was selected from a phage display peptide library as a Nogo-66 binding molecule. And PepII was synthesized as a potential NgR antagonist. The results indicated that PepIV and PepII decrease the mRNA levels of the small GTPase RhoA and partially neutralize CNS myelin inhibition to cultured cerebellar granule cells (CGCs). Moreover, treatment with both peptides was propitious to maintaining residual axons after SCI, thereby promoting regeneration and locomotion recovery. Because RhoA plays a role in stabilizing the cytoskeleton in growth cones and axons, enhanced neurite outgrowth might reflect a decrease in RhoA expression through PepIV and PepII treatment. Moreover, PepIV induced lower RhoA mRNA expression compared with PepII. Therefore, PepIV could block Nogo-66/NgR signalling and reduce RhoA mRNA level, and then contribute to neuronal survival and axonal regrowth after SCI, showing its ability to reverse CNS myelin inhibition to regeneration. Furthermore, selected small peptide might cover some unknown active sites on CNS myelin proteins, which could be potential targets for improving neurite outgrowth after injury.     

Small hairpin RNA interference of the Nogo receptor inhibits oxygen‐glucose deprivation‐induced damage in rat hippocampal slice cultures

In adult mammals, CNS damage does not repair well spontaneously. The Nogo receptor (NgR) signaling pathway prevents axonal regrowth and promotes neuronal apoptosis. This pathway, and pathways like it, may be part of the reason why nerves do not regrow. A number of preclinical experiments inhibiting portions of the NgR pathway have yielded limited induction of nerve repair. Here, we developed a small hairpin RNA (shRNA) to knock down NgR expression. With the use of rat hippocampal slices in tissue culture, we induced neuronal damage similar to that of ischemia‐reperfusion injury by exposing the cultured tissues to oxygen‐glucose deprivation. We then assayed the effect of NgR knockdown in this model system. Adenovirally delivered NgR shRNA decreased NgR mRNA and protein expression. Thirty minutes of oxygen‐glucose deprivation resulted in widespread tissue damage, including apoptosis and loss of neurite extension, 72 h after termination of oxygen‐glucose deprivation. The NgR shRNA knockdown reduced, but did not eliminate, the effects of oxygen‐glucose deprivation. Thus, NgR shRNA shows promise as a potential tool for the treatment of nerve damage.     

不同发育阶段大鼠CNS的NogO mRNA的表达

目的 研究神经再生抑制性蛋白Nogo在不同发育阶段大鼠中枢神经系统(CNS)中的表达。方法采用RT-PCR方法半定量分析不同发育阶段大鼠(胚胎期14d～成年，共7个时间点)脊髓中NogomRNA的表达变化。结果Nogo-A mRNA和Nogo-B mRNA在胚胎期14d就可以检测到，在所检查的时间点内，Nogo-A mRNA和Nogo-B mRSNA的扩增表现出很恒定的水平．其表达并未有任何特定的时间模式；Nogo-C mRNA的表达比前两者稍晚，在胚胎期21d检测到。结论Nogo在神经系统发育期就已存在，说明Nogo-A分子与髓鞘的成熟化进程关系可能并不很密切，提示Nogo分子在发育阶段具有不同于神经再生抑制作用的其他功能。     

Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration

The recently cloned gene Nogo, whose alternative splice products correspond to the antigenic target of the central nervous system (CNS) regeneration enhancing monoclonal antibody IN-1, codes for membrane proteins enriched in brain, particularly in oligodendrocytes. The 66-amino acid extracellular domain of Nogo (Nogo-66) interacts with a high-affinity receptor (NgR), a glycosylphosphatidylinositol (GPI)-linked protein with multiple leucine-rich repeats. The amino terminal cytoplasmic domain of Nogo appears to have a general cellular growth inhibitory effect. Nogo-66, on the other hand, specifically retards neurite outgrowth and induces growth cone collapse, possibly through its interaction with NgR and as yet unidentified transmembrane coreceptors. Recent results also suggest that Nogo expression may induce apoptosis in tumor cells. Together, these proteins provide new molecular handles for the design of therapeutic interventions for CNS injuries and neurodegenerative diseases, as well as possible leads to anticancer strategies.     

Detection of anti-Nogo receptor autoantibody in the serum of multiple sclerosis and controls

OBJECTIVES: A myelin-associated neurite outgrowth inhibitor Nogo-A plays a key role in inhibition of axonal regeneration. Axonal damage beginning at the early stage of multiple sclerosis (MS) is responsible for permanent neurological deficits, although its molecular mechanism remains unknown. The aim was to study the prevalence of autoantibodies against Nogo-A and Nogo receptor (NgR) in the serum of MS. METHODS: The antibodies were identified in the serum of 30 MS patients, 22 patients with non-MS other neurological diseases (OND), and 22 healthy control (HC) subjects by Western blot using recombinant human Nogo-A-specific segment (NAS), the shared segment of Nogo-A and -B (NAB), Nogo-66 (N66), the non-glycosylated form of NgR, the glycosylated NgR (NgR-Fc), and myelin oligodendrocyte glycoprotein (MOG). RESULTS: None showed immunoglobulin G (IgG) antibodies against NAS or NAB. In contrast, 30% of MS, 23% of OND and 32% of HC subjects exhibited anti-N66 IgG, while 27% of MS, 27% of OND and 18% of HC showed anti-MOG IgG. None of HC but 33% of MS and 14% of OND showed anti-non-glycosylated NgR IgG. Furthermore, 60% of MS, 18% of OND and 14% of HC showed anti-NgR-Fc IgG. CONCLUSIONS: Because IgG autoantibodies against N66, NgR and MOG are often detected in the serum of MS and controls, they do not serve as an MS-specific marker.     

Nogo-A and nogo receptor expression in demyelinating lesions of multiple sclerosis

A myelin-associated neurite outgrowth inhibitor, Nogo-A, plays a key role in inhibition of axonal regeneration following injury and ischemia in the central nervous system (CNS). Because axonal injury is a pathologic hallmark of multiple sclerosis (MS), we have investigated the expression of Nogo-A and its receptor NgR in four MS and 12 non-MS control brains by immunohistochemistry. Nogo-A expression was markedly upregulated in surviving oligodendrocytes at the edge of chronic active demyelinating lesions of MS and ischemic lesions of acute and old cerebral infarction, whereas NgR expression was greatly enhanced in reactive astrocytes and microglia/macrophages in these lesions when compared with their expression in the brains of neurologically normal controls. Nogo-A and NgR were also identified in a subpopulation of neurons. In contrast, Nogo-A was undetectable in reactive astrocytes and microglia/macrophages and NgR was not expressed on oligodendrocytes in any cases examined. Western blot analysis and double labeling immunocytochemistry identified the constitutive expression of NgR in cultured human astrocytes. These results suggest that Nogo-A expressed on oligodendrocytes might interact with NgR presented by reactive astrocytes and microglia/macrophages in active demyelinating lesions of MS, although biologic effects caused by Nogo-A/NgR interaction among glial cells remain unknown.     

Nogo signaling and non-physical injury-induced nervous system pathology

The Nogo gene products were described first as myelin-associated inhibitors that prevent neuronal regeneration upon injury. Recent findings have also implicated Nogo in several neuronal pathologies that are not induced by physical injury. Nogo-A may be an important determinant of autoimmune demyelinating diseases, as active immunization with Nogo-A fragments attenuates the symptoms of experimental autoimmune encephalomyelitis (EAE). Nogo-A levels are elevated markedly in hippocampal neurons of patients with temporal lobe epilepsy (TLE), in brain and muscle of patients with amyotrophic lateral sclerosis (ALS), and in schizophrenic patients. Concrete evidence for a direct role of Nogo-A in the latter neuropathies is not yet available, but such a role is logically in line with new findings associated with localization of Nogo-A and Nogo-Nogo-66 receptor (NgR)-mediated signaling. We speculate on possible linkages between the effect of aberrant elevation of Nogo levels and the signaling consequences that could lead to nervous system pathology.     

Experience-dependent expression of Nogo-A and Nogo receptor in the developing rat visual cortex

Nogo-A and Nogo receptor (NgR) expression in the visual cortex following a critical developmental period (postnatal days 20-60) has been previously shown. However, little is known regarding Nogo-A and NgR expression between postnatal day 0 and initiation of the critical period. The present study analyzed Nogo-A and NgR expression at four different time points: postnatal day 0 (P0), before critical period (P14), during critical period (P28), and after critical period (P60). Results showed significantly increased Nogo-A mRNA and protein expression levels in the visual cortex following birth, and expression levels remained steady between P28 and P60. NgR mRNA or protein expression was dramatically upregulated with age and peaked at P14 or P28, respectively, and maintained high expression to P60. In addition, Nogo-A and NgR expression was analyzed in each visual cortex layer in normal developing rats and rats with monocular deprivation. Monocular deprivation decreased Nogo-A and NgR mRNA and protein expression in the rat visual cortex, in particular in layers II-III and IV in the visual cortex contralateral to the deprived eye. These findings suggested that Nogo-A and NgR regulated termination of the critical period in experiencedependent visual cortical plasticity.     

Nogo A expression in the adult enteric nervous system

Neuronal plasticity plays an important role in physiological and pathological processes within the gastrointestinal (GI) tract. Nogo A is a major contributor to the negative effect central nervous system (CNS) myelin has on neurite outgrowth after injury and may also play a role in maintaining synaptic connections in the healthy CNS. Nogo A is highly expressed during neuronal development but in the CNS declines postnatally concomitantly with a loss of regenerative potential while ganglia of the Peripheral Nervous System (PNS) retain Nogo A. The enteric nervous system shares a number of features in common with the CNS, thus the peripheral distribution of factors affecting plasticity is of interest. We have investigated the distribution of Nogo in the adult mammalian gastrointestinal tract. Nogo A mRNA and protein are detectable in the adult rat GI tract. Nogo A is expressed heterogeneously in enteric neurons throughout the GI tract though expression levels appear not to be correlated with neuronal sub-type. The pattern of expression is maintained in cultured myenteric plexus from the guinea-pig small intestine. As is seen in developing neurons of the CNS, enteric Nogo A is present in both neuronal cell bodies and axons. Our results point to a hitherto unsuspected role for Nogo A in enteric neuronal physiology.     

Upregulation of axon guidance molecules in the adult central nervous system of Nogo‐A knockout mice restricts neuronal growth and regeneration

Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo‐A/Nogo receptor (NgR1) signaling pathway. Nogo‐A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody‐mediated neutralization of Nogo‐A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo‐A KO vs. wild‐type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4‐positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth‐inhibitory than WT but more than Nogo‐A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo‐A KO myelin as compared with WT neurons, supporting increased EphA4‐mediated growth inhibition in Nogo‐A KO mice. Consistently, in vivo, Nogo‐A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo‐A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo‐A.     

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.     

Regulation of Nogo and Nogo receptor during the development of the entorhino-hippocampal pathway and after adult hippocampal lesions

Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.     

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain.     

Neuronal activity-induced regulation of Lingo-1

Axonal regeneration after injury can be limited in the adult CNS by the presence of inhibitory proteins such as Nogo. Nogo binds to a receptor complex that consists of Nogo receptor (NgR), p75NTR, and Lingo-1. Nogo binding activates RhoA, which inhibits axonal outgrowth. Here we assessed Lingo-1 and NgR mRNA levels after delivery of BDNF into the rat hippocampal formation, Lingo-1 mRNA levels in rats subjected to kainic acid (KA) and running in running wheels. Lingo-1 mRNA was not changed by running. However, we found that Lingo-1 mRNA was strongly up-regulated while NgR mRNA was down-regulated in the dentate gyrus in both the BDNF and the KA experiments. Our data demonstrate inverse regulation of NgR and Lingo-1 in these situations, suggesting that Lingo-1 up-regulation is one characteristic of activity-induced neural plasticity responses.     

Overcoming inhibitors in myelin to promote axonal regeneration

The lack of axonal growth after injury in the adult central nervous system (CNS) is due to several factors including the formation of a glial scar, the absence of neurotrophic factors, the presence of growth-inhibitory molecules associated with myelin and the intrinsic growth-state of the neurons. To date, three inhibitors have been identified in myelin: Myelin-Associated Glycoprotein (MAG), Nogo-A, and Oligodendrocyte-Myelin glycoprotein (OMgp). In previous studies we reported that MAG inhibits axonal regeneration by high affinity interaction (K(D) 8 nM) with the Nogo66 receptor (NgR) and activation of a p75 neurotrophin receptor (p75NTR)-mediated signaling pathway. Similar to other axon guidance molecules, MAG is bifunctional. When cultured on MAG-expressing cells, dorsal root ganglia neurons (DRG) older than post-natal day 4 (PND4) extend neurites 50% shorter on average than when cultured on control cells. In contrast, MAG promotes neurite outgrowth from DRG neurons from animals younger than PND4. The response switch, which is also seen in retinal ganglia (RGC) and Raphe nucleus neurons, is concomitant with a developmental decrease in the endogenous neuronal cAMP levels. We report that artificially increasing cAMP levels in older neurons can alter their growth-state and induce axonal growth in the presence of myelin-associated inhibitors.