Molecular Characterization and Function of the Nogo-66 Receptor (NgR1) Gene in the Chinese Tree Shrew.

BACKGROUND: Nogo-66 receptor (NgR1) is a glycosylphosphatidylinositol-linked cell surface receptor with high affinity for Nogo-66. The binding of Nogo-66 to NgR1 plays a key role in inhibiting neurite growth, limiting synaptic plasticity and mediating Mammalian Reovirus (MRV) infection. The Chinese tree shrew (Tupaia belangeri chinensis) is, a new and valuable experimental animal that is widely used in biomedical research. Although susceptible to MRV, little is known about tree shrew NgR1 and its role in MRV infection. METHODS: In this study, we cloned NgR1 form the Chinese tree shrew by RACE technology and analyzed its characteristics, spatial structure and its tissue expression. We also examined the expression pattern of NgR1 in the response of tree shrew primary nerve cells (tNC) to MRV1/TS/2011 infection. RESULTS: Tree shrew NgR1 was found to have a closer relationship to human NgR1 (90.34%) than to mouse NgR1. Similar to the protein structure of human NgR1, the tree shrew NgR1 has the same leucine-rich repeat (LRR) domain structure that is capped by C-terminal and N-terminal cysteine-rich modules. The tree shrew NgR1 mRNAs were predominantly detected in the central nervous system (CNS), and tree shrew NgR1 can mediate infection by MRV1/TS/2011. CONCLUSIONS: Taken together, these results help to elucidate the function of NgR1 and provide a basis for using the tree shrew as an animal model for studies of the nervous system and infectious diseases.

NgR1 is an NK cell inhibitory receptor that destabilizes the immunological synapse.

The formation of an immunological synapse (IS) is essential for natural killer (NK) cells to eliminate target cells. Despite an advanced understanding of the characteristics of the IS and its formation processes, the mechanisms that regulate its stability via the cytoskeleton are unclear. Here, we show that Nogo receptor 1 (NgR1) has an important function in modulating NK cell-mediated killing by destabilization of IS formation. NgR1 deficiency or blockade resulted in improved tumor control of NK cells by enhancing NK-to-target cell contact stability and regulating F-actin dynamics during IS formation. Patients with tumors expressing abundant NgR1 ligand had poor prognosis despite high levels of NK cell infiltration. Thus, our study identifies NgR1 as an immune checkpoint in IS formation and indicates a potential approach to improve the cytolytic function of NK cells in cancer immunotherapy.

[Fasudil reduces apoptosis of SH-SY5Y cells induced by H2O2 and promotes synaptic plasticity by inhibiting neurite outgrowth inhibitor A and its receptor (NogoA/NgR) signaling pathway].

Objective To investigate the effect of Fasudil on H2O2-induced apoptosis and synaptic plasticity in human neuroblastoma SY5Y cells and its mechanism. Methods The cells were divided into three groups: PBS control group, H2O2 model group (250 µmol/L H2O2 treatment) and Fasudil intervention group (250 µmol/L H2O2 combined with 15 µg/mL Fasudil treatment). MTT assay was applied to detect cell activity and TUNEL was performed to detect cell apoptosis respectively. Immunofluorescence cytochemical staining was used to determine the expression of neurite outgrowth inhibitor A (NogoA), Nogo receptor (NgR) and synaptophysin (Syn). Western blotting was then conducted to detect the expression of NogoA, NgR, p75 neurotrophin receptor (p75NTR), leucine-rich repeat Ig domain-containing Nogo-interacting protein 1 (LINGO-1), Syn and postsynaptic density protein-95 (PSD-95). Results Compared with the PBS group, the H2O2 group showed decreased cell viability and increased apoptosis rate while Fasudil treatment significantly increased the cell viability and reduced the apoptosis rate. Compared with the H2O2 model group, Fasudil intervention increased expressions of Syn and PSD-95. Compared with the PBS group, the expression of NogoA and its receptor complex NgR/p75NTR/LINGO-1 grew significantly in the H2O2 group, suggesting Fasudil treatment could inhibit the expression of NogoA and its receptor complex NgR/p75NTR/LINGO-1. Conclusion Fasudil may inhibit the activation of the NogoA/NgR signaling pathway, therefore reducing the apoptosis induced by H2O2 in SH-SY5Y cells and enhancing the plasticity of the synapses.

Ameliorative effect of Gastrodia elata Blume extracts on depression in zebrafish and cellular models through modulating reticulon 4 receptors and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume (G. elata), a traditional Chinese herb, known as "Tian Ma", is widely used as a common medicine and diet ingredient for treating or preventing neurological disorders for thousands of years in China. However, the anti-depressant effect of G. elata and the underlying mechanism have not been fully evaluated. AIM OF THE STUDY: The study is aimed to investigate the anti-depressant effect and the molecular mechanism of G. elata in vitro and in vivo using PC12 cells and zebrafish model, respectively. MATERIAL AND METHODS: Network pharmacology was performed to explore the potential active ingredients and action targets of G. elata Blume extracts (GBE) against depression. The cell viability and proliferation were determined by MTT and EdU assay, respectively. TUNEL assay was used to examine the anti-apoptotic effect of GBE. Immunofluorescence and Western blot were used to detect the protein expression level. In addition, novel tank diving test was used to investigate the anti-depressant effect in zebrafish depression model. RT-PCR was used to analyze the mRNA expression levels of genes. RESULTS: G. elata against depression on the reticulon 4 receptors (RTN4R) and apoptosis-related targets, which were predicted by network pharmacology. Furthermore, GBE enhanced cell viability and inhibited the apoptosis in PC12 cells against CORT treatment. GBE relieved depression-like symptoms in adult zebrafish, included increase of exploratory behavior and regulation of depression related genes. Mechanism studies showed that the GBE inhibited the expression of RTN4R-related and apoptosis-related genes. CONCLUSION: Our studies show the ameliorative effect of G. elata against depression. The mechanism may be associated with the inhibition of RTN4R-related and apoptosis pathways.

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.

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.

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.

SATB2 and NGR1: potential upstream regulatory factors in uterine leiomyomas.

PURPOSE: We attempted to identify the genes involved in the pathogenesis of uterine leiomyomas, under a hypothesis that the aberrant expression of upstream regulatory genes caused by aberrant DNA methylation is involved in the onset and development of uterine leiomyomas. METHODS: To find such genes, we compared genome-wide mRNA expression and DNA methylation in uterine leiomyomas and adjacent normal myometrium. Analysis of the data by Ingenuity Pathway Analysis software identified SATB2 which is known to be an epigenetic regulator, and NRG1 as candidate upstream regulatory genes. To infer the functions of these genes, human uterine smooth muscle cell lines overexpressing SATB2 or NRG1 genes were established (SATB2 or NRG1 lines), and their transcriptomes and pathways were analyzed. RESULTS: SATB2 and NRG1 were confirmed to be hypermethylated and upregulated in most uterine leiomyoma specimens (nine to 11 of the 11 cases). Among the established cell lines, morphological changes from spindle-like forms to fibroblast-like forms with elongated protrusions were observed in only the SATB2 line. Pathway analysis revealed that WNT/ß-catenin and TGF-ß signaling pathways which are related to the pathogenesis of uterine leiomyomas were activated in both SATB2 and NRG1 lines. In addition, signaling of growth factors including VEGF, PDGF, and IGF1, and retinoic acid signaling were activated in the SATB2 and NRG1 lines, respectively. CONCLUSIONS: These results indicate that SATB2 and NRG1 overexpression induced many of the signaling pathways that are considered to be involved in the pathogenesis of uterine leiomyomas, suggesting that these genes have roles as upstream regulatory factors.

Nogo receptor-vimentin interaction: a novel mechanism for the invasive activity of glioblastoma multiforme.

Nogo receptor (NgR) has been shown to inhibit the migration and invasion of human glioma cells. However, little is known regarding the regulatory mechanisms of NgR in glioblastoma multiforme (GBM). In this study, we propose a novel mechanism that regulates the maturation process of NgR through an interaction with vimentin. The inhibition of TGFß1 activity by LY2109761 attenuated the migration/invasion of GBM cells by upregulating cell-surface NgR. Conversely, the treatment of GBM cells with TGFß1 suppressed NgR maturation. We showed that NgR and vimentin interact, which could be a possible mechanism for the suppression of NgR maturation. The knockdown of vimentin suppressed the migration/invasion of GBM cells through the increased maturation of NgR. Finally, TCGA (The Cancer Genome Atlas) analysis also supported the association of NgR and vimentin. The maturation of NgR is regulated by the interaction of vimentin and NgR, which attenuates the invasive activity of GBM, and might be a potential therapeutic target for brain cancer.

Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination.

We previously identified that ngr1 allele deletion limits the severity of experimental autoimmune encephalomyelitis (EAE) by preserving axonal integrity. However, whether this favorable outcome observed in EAE is a consequence of an abrogated neuronal-specific pathophysiological mechanism, is yet to be defined. Here we show that, Cre-loxP-mediated neuron-specific deletion of ngr1 preserved axonal integrity, whereas its re-expression in ngr1-/- female mice potentiated EAE-axonopathy. As a corollary, myelin integrity was preserved under Cre deletion in ngr1flx/flx , retinal ganglion cell axons whereas, significant demyelination occurred in the ngr1-/- optic nerves following the re-introduction of NgR1. Moreover, Cre-loxP-mediated axon-specific deletion of ngr1 in ngr1flx/flx mice also demonstrated efficient anterograde transport of fluorescently-labeled ChTxß in the optic nerves of EAE-induced mice. However, the anterograde transport of ChTxß displayed accumulation in optic nerve degenerative axons of EAE-induced ngr1-/- mice, when NgR1 was reintroduced but was shown to be transported efficiently in the contralateral non- recombinant adeno-associated virus serotype 2-transduced optic nerves of these mutant mice. We further identified that the interaction between the axonal motor protein, Kinesin-1 and collapsin response mediator protein 2 (CRMP2) was unchanged upon Cre deletion of ngr1 Whereas, this Kinesin-1/CRMP2 association was reduced when NgR1 was re-expressed in the ngr1-/- optic nerves. Our data suggest that NgR1 governs axonal degeneration in the context of inflammatory-mediated demyelination through the phosphorylation of CRMP2 by stalling axonal vesicular transport. Moreover, axon-specific deletion of ngr1 preserves axonal transport mechanisms, blunting the induction of inflammatory demyelination and limiting the severity of EAE.SIGNIFICANCE STATEMENT Multiple sclerosis (MS) is commonly induced by aberrant immune-mediated destruction of the protective sheath of nerve fibers (known as myelin). However, it has been shown that MS lesions do not only consist of this disease pattern, exhibiting heterogeneity with continual destruction of axons. Here we investigate how neuronal NgR1 can drive inflammatory-mediated axonal degeneration and demyelination within the optic nerve by analyzing its downstream signaling events that govern axonal vesicular transport. We identify that abrogating the NgR1/pCRMP2 signaling cascade can maintain Kinesin-1-dependent anterograde axonal transport to limit inflammatory-mediated axonopathy and demyelination. The ability to differentiate between primary and secondary mechanisms of axonal degeneration may uncover therapeutic strategies to limit axonal damage and progressive MS.

The Nogo Receptor Ligand LGI1 Regulates Synapse Number and Synaptic Activity in Hippocampal and Cortical Neurons.

Leucine-rich glioma-inactivated protein 1 (LGI1) is a secreted neuronal protein and a Nogo receptor 1 (NgR1) ligand. Mutations in LGI1 in humans causes autosomal dominant lateral temporal lobe epilepsy and homozygous deletion of LGI1 in mice results in severe epileptic seizures that cause early postnatal death. NgR1 plays an important role in the development of CNS synapses and circuitry by limiting plasticity in the adult cortex via the activation of RhoA. These relationships and functions prompted us to examine the effect of LGI1 on synapse formation in vitro and in vivo. We report that application of LGI1 increases synaptic density in neuronal culture and that LGI1 null hippocampus has fewer dendritic mushroom spines than in wild-type (WT) littermates. Further, our electrophysiological investigations demonstrate that LGI1 null hippocampal neurons possess fewer and weaker synapses. RhoA activity is significantly increased in cortical cultures derived from LGI1 null mice and using a reconstituted system; we show directly that LGI1 antagonizes NgR1-tumor necrosis factor receptor orphan Y (TROY) signaling. Our data suggests that LGI1 enhances synapse formation in cortical and hippocampal neurons by reducing NgR1 signaling.

The soluble form of LOTUS inhibits Nogo receptor type 1-mediated signaling induced by B lymphocyte stimulator and chondroitin sulfate proteoglycans.

There are global efforts in developing therapeutic strategies for central nervous system (CNS) injuries using multimodal approaches. Nogo receptor type 1 (NgR1) has been known as a primary molecule limiting neuronal regeneration in the adult CNS. We identified lateral olfactory tract usher substance (LOTUS) as an endogenous NgR1 antagonist. Membrane-bound LOTUS interacts with NgR1 and inhibits its function by blocking its ligand binding. Five molecules including Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), B lymphocyte stimulator (BLyS) and chondroitin sulfate proteoglycans (CSPGs) have been identified as NgR1 ligands. These ligands bind to NgR1 and activate NgR1 signaling, leading to axon growth inhibition such as growth cone collapse and neurite outgrowth inhibition. We have recently reported that the soluble form of LOTUS (s-LOTUS) also suppressed NgR1-mediated signaling induced by myelin axonal inhibitors (MAIs) including Nogo, MAG and OMgp by binding with both NgR1 and its co-receptor p75 neurotrophin receptor (p75NTR). Though s-LOTUS has been reported to suppress MAIs, whether s-LOTUS also suppresses NgR1 signaling induced by BLyS and CSPGs remains to be elucidated. Here, we show that s-LOTUS inhibits NgR1-mediated signaling induced by BLyS and CSPGs. Although treatment with s-LOTUS did not suppress BLyS-NgR1 interaction, s-LOTUS inhibited growth cone collapse and neurite outgrowth inhibition induced by BLyS and CSPGs in chick dorsal root ganglion (DRG) neurons. Furthermore, s-LOTUS compensated for the suppressive function of endogenous LOTUS in NgR1-mediated signaling in olfactory bulb neurons of lotus-knockout mice. These findings suggest that s-LOTUS is a potent therapeutic agent for neuronal regeneration in the CNS injuries.

Axonal Guidance Signaling Pathway Is Suppressed in Human Nasal Polyps.

Background Dysfunctional innervation might contribute to the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP), but the state of the axonal outgrowth signaling in CRSwNP is unknown. The purpose of this study was to explore the axonal outgrowth pathway-related protein expression in CRSwNP. Methods Institutional review board approved study in which tissue proteomes were compared between control and CRSwNP patients (n = 10/group) using an aptamer-based proteomic array and confirmed by whole transcriptomic analysis. Results Compared with controls, proteins associated with axonal guidance signaling pathway such as beta-nerve growth factor, semaphorin 3A, Ras-related C3 botulinum toxin substrate 1, Bcl-2, protein kinase C delta type, and Fyn were significantly decreased in patients with CRSwNP (fold change [FC] = -1.17, P = .002; FC = -1.09, P < .001; FC = -1.33, P < .001; FC = -1.31, P < .001; FC = -1.31, P = .004; and FC = -1.20, P = 0.012, respectively). In contrast, reticulon-4 receptor, an inhibitory factor, was significantly increased in patients with CRSwNP (FC = 1.25, P < .001). Furthermore, neuronal growth-associated proteins such as ciliary neurotrophic factor receptor subunit alpha, neuronal growth regulator 1, neuronal cell adhesion molecule, neural cell adhesion molecule L1, platelet-derived growth factor subunit A, and netrin-4 were all significantly decreased in patients with CRSwNP (FC = -1.25, P < .001; FC = -1.27, P = .002; FC = -1.65, P = .013; FC = -4.20, P < .001; FC = -1.28, P < .001; and FC = -2.31, P < .001, respectively). In contrast, tissue eosinophil count ( P < .001) and allergic inflammation factors such as IgE, periostin, and galectin-10 were all significantly increased in patients with CRSwNP (FC = 12.28, P < .001; FC = 3.95, P < .001; and FC = 2.44, P < .001, respectively). Furthermore, the log FC of the studied proteins expression significantly and positively correlated with log FC of their mRNA expression ( P < .001, r = .88). Conclusions Axonal guidance signaling and neural growth factors pathways proteins are significantly suppressed in eosinophilic CRSwNP.

The nociceptin receptor inhibits axonal regeneration and recovery from spinal cord injury.

Axonal growth after traumatic spinal cord injury is limited by endogenous inhibitors, selective blockade of which promotes partial neurological recovery. The partial repair phenotypes suggest that compensatory pathways limit improvement. Gene expression profiles of mice deficient in Ngr1, which encodes a receptor for myelin-associated inhibitors of axonal regeneration such as Nogo, revealed that trauma increased the mRNA expression of ORL1, which encodes the receptor for the opioid-related peptide nociceptin. Endogenous and overexpressed ORL1 coimmunoprecipitated with immature NgR1 protein, and ORL1 enhanced the O-linked glycosylation and surface expression of NgR1 in HEK293T and Neuro2A cells and primary neurons. ORL1 overexpression inhibited cortical neuron axon regeneration independently of NgR1. Furthermore, regeneration was inhibited by an ORL1 agonist and enhanced by the ORL1 antagonist J113397 through a ROCK-dependent mechanism. Mice treated with J113397 after dorsal hemisection of the mid-thoracic spinal cord recovered greater locomotor function and exhibited lumbar raphespinal axon sprouting. These effects were further enhanced by combined Ngr1 deletion and ORL1 inhibition. Thus, ORL1 limits neural repair directly and indirectly by enhancing NgR1 maturation, and ORL1 antagonists enhance recovery from traumatic CNS injuries in wild-type and Ngr1 null mice.

The Adiponectin Homolog Osmotin Enhances Neurite Outgrowth and Synaptic Complexity via AdipoR1/NgR1 Signaling in Alzheimer's Disease.

Alzheimer's disease is a major neurodegenerative disease characterized by memory loss and cognitive deficits. Recently, we reported that osmotin, which is a homolog of adiponectin, improved long-term potentiation and cognitive functions in Alzheimer's disease mice. Several lines of evidence have suggested that Nogo-A and the Nogo-66 receptor 1 (NgR1), which form a complex that inhibits long-term potentiation and cognitive function, might be associated with the adiponectin receptor 1 (AdipoR1), which is a receptor for osmotin. Here, we explore whether osmotin's effects on long-term potentiation and memory function are associated with NgR1 signaling via AdipoR1 in Alzheimer's disease. Osmotin reduced the expression of NgR1 without affecting Nogo-A expression. Furthermore, osmotin inhibited NgR1 signaling by prohibiting the formation of the Nogo-A and NgR1 ligand-receptor complex, resulting in enhanced neurite outgrowth; these effects disappeared in the presence of AdipoR1 interference. In addition, osmotin increased the expression of the pre- and postsynaptic markers synaptophysin and PSD-95, as well as the activation of the memory-associated markers AMPA receptor and CREB; these effects occurred in an AdipoR1- and NgR1-dependent manner. Osmotin was also found to enhance dendritic complexity and spine density in the hippocampal region of Alzheimer's disease mouse brains. These results suggest that osmotin can enhance neurite outgrowth and synaptic complexity through AdipoR1 and NgR1 signaling, implying that osmotin might be an effective therapeutic agent for Alzheimer's disease and that AdipoR1 might be a crucial therapeutic target for neurodegenerative diseases such as Alzheimer's.

PirB inhibits axonal outgrowth via the PI3K/Akt/mTOR signaling pathway.

Accumulating data strongly suggests that leukocyte immunoglobulin like receptor B1 (PirB) inhibits axonal outgrowth. However, the underlying mechanisms remain unclear. In the present study, cortical neurons of newborn mice were cultured with Nogo­66 (Nogo­p4; 4 µmol/l; a PirB ligand) together with NEP1­40 (Nogo inhibitory peptide) and/or anti­PirB body (50 mg/ml). PirB mRNA and protein was higher in cultured neurons induced by Nogo­66 compared with untreated cells. Neurite outgrowth assays demonstrated that the inhibitory effects of Nogo­66 on axonal outgrowth were reversed by anti­PirB body. Reverse transcription­quantitative polymerase chain reaction and western blot assays demonstrated that anti­PirB treatment led to reduced mRNA and protein expression of phosphoinositide 3­kinase (PI3K), Akt serine/threonine kinase (Akt), mechanistic target of rapamycin kinase (mTOR), myosin IIA and cofilin, which are involved in axonal outgrowth. Furthermore, blockade of the PI3K/Akt/mTOR pathway using a PI3K inhibitor or an mTOR inhibitor diminished the stimulatory effect of anti­PirB on axonal outgrowth, and the reduced effect of anti­PirB on factors that were activation by anti­PirB. In addition, blockade of PI3K/Akt/mTOR enhanced anti­PirB­induced gene and protein expression. These results revealed that PirB functions as a potential suppressor in axonal outgrowth via repressing PI3K/Akt/mTOR signaling pathway, and PirB/PI3K/Akt/mTOR may be a novel target for enhancing axonal outgrowth for developing rational therapeutic strategies.

Resistance of interleukin-6 to the extracellular inhibitory environment promotes axonal regeneration and functional recovery following spinal cord injury.

Interleukin-6 (IL)-6 was originally discovered as a factor that contributes to the secondary pathological and inflammatory response in the central nervous system (CNS) following injury. However, accumulating evidence suggests that IL-6 is also involved in functional and structural recovery following CNS injury by promoting axonal sprou-ting. This suggests a potential dual role of IL-6 in CNS injury. However, the definitive function of IL-6 in neural injury and the corresponding underlying mechanisms are still topics of controversy. The present study was carried out to examine the potential function of IL-6 in resistance to neurite growth­inhibitory effects via regulation of the expression of growth associated protein-43 (GAP-43), myelin-associated neurite outgrowth inhibitor (Nogo-A) and its receptor (NgR). Rat dorsal root ganglion (DRG) neurons cultured in an inhibitory microenvironment mimicking injured CNS were used to investigate the effects of IL-6 on the outgrowth of neuronal processes. Additionally, IL-6 was subarachnoidally injected into rats to establish a spinal cord injury (SCI) model, and the neurobehavioral manifestations and neural morphology were subsequently evaluated to determine the effect of IL-6 on neural regeneration. Finally, the potential molecular mechanisms of IL-6-mediated rege-neration and functional recovery following CNS injury are discussed. The results of the present study demonstrated that the in vitro administration of IL-6 enhanced the neurite outgrowth of DRG neurons in a dose-dependent manner via resisting the inhibitory function of myelin proteins. All doses of the IL-6 subarachnoid injection improved the Basso, Beattie and Bresnahan scores following SCI, with a large number of axonal sproutings observed at the spinal lesion site, and several sprouting fibers being elongated and bypassing the lesion and entered the caudal spinal cord. Additionally, a significantly increased density area of diaminobenzidine-labeled neural fiber was observed in rats that received a subarachnoid injection of IL-6, and the rats exhibited increased expression of GAP-43 and decreased expression of Nogo-A. In conclusion, the results of the present study indicated that IL-6 interferes with the inhibitory functions of myelin proteins by upregulating the expression of GAP-43 and simultaneously downregulating the expression of Nogo-A and NgR to promote axonal sprouting and functional recovery following SCI.

Panax notoginseng saponins provide neuroprotection by regulating NgR1/RhoA/ROCK2 pathway expression, in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng saponins (PNS) extracted from a traditional Chinese herbal medicine, Panax notoginseng (Burkill) F.H. Chen (Araliaceae), which has been extensively used in treating coronary heart disease, ischemic cerebrovascular disease and hemorrhagic disorders in China over hundreds of years. AIMS OF THE STUDY: This study explored whether panax notoginseng saponins (PNS) provided neuroprotective effects by inhibiting the expressions of NgR1, RhoA, and ROCK2 following middle cerebral artery occlusion in rats and oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SH-SY5Y cells. MATERIALS AND METHODS: 2,3,5-Triphenyltetrazolium chloride staining was used to determine successful middle cerebral artery occlusion establishment in sham-operated and operated Sprague-Dawley rats 1 day after injury. The rats were randomly separated into sham, model, NEP1-40, PNS, and NEP1-40 plus PNS (N+P) groups. After 7 days of treatment, body mass and neurological deficit scores were analyzed. Tissues were harvested and analyzed by hematoxylin-eosin staining and immunohistochemical analysis, western blotting, and quantitative real-time PCR (qRT-PCR). The optimal drug concentration of NEP1-40 and PNS on SH-SY5Y cells exposed to OGD/R injury was determined by CCK8 analysis. qRT-PCR was used to measure mRNA expression profiles of NgR1, RhoA, and ROCK2 in SH-SY5Y cells subjected to OGD/R. RESULTS: The results showed that MCAO surgery successfully produced an infarct, and the PNS, NEP1-40, and N+P groups exhibited increased body mass and ameliorated neurological deficits compared with the model group. NEP1-40 treatment markedly reduced NgR1 and RhoA overexpression when compared to the model group, although there was no significant difference in ROCK2 expression. PNS and N+P treatment significantly decreased NgR1, RhoA, and ROCK2 overexpression compared with the model group. However, N+P treatment did not result in a synergistic effect, as assessed by immunohistochemistry, western blotting, and qRT-PCR. Following optimal administration of PNS (160µg/ml) and NEP1-40 (10ng/ml) on SH-SY5Y cells exposed to OGD/R injury, cell viability in the NEP1-40, PNS, and N+P groups significantly increased compared with the model group, as assessed by CCK8 analysis. Additionally, NgR1, RhoA, and ROCK2 mRNA expression profiles were significantly less in the NEP1-40, PNS, and N+P groups compared with the model group. CONCLUSION: PNS provided neuroprotective effects in a rat model of cerebral ischemia and SH-SY5Y cells exposed to oxygen/glucose deprivation injury by inhibiting the overexpression of NgR1, RhoA, and ROCK2.