Development of neural repair therapy for chronic spinal cord trauma: soluble Nogo receptor decoy from discovery to clinical trial.

PURPOSE OF REVIEW: After traumatic spinal cord injury (SCI), neurological deficits persist due to the disconnection of surviving neurons. While repair of connectivity may restore function, no medical therapy exists today.This review traces the development of the neural repair-based therapeutic AXER-204 from animal studies to the recent clinical trial for chronic cervical SCI. RECENT FINDINGS: Molecular studies reveal a Nogo-66 Receptor 1 (NgR1, RTN4R) pathway inhibiting axon regeneration, sprouting, and plasticity in the adult mammalian central nervous system (CNS). Rodent and nonhuman primate studies demonstrate that the soluble receptor decoy NgR(310)ecto-Fc or AXER-204 promotes neural repair and functional recovery in transection and contusion SCI. Recently, this biological agent completed a first-in-human and randomized clinical trial for chronic cervical SCI. The intervention was safe and well tolerated. Across all participants, upper extremity strength did not improve with treatment. However, posthoc and biomarker analyses suggest that AXER-204 may benefit treatment-naïve patients with incomplete SCI in the chronic stage. SUMMARY: NgR1 signaling restricts neurological recovery in animal studies of CNS injury. The recent clinical trial of AXER-204 provides encouraging signals supporting future focused trials of this neural repair therapeutic. Further, AXER-204 studies provide a roadmap for the development of additional and synergistic therapies for chronic SCI.

NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein.

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

Reduction of NgR in perforant path protects neuronal morphology and function in APP/PS1 transgenic mice.

Neuronal loss is the central abnormality occurring in brains suffering from Alzheimer's disease (AD). The notion that AD causes the death of neurons point towards protection of neuronal morphology and function as important therapeutic strategies. The perforant path projections from the entorhinal cortex to the dentate gyrus is the most vulnerable circuit with respect to AD. It's known that the perforant path is a very important structure for synaptic plasticity and cognitive functions. NgR (Nogo receptor) is not only involved in limiting injury-induced axonal growth but also in pathological features of AD. So, the mechanism of how NgR affects the perforant path needs further investigation. In this study, the effect of NgR in the perforant path on the neuronal morphology and function in APP/PS1 transgenic mice was studied. The results showed that downregulation of NgR in perforant path ameliorate the damaged morphology and decreased number of neurons in APP/PS1 mice. Concurrently, NgR knockdown enhanced dendritic complexity and increased postsynaptic protein density in APP/PS1 mice. Furthermore, the RT-PCR results indicated that there is downregulation of M1 phenotypes of microglial gene expression in the hippocampus of TG-shNgR mice. Our study suggests that NgR plays a critical role in microglial phenotype polarization, which might account for the NgR knockdown in the perforant path initiated a decrease in neuronal death and improved synaptic function. Our study provided a better understanding of the perforant path and the role of NgR in AD pathogenesis, thus offering the potential application of hippocampal neurons in treatment of AD.

Myelin-associated glycoprotein alters the neuronal secretome and stimulates the release of TGFß and proteins that affect neural plasticity.

Myelin-associated glycoprotein (MAG) and Nogo inhibit neurite outgrowth by binding to receptors such as NgR1, PirB and LRP1, and they have also been shown to induce phosphorylation of Smad2, a key intermediate in the transforming growth factor ß (TGFß) signalling pathway. In this study, we determined that MAG and Nogo do not transactivate the TGFß receptor through their canonical receptors or discoidin domain receptor 1, which we identified as a novel receptor for MAG and Nogo. Instead, MAG and Nogo promoted Smad2 phosphorylation by stimulating secretion of TGFß. Proteomic analysis of the neuronal secretome revealed that MAG also regulated the secretion of proteins that affect central nervous system plasticity-inducing the secretion of S100A6, septin-7 and neurofascin 186, while inhibiting the secretion of frataxin, MAP6, syntenin-1 and GAP-43. This represents a novel function for MAG that has broad implications for the treatment for spinal cord injury.

The Nogo-66 Receptors NgR1 and NgR3 Are Required for Commissural Axon Pathfinding.

Nogo-66 receptors (NgR1-3) are glycosylphosphatidyl inositol-linked proteins that belong to the leucine-rich repeat superfamily. Through binding to myelin-associated inhibitors, NgRs contribute to the inhibition of axonal regeneration after spinal cord injury. Their role in limiting synaptic plasticity and axonal outgrowth in the adult CNS has been described previously, but not much is known about their role during the development of the nervous system. Here, we show that NgR1 and NgR3 mRNAs are expressed during spinal cord development of the chicken embryo. In particular, they are expressed in the dI1 subpopulation of commissural neurons during the time when their axons navigate toward and across the floorplate, the ventral midline of the spinal cord. To assess a potential role of NgR1 and NgR3 in axon guidance, we downregulated them using in ovo RNAi and analyzed the trajectory of commissural axons by tracing them in open-book preparations of spinal cords. Our results show that loss of either NgR1 or NgR3 causes axons to stall in the midline area and to interfere with the rostral turn of postcrossing axons. In addition, we also show that NgR1, but not NgR3, requires neuronal PlexinA2 for the regulation of commissural axon guidance.SIGNIFICANCE STATEMENT Over the last decades, many studies have focused on the role of NgRs, particularly NgR1, in axonal regeneration in the injured adult CNS. Here, we show a physiological role of NgRs in guiding commissural axons during early development of the chicken spinal cord in vivo Both NgR1 and NgR3 are required for midline crossing and subsequent turning of postcrossing axons into the longitudinal axis of the spinal cord. NgR1, but not NgR3, forms a receptor complex with PlexinA2 during axon guidance. Overall, these findings provide a link between neural regenerative mechanisms and developmental processes.

The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis.

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host range, tissue tropism, and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by the viral attachment protein σ1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule A (JAM-A) and Nogo-66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread and infection of the CNS. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease in wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter reovirus replication in the intestine or transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of NgR1 also did not alter reovirus replication, neural tropism, and virulence following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. Thus, neither JAM-A nor NgR1 is required for reovirus CNS tropism in mice, suggesting that other unidentified receptors support this function. IMPORTANCE Viruses engage diverse molecules on host cell surfaces to navigate barriers, gain cell entry, and establish infection. Despite discovery of several reovirus receptors, host factors responsible for reovirus neurotropism are unknown. Human NgR1 functions as a reovirus receptor in vitro and is expressed in CNS neurons in a pattern overlapping reovirus tropism. We used mice lacking NgR1 to test whether NgR1 functions as a reovirus neural receptor. Following different routes of inoculation, we found that murine NgR1 is dispensable for reovirus dissemination to the CNS, tropism and replication in the brain, and resultant disease. Concordantly, expression of human but not murine NgR1 confers reovirus binding and infection of nonsusceptible cells in vitro. These results highlight species-specific use of alternate receptors by reovirus. A detailed understanding of species- and tissue-specific factors that dictate viral tropism will inform development of antiviral interventions and targeted gene delivery and therapeutic viral vectors.

A Novel Role of Nogo Proteins: Regulating Macrophages in Inflammatory Disease.

Nogo proteins, also known as Reticulon-4, have been identified as myelin-derived inhibitors of neurite outgrowth in the central nervous system (CNS). There are three Nogo variants, Nogo-A, Nogo-B and Nogo-C. Recent studies have shown that Nogo-A/B is abundant in macrophages and may have a wider effect on inflammation. In this review, we focus mainly on the possible roles of Nogo-A/B on polarization and recruitment of macrophages and their involvement in a variety of inflammatory diseases. We then discuss the Nogo receptor1 (NgR1), a common receptor for Nogo proteins that is also abundant in microglia/macrophage in the CNS. Interaction of Nogo and NgR1 in microglia/macrophage may affect the adhesion and polarization of macrophages that are involved in multiple neurodegenerative diseases, including Alzheimer's disease and multiple sclerosis. Overall, this review provides insights into the roles of Nogo proteins in regulating macrophage functions and suggests that, potentially, Nogo proteins maybe a new target in the treatment of inflammatory diseases.

A Kalirin missense mutation enhances dendritic RhoA signaling and leads to regression of cortical dendritic arbors across development.

Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

NEP1­40 promotes myelin regeneration via upregulation of GAP­43 and MAP­2 expression after focal cerebral ischemia in rats.

Axon regeneration after lesions to the central nervous system (CNS) is largely limited by the presence of growth inhibitory molecules expressed in myelin. Nogo­A is a principal inhibitor of neurite outgrowth, and blocking the activity of Nogo­A can induce axonal sprouting and functional recovery. However, there are limited data on the expression of Nogo­A after CNS lesions, and the mechanism underlying its influences on myelin growth remains unknown. The aim of the present study was to observe the time course of Nogo­A after cerebral ischemia/reperfusion in rats using immunohistochemistry and western blot techniques, and to test the effect of its inhibitor Nogo extracellular peptide 1­40 (NEP1­40) on neural plasticity proteins, growth­associated binding protein 43 (GAP­43) and microtubule associated protein 2 (MAP­2), as a possible mechanism underlying myelin suppression. A classic model of middle cerebral artery occlusion (MCAO) was established in Sprague­Dawley rats, which were divided into three groups: i) MCAO model group; ii) MCAO + saline group; and iii) MCAO + NEP1­40 group. Rats of each group were divided into five subgroups by time points as follows: days 1, 3, 7, 14 and 28. Animals that only received sham operation were used as controls. The Nogo­A immunoreactivity was located primarily in the cytoplasm of oligodendrocytes. The number of Nogo­A immunoreactive cells significantly increased from day 1 to day 3 after MCAO, nearly returning to the control level at day 7, increased again at day 14 and decreased at day 28. Myelin basic protein (MBP) immunoreactivity in the ipsilateral striatum gradually decreased from day 1 to day 28 after ischemia, indicating myelin loss appeared at early time points and continuously advanced during ischemia. Then, intracerebroventricular infusion of NEP1­40, which is a Nogo­66 receptor antagonist peptide, was administered at days 1, 3 and 14 after MCAO. It was observed that GAP­43 considerably increased from day 1 to day 7 and then decreased to a baseline level at day 28 compared with the control. MAP­2 expression across days 1­28 significantly decreased after MCAO. Administration of NEP1­40 attenuated the reduction of MBP, and upregulated GAP­43 and MAP­2 expression at the corresponding time points after MCAO compared with the MCAO + saline group. The present results indicated that NEP1­40 ameliorated myelin damage and promoted regeneration by upregulating the expression of GAP­43 and MAP­2 related to neuronal and axonal plasticity, which may aid with the identification of a novel molecular mechanism of restriction in CNS regeneration mediated by Nogo­A after ischemia in rats.

LOTUS, an endogenous Nogo receptor antagonist, is involved in synapse and memory formation.

The Nogo signal is involved in impairment of memory formation. We previously reported the lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of the Nogo receptor 1 that mediates the inhibition of axon growth and synapse formation. Moreover, we found that LOTUS plays an essential role in neural circuit formation and nerve regeneration. However, the effects of LOTUS on synapse formation and memory function have not been elucidated. Here, we clearly showed the involvement of LOTUS in synapse formation and memory function. The cultured hippocampal neurons derived from lotus gene knockout (LOTUS-KO) mice exhibited a decrease in synaptic density compared with those from wild-type mice. We also found decrease of dendritic spine formation in the adult hippocampus of LOTUS-KO mice. Finally, we demonstrated that LOTUS deficiency impairs memory formation in the social recognition test and the Morris water maze test, indicating that LOTUS is involved in functions of social and spatial learning and memory. These findings suggest that LOTUS affects synapse formation and memory function.

B-cells expressing NgR1 and NgR3 are localized to EAE-induced inflammatory infiltrates and are stimulated by BAFF.

We have previously reported evidence that Nogo-A activation of Nogo-receptor 1 (NgR1) can drive axonal dystrophy during the neurological progression of experimental autoimmune encephalomyelitis (EAE). However, the B-cell activating factor (BAFF/BlyS) may also be an important ligand of NgR during neuroinflammation. In the current study we define that NgR1 and its homologs may contribute to immune cell signaling during EAE. Meningeal B-cells expressing NgR1 and NgR3 were identified within the lumbosacral spinal cords of ngr1+/+ EAE-induced mice at clinical score 1. Furthermore, increased secretion of immunoglobulins that bound to central nervous system myelin were shown to be generated from isolated NgR1- and NgR3-expressing B-cells of ngr1+/+ EAE-induced mice. In vitro BAFF stimulation of NgR1- and NgR3-expressing B cells, directed them into the cell cycle DNA synthesis phase. However, when we antagonized BAFF signaling by co-incubation with recombinant BAFF-R, NgR1-Fc, or NgR3 peptides, the B cells remained in the G0/G1 phase. The data suggest that B cells express NgR1 and NgR3 during EAE, being localized to infiltrates of the meninges and that their regulation is governed by BAFF signaling.

Modulation of Nogo receptor 1 expression orchestrates myelin-associated infiltration of glioblastoma.

As the clinical failure of glioblastoma treatment is attributed by multiple components, including myelin-associated infiltration, assessment of the molecular mechanisms underlying such process and identification of the infiltrating cells have been the primary objectives in glioblastoma research. Here, we adopted radiogenomic analysis to screen for functionally relevant genes that orchestrate the process of glioma cell infiltration through myelin and promote glioblastoma aggressiveness. The receptor of the Nogo ligand (NgR1) was selected as the top candidate through Differentially Expressed Genes (DEG) and Gene Ontology (GO) enrichment analysis. Gain and loss of function studies on NgR1 elucidated its underlying molecular importance in suppressing myelin-associated infiltration in vitro and in vivo. The migratory ability of glioblastoma cells on myelin is reversibly modulated by NgR1 during differentiation and dedifferentiation process through deubiquitinating activity of USP1, which inhibits the degradation of ID1 to downregulate NgR1 expression. Furthermore, pimozide, a well-known antipsychotic drug, upregulates NgR1 by post-translational targeting of USP1, which sensitizes glioma stem cells to myelin inhibition and suppresses myelin-associated infiltration in vivo. In primary human glioblastoma, downregulation of NgR1 expression is associated with highly infiltrative characteristics and poor survival. Together, our findings reveal that loss of NgR1 drives myelin-associated infiltration of glioblastoma and suggest that novel therapeutic strategies aimed at reactivating expression of NgR1 will improve the clinical outcome of glioblastoma patients.

Nogo­66 promotes ß­amyloid protein secretion via NgR/ROCK­dependent BACE1 activation.

The generation of ß­amyloid protein (Aß) is considered a key step in the pathogenesis of Alzheimer's disease (AD) and the regulation of its production is an important therapeutic strategy. It was hypothesized in the present study that Nogo­A may be involved in AD and may regulate the generation of Aß. Nogo­A is known to act as a major inhibitor of neuron regeneration in the adult central nervous system. A recent study indicated that Nogo­A is associated with AD; however, the underlying effect and molecular mechanisms remain largely elusive. In the present study, the potential effects of Nogo­A on AD were investigated. ELISA was used to detect the levels of Aß, enzymatic activity detection kits were used to determine the activity of secretase enzymes in amyloid precursor protein (APP) metabolism, and western blot analysis was used to detect the expression levels of proteins associated with the APP processing and Nogo­A/Nogo­66 receptor (NgR) signaling pathways. The results revealed that Nogo­66, the major inhibitory region of Nogo­A, promoted neuronal Aß secretion by increasing the activity of ß­secretase 1 via the NgR/Rho­associated coiled­coil containing kinases pathway in a dose­dependent manner. The present data suggested that Nogo­A may facilitate the onset and development of AD by promoting Aß secretion, providing information on a potential novel target for AD therapy.

Tanshinone IIA Promotes Axonal Regeneration in Rats with Focal Cerebral Ischemia Through the Inhibition of Nogo-A/NgR1/RhoA/ROCKII/MLC Signaling.

PURPOSE: The aim of this study was to evaluate the neuroprotective effect of tanshinone IIA (TSA) on focal cerebral ischemia in rats and to investigate whether it was associated with Nogo-A/NgR1/RhoA/Rho-associated protein kinase 2 (ROCKII)/myosin light chain (MLC) signaling. METHODS: In this study, focal cerebral ischemia animal model was used. Neurological deficit scores and infarction volume were investigated to evaluate the neuroprotection of TSA. Hematoxylin-eosin staining, Nissl staining, and immunofluorescence staining were conducted to detect ischemic changes in brain tissue and changes in neurofilament protein 200 (NF200) and growth-associated protein-43 (GAP-43) expression, respectively. Western blotting and qRT-PCR analyses were used to detect the expression levels of NF200, GAP-43 and Nogo-A/NgR1/RhoA/ROCKII/MLC pathway-related signaling molecules. RESULTS: TSA treatment can improve the survival rate of rats, reduce the neurological score and infarct volume, and reduce neuron damage. In addition, TSA also increased axon length and enhanced expression of NF200 and GAP-43. Importantly, TSA significantly attenuated the expression of Nogo-A, NgR1, RhoA, ROCKII, and p-MLC, and thus inhibiting the activation of this signaling pathway. CONCLUSION: TSA promoted axonal regeneration by inhibiting the Nogo-A/NgR1/RhoA/ROCKII/MLC signaling pathway, thereby exerting neuroprotective effects in cerebral ischemia rats, which provided support for the clinical application of TSA in stroke treatment.

Identification of a new functional domain of Nogo-A that promotes inflammatory pain and inhibits neurite growth through binding to NgR1.

Nogo-A is a key inhibitory molecule to axon regeneration, and plays diverse roles in other pathological conditions, such as stroke, schizophrenia, and neurodegenerative diseases. Nogo-66 and Nogo-Δ20 fragments are two known functional domains of Nogo-A, which act through the Nogo-66 receptor (NgR1) and sphingosine-1-phosphate receptor 2 (S1PR2), respectively. Here, we reported a new functional domain of Nogo-A, Nogo-A aa 846-861, was identified in the Nogo-A-specific segment that promotes complete Freund's adjuvant (CFA)-induced inflammatory pain. Intrathecal injection of its antagonist peptide 846-861PE or the specific antibody attenuated the CFA-induced inflammatory heat hyperalgesia. The 846-861 PE reduced the content of transient receptor potential vanilloid subfamily member 1 (TRPV1) in dorsal root ganglia (DRG) and decreased the response of DRG neurons to capsaicin. These effects were accompanied by a reduction in LIMK/cofilin phosphorylation and actin polymerization. GST pull-down and fluorescence resonance energy transfer (FRET) assays both showed that Nogo-A aa 846-861 bound to NgR1. Moreover, we demonstrated that Nogo-A aa 846-861 inhibited neurite outgrowth from cortical neurons and DRG explants. We concluded that Nogo-A aa 846-861 is a novel ligand of NgR1, which activates the downstream signaling pathways that inhibit axon growth and promote inflammatory pain.

Integrative analysis of in vivo recording with single-cell RNA-seq data reveals molecular properties of light-sensitive neurons in mouse V1.

Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na+/K+ ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.

Nogo receptor decoy promotes recovery and corticospinal growth in non-human primate spinal cord injury.

After CNS trauma such as spinal cord injury, the ability of surviving neural elements to sprout axons, reorganize neural networks and support recovery of function is severely restricted, contributing to chronic neurological deficits. Among limitations on neural recovery are myelin-associated inhibitors functioning as ligands for neuronal Nogo receptor 1 (NgR1). A soluble decoy (NgR1-Fc, AXER-204) blocks these ligands and provides a means to promote recovery of function in multiple preclinical rodent models of spinal cord injury. However, the safety and efficacy of this reagent in non-human primate spinal cord injury and its toxicological profile have not been described. Here, we provide evidence that chronic intrathecal and intravenous administration of NgR1-Fc to cynomolgus monkey and to rat are without evident toxicity at doses of 20 mg and greater every other day (≥2.0 mg/kg/day), and far greater than the projected human dose. Adult female African green monkeys underwent right C5/6 lateral hemisection with evidence of persistent disuse of the right forelimb during feeding and right hindlimb during locomotion. At 1 month post-injury, the animals were randomized to treatment with vehicle (n = 6) or 0.10-0.17 mg/kg/day of NgR1-Fc (n = 8) delivered via intrathecal lumbar catheter and osmotic minipump for 4 months. One animal was removed from the study because of surgical complications of the catheter, but no treatment-related adverse events were noted in either group. Animal behaviour was evaluated at 6-7 months post-injury, i.e. 1-2 months after treatment cessation. The use of the impaired forelimb during spontaneous feeding and the impaired hindlimb during locomotion were both significantly greater in the treatment group. Tissue collected at 7-12 months post-injury showed no significant differences in lesion size, fibrotic scar, gliosis or neuroinflammation between groups. Serotoninergic raphespinal fibres below the lesion showed no deficit, with equal density on the lesioned and intact side below the level of the injury in both groups. Corticospinal axons traced from biotin-dextran-amine injections in the left motor cortex were equally labelled across groups and reduced caudal to the injury. The NgR1-Fc group tissue exhibited a significant 2-3-fold increased corticospinal axon density in the cervical cord below the level of the injury relative to the vehicle group. The data show that NgR1-Fc does not have preclinical toxicological issues in healthy animals or safety concerns in spinal cord injury animals. Thus, it presents as a potential therapeutic for spinal cord injury with evidence for behavioural improvement and growth of injured pathways in non-human primate spinal cord injury.

Nogo receptor antagonist LOTUS exerts suppression on axonal growth-inhibiting receptor PIR-B.

Damaged axons in the adult mammalian central nervous system have a restricted regenerative capacity mainly because of Nogo protein, which is a major myelin-associated axonal growth inhibitor with binding to both receptors of Nogo receptor-1 (NgR1) and paired immunoglobulin-like receptor (PIR)-B. Lateral olfactory tract usher substance (LOTUS) exerts complete suppression of NgR1-mediated axonal growth inhibition by antagonizing NgR1. However, the regulation of PIR-B functions in neurons remains unknown. In this study, protein-protein interactions analyses found that LOTUS binds to PIR-B and abolishes Nogo-binding to PIR-B completely. Reverse transcription-polymerase chain reaction and immunocytochemistry revealed that PIR-B is expressed in dorsal root ganglions (DRGs) from wild-type and Ngr1-deficient mice (male and female). In these DRG neurons, Nogo induced growth cone collapse and neurite outgrowth inhibition, but treatment with the soluble form of LOTUS completely suppressed them. Moreover, Nogo-induced growth cone collapse and neurite outgrowth inhibition in Ngr1-deficient DRG neurons were neutralized by PIR-B function-blocking antibodies, indicating that these Nogo-induced phenomena were mediated by PIR-B. Our data show that LOTUS negatively regulates a PIR-B function. LOTUS thus exerts an antagonistic action on both receptors of NgR1 and PIR-B. This may lead to an improvement in the defective regeneration of axons following injury.

Relationship between NogoA/NgR1/RhoA signaling pathway and the apoptosis of cerebral neurons after cerebral infarction in rats.

OBJECTIVE: The aim of this study was to investigate the effect and the mechanism of the NogoA/NgR1/RhoA signaling pathway on the apoptosis of neurons in cerebral infarction (CI) rats. Our findings might provide references for clinical prevention and treatment of CI. MATERIALS AND METHODS: A total of 60 adult male Wistar rats were randomly divided into 3 groups, including: Sham operation group (Sham group), CI group, and CI + NogoA gene knockout group (CI + NogoA KO group) using a random number table. The model of CI was successfully constructed using suture method in rats of CI group and CI + NogoA KO group. Only blood vessels were exposed in Sham group. At 2 days after CI operation, the rats were killed, and brain tissues were collected. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting were used to detect the messenger ribonucleic acid (mRNA) and protein expression levels of NogoA/NgR1/RhoA in brain lesion tissues of rats in the three groups, respectively. Subsequently, the pathological damage of brain tissues was detected via hematoxylin and eosin (H&E) staining. TTC staining was carried out to evaluate the infarction area in each group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted to measure the apoptosis level of neurons in brain tissues of rats in each group. Additionally, the level of Nissl's body in brain tissues of each group was examined by Nissl staining. Furthermore, the expression level of the platelet-derived growth factor (PDGF) in brain tissues of rats in the three groups was measured via immunohistochemistry. RESULTS: The mRNA and protein expression levels of the NogoA/NgR1/RhoA signaling pathway in brain tissues of rats in CI group increased significantly (p<0.05). NogoA KO could significantly reduce the infarction area of brain tissues in rats (p<0.05). H&E staining and Nissl's body staining revealed that neurons in the brain tissues of rats showed evident edema and disordered arrangement after CI. Meanwhile, the number of Nissl's body was remarkably reduced. However, after KO of NogoA, brain tissue damage was significantly alleviated in rats, and the number of Nissl's body increased remarkably at the same time (p<0.05). According to TUNEL staining results, inhibiting NogoA could notably reverse CI-induced apoptosis of neurons in brain tissues of rats (p<0.05). Immunohistochemical staining results demonstrated that the expression of PDGF in brain tissues of rats in CI group decreased markedly, whereas was significantly elevated in rats of CI + NogoA KO group (p<0.05). CONCLUSIONS: The expression of the NogoA/NgR1/RhoA signaling pathway was significantly elevated in brain tissues of CI rats. Furthermore, suppressing the NogoA/NgR1/RhoA signaling pathway could reduce CI-induced apoptosis of neurons in rats.

Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone.

The promotion of axonal regeneration is required for functional recovery from stroke and various neuronal injuries. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. Nogo-66, a C-terminal domain of Nogo-A, binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibits neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors, suggesting it is an important target for the prevention of axonal growth inhibition. By using the indirect immunofluorescence method, we show for the first time that a cell-permeable cAMP analog (dibutyryl-cAMP) induced a rapid decrease in the cell surface expression of NgR1 in Neuroscreen-1 (NS-1) cells. The biotinylation method revealed that cAMP indeed induced internalization of NgR1 within minutes. Other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, also induced this process. This internalization was found to be reversible and influenced by intracellular levels of cAMP. Using selective activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA, but dependent on Epac. The decrease in cell surface expression of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, it is likely that besides axonal growth inhibitors affecting neurons, neurons themselves also self-regulate their sensitivity to axonal growth inhibitors, as influenced by intracellular cAMP/Epac. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors, and enhance functional recovery after stroke and neuronal injuries.