Nogo Receptor Research Articles

Assessment of Nogo-66 Receptor 1 Function In Vivo After Spinal Cord Injury

Neuronal Nogo-66 receptor 1 (NgR1) has attracted attention as a converging point for mediating the effects of myelin-associate inhibitory ligands in the central nervous system, establishing the growth-restrictive environment, and limiting axon regeneration after traumatic injury. To investigate the factors that may be contributing to the discrepancy in the importance of NgR1, which has been undermined by several studies that have shown the lack of substantial axon regeneration after spinal cord injury (SCI) in NgR1-knockout or -knockdown animal models. We used mice carrying either a homozygous or heterozygous null mutation in the NgR1 gene and subjected them to either a moderate or severe SCI. Locomotor function assessments revealed that the level of functional recovery is affected by the degree of injury suffered. NgR1 ablation enhanced local collateral sprouting in the mutant mice. Reactive astrocytes and chondroitin sulfate proteoglycans (CSPGs) are upregulated surrounding the injury site. Matrix metalloproteinase-9, which has been shown to degrade CSPGs, was significantly upregulated in the homozygous mutant mice compared with the heterozygous or wild-type mice. However, CSPG levels remained higher in the homozygous compared with the heterozygous mice, suggesting that CSPG-degrading activity of matrix metalloproteinase-9 may require the presence of NgR1. Genetic ablation of NgR1 may lead to significant recovery in locomotor function after SCI. The difference in locomotor recovery we observed between the groups that suffered various degrees of injury suggests that injury severity may be a confounding factor in functional recovery after SCI.

The P75 neurotrophin receptor regulates proliferation of the human MG63 osteoblast cell line

The 75kDa transmembrane protein, p75NTR, is a marker of mesenchymal stem cells (MSCs). Isolated MSCs are capable of differentiating into osteoblasts, but the molecular function of p75NTR in MSCs and osteoblasts is poorly understood. The aim of this study was to examine the function of p75NTR in the human MG63 osteoblast cell line compared to the murine MC3T3E-1 pre-osteoblast cell line.MG63 cells and MC3T3-E1 cells expressing exogenous p75NTR protein (denoted as p75-MG63 and p75GFP-E1, respectively) were generated to compare osteogenic differentiation and cell proliferation abilities. Overexpression of p75NTR induced alkaline phosphatase activity and the mRNA expression of osteoblast-related genes such as osterix and bone sialoprotein in both p75-MG63 and p75GFP-E1. Interestingly, exogenous p75NTR stimulated cell proliferation and cell cycle progression in p75GFP-E1, but not in p75-MG63.To elucidate any different effects of p75NTR expression on osteogenic differentiation and cell proliferation, we examined the mRNA expression of tropomyosin receptor kinase (trk) genes (trkA, trkB, trkC) and Nogo receptor (NgR), which are binding partners of p75NTR. Although trkA, trkB, and trkC were detected in both p75-MG63 and p75GFP-E1, only NgR was detected in p75-MG63. We then used the K252a inhibitor of the trks to identify the signaling pathway for osteogenic differentiation and cell proliferation. Inhibition of trks by K252a suppressed p75NTR-mediated osteogenic differentiation of p75GFP-E1, whereas deletion of the GDI domain in P75NTR from the p75-MG63 produced enhanced cell proliferation compared to p75-MG63. These results suggest that p75NTR signaling associated with trk receptors promotes both cell proliferation and osteoblast differentiation, but that p75NTR-mediated proliferation may be suppressed by signaling from the p75NTR/NgR complex.

The mechanism of the NgR1 antagonist for promoting the neural precursor cells differentiate into neurons

Objective To study the effect of neuronal Nogo-66 receptor (NgR1) antagonist,soluble Nogo-66 receptor (sNgR1-Fc),on promoting the endogenous neural precursor cells (NPCs) differentiating into neurons in order to clarify the mechanism.Methods The cortical infarction was induced by photochemistry,named photothrombotic cortical injury (PCI).Twelve Sprague Dawley rats were randomly divided (random number) into three groups:Sham-operated group,PBS group,and sNgR1-Fc group.PBS (PBS group) or sNgR1-Fc (sNgR1-Fc group) was injected into the lateral ventricle of brain with a minipump.BrdU (Bromodeoxyuridine) was injected into the peritoneal cavity 4-6 days after PCI.The subdentate gyrus zone (SGZ) of brain from sacrificed rat was harvested for Immunohistochemistry to observe the ratio of NeuN +/BrdU + cells 35 days after PCI.Proteins including Nestin、Notch1 and Mash1 were detected by Western Blot.Results The cortical infarction in rat was successfully induced by photochemistry.Thirty-five days after PCI,the BrdU + cells number and theratio of NeuN +/BrdU + in the SGZ of the ipsilateral cerebrum hemisphere with PCI were significantly higher in sNgR1-Fc group than those in PBS group (P ＜ 0.05).The levels of Notch1,Mash1 and Neuro D in the sNgR1-Fc group were significantly higher than those in the PBS group (P ＜ 0.05),which were significantly higher than those in the Sham-operated group.Conclusions sNgR1-Fc could promote the endogenous NPCs differentiating into neurons in a cortical infarction model.The mechanisms may be attributed to the Notch/bHLH (proneural basic helix-loop-helix genes) signaling way. Key words: Nogo-66 receptor; Cortical infarction; Neural precursor cells; Differentiation; Proliferation; Axon ; Regeneration; Signal pathway

Nogo Receptor Homolog NgR2 Expressed in Sensory DRG Neurons Controls Epidermal Innervation by Interaction with Versican

Primary sensory afferents of the dorsal root ganglion (DRG) that innervate the skin detect a wide range of stimuli, such as touch, temperature, pain, and itch. Different functional classes of nociceptors project their axons to distinct target zones within the developing skin, but the molecular mechanisms that regulate target innervation are less clear. Here we report that the Nogo66 receptor homolog NgR2 is essential for proper cutaneous innervation. NgR2(-/-) mice display increased density of nonpeptidergic nociceptors in the footpad and exhibit enhanced sensitivity to mechanical force and innocuous cold temperatures. These sensory deficits are not associated with any abnormality in morphology or density of DRG neurons. However, deletion of NgR2 renders nociceptive nonpeptidergic sensory neurons insensitive to the outgrowth repulsive activity of skin-derived Versican. Biochemical evidence shows that NgR2 specifically interacts with the G3 domain of Versican. The data suggest that Versican/NgR2 signaling at the dermo-epidermal junction acts in vivo as a local suppressor of axonal plasticity to control proper density of epidermal sensory fiber innervation. Our findings not only reveal the existence of a novel and unsuspected mechanism regulating epidermal target innervation, but also provide the first evidence for a physiological role of NgR2 in the peripheral nervous system.

The Nogo-C2/Nogo Receptor Complex Regulates the Morphogenesis of Zebrafish Lateral Line Primordium through Modulating the Expression of dkk1b, a Wnt Signal Inhibitor

The fish lateral line (LL) is a mechanosensory system closely related to the hearing system of higher vertebrates, and it is composed of several neuromasts located on the surface of the fish. These neuromasts can detect changes in external water flow, to assist fish in maintaining a stationary position in a stream. In the present study, we identified a novel function of Nogo/Nogo receptor signaling in the formation of zebrafish neuromasts. Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors, as well as three co-receptors (TROY, p75, and LINGO-1). We first demonstrated that Nogo-C2, NgRH1a, p75, and TROY are able to form a Nogo-C2 complex, and that disintegration of this complex causes defective neuromast formation in zebrafish. Time-lapse recording of the CldnB::lynEGFP transgenic line revealed that functional obstruction of the Nogo-C2 complex causes disordered morphogenesis, and reduces rosette formation in the posterior LL (PLL) primordium during migration. Consistent with these findings, hair-cell progenitors were lost from the PLL primordium in p75, TROY, and Nogo-C2/NgRH1a morphants. Notably, the expression levels of pea3, a downstream marker of Fgf signaling, and dkk1b, a Wnt signaling inhibitor, were both decreased in p75, TROY, and Nogo-C2/NgRH1a morphants; moreover, dkk1b mRNA injection could rescue the defects in neuromast formation resulting from knockdown of p75 or TROY. We thus suggest that a novel Nogo-C2 complex, consisting of Nogo-C2, NgRH1a, p75, and TROY, regulates Fgf signaling and dkk1b expression, thereby ensuring stable organization of the PLL primordium.

Altered expression of myelin-associated inhibitors and their receptors after traumatic brain injury in the mouse.

When central nervous system axons are injured, regeneration is partly inhibited by myelin-associated inhibitors (MAIs). Following traumatic brain injury (TBI) in the rat, pharmacological neutralisation of the MAIs Nogo-A and myelin-associated glycoprotein (MAG) resulted in improved functional outcome. In contrast, genetic or pharmacological neutralization of the MAI receptors Nogo-66 receptor 1 (NgR1) or paired-immunoglobulin like receptor-B (PirB) showed an unaltered or impaired outcome following TBI in mice. The aim of the present study was thus to evaluate the MAI expression levels following TBI in mice. Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure total RNA isolated from brains of young adult male C57BL/6 mice at one, three or seven days following controlled cortical impact TBI or sham injury. Hippocampal and neocortical tissue ipsi- and contralateral to the injury was analyzed for Nogo-A, oligodendrocyte-myelin glycoprotein (OMgp), MAG, and the MAI receptors PirB and NgR1, including its co-receptor Lingo1. Compared to sham-injured controls, PirB neocortical expression was significantly upregulated at one day and NgR1 expression downregulated at seven days post-TBI. In the hippocampus, transcriptional upregulation was observed in Nogo-A (one day post-injury), MAG and PirB at seven days post-injury. In contrast, the hippocampal transcripts of NgR1 and Lingo1 were decreased at seven days post-injury. The expression of OMgp was unaltered at all time points post-injury. These results suggest that early dynamic changes in MAI gene expression occur following TBI in the mouse, particularly in the hippocampus, which may play an inhibitory role for post-injury regeneration and plasticity.

Novel implications of Lingo-1 and its signaling partners in schizophrenia

Myelination and neurite outgrowth both occur during brain development, and their disturbance has been previously been implicated in the pathophysiology of schizophrenia. Leucine-rich repeat and immunoglobulin domain-containing protein (Lingo-1) is a potent negative regulator of axonal myelination and neurite extension. As co-factors of Lingo-1 signaling (Nogo receptor (NgR), With No Lysine (K) (WNK1) and Myelin transcription factor 1 (Myt1)) have been implicated in the genetics of schizophrenia, we explored for the first time the role of Lingo-1 signaling pathways in this disorder. Lingo-1 protein, together with its co-receptor and co-factor proteins NgR, tumor necrosis factor (TNF) receptor orphan Y (TROY), p75, WNK1 and Myt1, have never been explored in the pathogenesis of schizophrenia. We examined protein levels of Lingo-1, NgR, TROY, p75, WNK1, Myt1 and myelin basic protein (MBP) (as a marker of myelination) within the post-mortem dorsolateral prefrontal cortex (DLPFC) (37 schizophrenia patients versus 37 matched controls) and hippocampus (Cornu Ammonis, CA1 and CA3) (20 schizophrenia patients versus 20 matched controls from the same cohort). Both of these brain regions are highly disrupted in the schizophrenia pathophysiology. There were significant increases in Lingo-1 (P<0.001) and Myt1 (P=0.023) and a reduction in NgR (P<0.001) in the DLPFC in schizophrenia subjects compared with controls. There were also increases in both TROY (P=0.001) and WNK1 (P=0.011) in the CA1 of schizophrenia subjects and, in contrast to the DLPFC, there was an increase in NgR (P=0.006) in the CA3 of schizophrenia subjects compared with controls. No significant difference was reported for MBP levels (P>0.05) between the schizophrenia and control groups in the three tested regions. This is the first time that a study has shown altered Lingo-1 signaling in the schizophrenia brain. Our novel findings may present a direct application for the use of a Lingo-1 antagonist to complement current and future schizophrenia therapies.

Upregulation of Nogo receptor expression induces apoptosis of retinal ganglion cells in diabetic rats.

The Nogo receptor is an essential factor for neuronal apoptosis, but the changes in Nogo receptor expression in the retina and the effects of the Nogo receptor on retinal ganglion cell apoptosis in diabetes mellitus remain unclear. We found that Nogo receptor expression was mainly visible in retinal ganglion cells of a rat model of diabetes mellitus induced by streptozotocin. At 12 weeks after onset of diabetes mellitus, Nogo receptor and Rho kinase expression significantly increased in the retina, and retinal ganglion cell apoptosis was apparent. When RNA interference was used to suppress Nogo receptor expression in rat retina, Rho kinase expression was obviously inhibited, and retinal ganglion cell apoptosis was evidently reduced in rats with diabetes mellitus. These results indicate that upregulation of Nogo receptor expression is an important mechanism of retinal ganglion cell apoptosis in rats with diabetes mellitus.

Mild hypothermia combined with a scaffold of NgR-silenced neural stem cells/Schwann cells to treat spinal cord injury.

Because the inhibition of Nogo proteins can promote neurite growth and nerve cell differentiation, a cell-scaffold complex seeded with Nogo receptor (NgR)-silenced neural stem cells and Schwann cells may be able to improve the microenvironment for spinal cord injury repair. Previous studies have found that mild hypothermia helps to attenuate secondary damage in the spinal cord and exerts a neuroprotective effect. Here, we constructed a cell-scaffold complex consisting of a poly(D,L-lactide-co-glycolic acid) (PLGA) scaffold seeded with NgR-silenced neural stem cells and Schwann cells, and determined the effects of mild hypothermia combined with the cell-scaffold complexes on the spinal cord hemi-transection injury in the T9 segment in rats. Compared with the PLGA group and the NgR-silencing cells + PLGA group, hindlimb motor function and nerve electrophysiological function were clearly improved, pathological changes in the injured spinal cord were attenuated, and the number of surviving cells and nerve fibers were increased in the group treated with the NgR-silenced cell scaffold + mild hypothermia at 34°C for 6 hours. Furthermore, fewer pathological changes to the injured spinal cord and more surviving cells and nerve fibers were found after mild hypothermia therapy than in injuries not treated with mild hypothermia. These experimental results indicate that mild hypothermia combined with NgR gene-silenced cells in a PLGA scaffold may be an effective therapy for treating spinal cord injury.

Effects of curcumin on hippocampal expression of NgR and axonal regeneration in Aβ-induced cognitive disorder rats.

Curcumin has been widely used for the prevention and treatment of Alzheimer's disease (AD), but its mechanism is still not clear. Inhibitory factors of axonal regeneration have been shown to cause a series of pathophysiological changes in the early period of AD. In this study, the co-receptor (Nogo receptor; NgR) of three axonal growth-inhibitory proteins was examined, and effects of curcumin on spatial learning and memory abilities and hippocampal axonal growth were investigated in amyloid β-protein (Aβ)1-40-induced AD rats. Results showed that the expression of NgR in the AD group significantly increased and the number of axonal protein-positive fibers significantly reduced. The spatial learning and memory abilities of AD rats were significantly improved in the curcumin group. Furthermore, hippocampal expressions of NgR mRNA and protein decreased, and the expression of axonal protein significantly increased. There was a negative correlation between the expression of NgR and axonal growth. Together, these results suggested that curcumin could improve the spatial learning and memory abilities of AD rats. The mechanism might be related with its lowering of hippocampal NgR expression and promoting axonal regeneration.

Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury.

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was significantly enhanced in the model group. After 8 weeks, the number of horseradish peroxidase-labeled nerve fibers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and significantly higher than in the model group. The newly formed nerve fibers and myelinated nerve fibers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

Loss of Nogo receptor homolog NgR2 alters spine morphology of CA1 neurons and emotionality in adult mice.

Molecular mechanisms which stabilize dendrites and dendritic spines are essential for regulation of neuronal plasticity in development and adulthood. The class of Nogo receptor proteins, which are critical for restricting neurite outgrowth inhibition signaling, have been shown to have roles in developmental, experience and activity induced plasticity. Here we investigated the role of the Nogo receptor homolog NgR2 in structural plasticity in a transgenic null mutant for NgR2. Using Golgi-Cox staining to analyze morphology, we show that loss of NgR2 alters spine morphology in adult CA1 pyramidal neurons of the hippocampus, significantly increasing mushroom-type spines, without altering dendritic tree complexity. Furthermore, this shift is specific to apical dendrites in distal CA1 stratum radiatum (SR). Behavioral alterations in NgR2−/− mice were investigated using a battery of standardized tests and showed that whilst there were no alterations in learning and memory in NgR2−/− mice compared to littermate controls, NgR2−/− displayed reduced fear expression in the contextual conditioned fear test, and exhibited reduced anxiety- and depression-related behaviors. This suggests that the loss of NgR2 results in a specific phenotype of reduced emotionality. We conclude that NgR2 has role in maintenance of mature spines and may also regulate fear and anxiety-like behaviors.

The experimental therapy on brain ischemia by improvement of local angiogenesis with tissue engineering in the mouse.

Neural restoration has proven to be difficult after brain stroke, especially in its chronic stage. This is mainly due to the generation of an unpropitious niche in the injured area, including loss of vascular support but production of numerous inhibitors against neuronal regeneration. Reconstruction of a proper niche for promoting local angiogenesis, therefore, should be a key approach for neural restoration after stroke. In the present study, a new biomaterial composite that could be implanted in the injured area of the brain was created for experimental therapy of brain ischemia in the mouse. This composite was made using a hyaluronic acid (HA)-based biodegradable hydrogel scaffold, mixed with poly(lactic-co-glycolic acid) (PLGA) microspheres containing vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1), two factors that stimulate angiogenesis. In addition, the antibody of Nogo receptor (NgR-Ab), which can bind to multiple inhibitory myelin proteins and promote neural regeneration, was covalently attached to the hydrogel, making the hydrogel more bioactive and suitable for neural survival. This composite (HA-PLGA) was implanted into the mouse model with middle cerebral artery occlusion (MCAO) to explore a new approach for restoration of brain function after ischemia. A good survival and proliferation of human umbilical artery endothelial cells (HUAECs) and neural stem cells (NSCs) were seen on the HA hydrogel with PLGA microspheres in vitro. This new material was shown to have good compatibility with the brain tissue and inhibition to gliosis and inflammation after its implantation in the normal or ischemic brain of mice. Particularly, good angiogenesis was found around the implanted HA-PLGA hydrogel, and the mouse models clearly showed a behavioral improvement. The results in this present study indicate, therefore, that the HA-PLGA hydrogel is a promising material, which is able to induce angiogenesis in the ischemic region by releasing VEGF and Ang1, thus creating a suitable niche for neural restoration in later stages of stroke. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Antagonizing Nogo-receptor 1 promotes the number of cultured dopaminergic neurons and elongates their neurites

The myelin-associated protein Nogo-A and its receptor Nogo-receptor 1 (NgR1) are known as potent growth inhibitors of the adult central nervous system (CNS). Nogo-A is mostly expressed on the surface of oligodendrocytes, but is also found in neurons of the adult and developing CNS. This observation suggests that Nogo-A serves additional functions in the brain. Hence, in the present study, we investigated the effects of antagonizing NgR1 on cultured organotypic and dissociated dopaminergic neurons. For that purpose ventral mesencephalic cultures from E14 rat embryos were grown in absence or presence of the NgR1 antagonist NEP1-40 for 1 week. Treatment with NEP1-40 significantly increased cell densities of tyrosine hydroxylase-immunoreactive neurons. Moreover, organotypic ventral mesencephalic cultures displayed a significantly bigger volume after NEP1-40 treatment. Morphological analysis of tyrosine hydroxylase-positive neurons disclosed longer neurites and higher numbers of primary neurites in dissociated cultures incubated with NEP1-40, whereas soma size was not changed. In conclusion, our findings demonstrate that interfering with Nogo-A signaling by antagonizing NgR1 modulates dopaminergic neuron properties during development. These observations highlight novel aspects of the role of Nogo-A in the CNS and might have an impact in the context of Parkinson's disease.

White matter abnormalities in 22q11.2 deletion syndrome: Preliminary associations with the Nogo-66 receptor gene and symptoms of psychosis

BackgroundThis study utilized diffusion tensor imaging (DTI) to analyze white matter tractography in the anterior limb of the internal capsule (ALIC), fornix, and uncinate fasciculus (UF) of individuals with 22q11.2 deletion syndrome and controls. Aberrations in these tracts have been previously associated with schizophrenia. With up to 25% of individuals with 22q11.2DS developing schizophrenia in adulthood, we hypothesized reduction in structural integrity of these tracts, including an association with prodromal symptoms of psychosis. We further predicted an association between allelic variation in a functional polymorphism of the Nogo-66 receptor gene and 22q11.2DS white matter integrity. MethodsTractography was conducted using fiber assignment by streamline tracking algorithm in DTI Studio. Subjects were genotyped for the rs701428 SNP of the Nogo-66 receptor gene, and assessed for presence of prodromal symptoms. ResultsWe found significant group differences between 22q11.2DS and controls in DTI metrics for all three tracts. DTI metrics of ALIC and UF were associated with prodromal symptoms in 22q11.2DS. Further, ALIC DTI metrics were associated with allelic variation of the rs701428 SNP of the Nogo-66 receptor gene in 22q11.2DS. ConclusionsAlterations in DTI metrics suggest white matter microstructural anomalies of the ALIC, fornix, and UF in 22q11.2DS. Structural differences in ALIC appear to be associated with the Nogo-66 receptor gene, which has been linked to myelin-mediated axonal growth inhibition. Moreover, the association between psychosis symptoms and ALIC and UF metrics suggests that the Nogo-66 receptor gene may represent a susceptibility gene for psychosis through its disruption of white matter microstructure and myelin-associated axonal growth.

Nogo-Receptor 1 Deficiency Has No Influence on Immune Cell Repertoire or Function during Experimental Autoimmune Encephalomyelitis

The potential role of Nogo-66 Receptor 1 (NgR1) on immune cell phenotypes and their activation during neuroinflammatory diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), is unclear. To further understand the function of this receptor on haematopoietically-derived cells, phenotypic and functional analyses were performed using NgR1-deficient (ngr1-/-) animals. Flow cytometry-based phenotypic analyses performed on blood, spleen, thymus, lymph nodes, bone marrow and central nervous-system (CNS)-infiltrating blood cells revealed no immunological defects in naïve ngr1-/- animals versus wild-type littermate (WTLM) controls. EAE was induced by either recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or by MOG35–55 peptide, a B cell-independent model. We have demonstrated that in ngr1-/- mice injected with MOG35–55, a significant reduction in the severity of EAE correlated with reduced axonal damage present in the spinal cord when compared to their WTLM controls. However, despite a reduction in axonal damage observed in the CNS of ngr1-/- mice at the chronic stage of disease, no clinical differences could be attributed to a specific genotype when rMOG was used as the encephalitogen. Following MOG35–55-induction of EAE, we could not derive any major changes to the immune cell populations analyzed between ngr1-/- and WTLM mice. Collectively, these data demonstrate that NgR1 has little if any effects on the repertoire of immune cells, their activation and trafficking to the CNS.

Orchestrated Regulation of Nogo Receptors, Lotus, AMPA Receptors and BDNF in an ECT Model Suggests Opening and Closure of a Window of Synaptic Plasticity

Electroconvulsive therapy (ECT) is an efficient and relatively fast acting treatment for depression. However, one severe side effect of the treatment is retrograde amnesia, which in certain cases can be long-term. The mechanisms behind the antidepressant effect and the amnesia are not well understood. We hypothesized that ECT causes transient downregulation of key molecules needed to stabilize synaptic structure and to prevent Ca2+ influx, and a simultaneous increase in neurotrophic factors, thus providing a short time window of increased structural synaptic plasticity. Here we followed regulation of NgR1, NgR3, LOTUS, BDNF, and AMPA subunits GluR1 and GluR2 flip and flop mRNA levels in hippocampus at 2, 4, 12, 24, and 72 hours after a single episode of induced electroconvulsive seizures (ECS) in rats. NgR1 and LOTUS mRNA levels were transiently downregulated in the dentate gyrus 2, 4, 12 and 4, 12, 24 h after ECS treatment, respectively. GluR2 flip, flop and GluR1 flop were downregulated at 4 h. GluR2 flip remained downregulated at 12 h. In contrast, BDNF, NgR3 and GluR1 flip mRNA levels were upregulated. Thus, ECS treatment induces a transient regulation of factors important for neuronal plasticity. Our data provide correlations between ECS treatment and molecular events compatible with the hypothesis that both effects and side effects of ECT may be caused by structural synaptic rearrangements.

TROY Interacts with Rho Guanine Nucleotide Dissociation Inhibitor α (RhoGDIα) to Mediate Nogo-induced Inhibition of Neurite Outgrowth

TROY can functionally substitute p75 to comprise the Nogo receptor complex, which transduces the inhibitory signal of myelin-associated inhibitory factors on axon regeneration following CNS injury. The inhibition of neurite extension relies on TROY-dependent RhoA activation, but how TROY activates RhoA remains unclear. Here, we firstly identified Rho guanine nucleotide dissociation inhibitor α (RhoGDIα) as a binding partner of TROY using GST pull-down combined with two-dimensional gel electrophoresis and mass spectra analysis. The interaction was further confirmed by coimmunoprecipitation in vitro and in vivo. Deletion mutagenesis revealed that two regions of the TROY intracellular domain (amino acids 234-256 and 321-350) were essential for the interaction with RhoGDIα. Secondly, TROY and RhoGDIα were coexpressed in postnatal dorsal root ganglion neurons, cortex neurons, and cerebellar granule neurons (CGNs). Thirdly, TROY/RhoGDIα association was potentiated by Nogo-66 and was independent of p75/RhoGDIα interaction. Fourthly, TROY/RhoGDIα interaction was still able to activate RhoA when p75 was deficient. Furthermore, RhoA activation was decreased dramatically when TROY was knocked down in p75-deficient CGNs cells. Finally, RhoGDIα overexpression abolished RhoA activation and following neurite outgrowth inhibition by Nogo-66 in both wild-type and p75-deficient CGNs. These results showed that the association of RhoGDIα with TROY contributed to TROY-dependent RhoA activation and neurite outgrowth inhibition after Nogo-66 stimulation.

Neuroprotection by Sildenafil: Neuronal Networks Potentiation in Acute Experimental Stroke

Sildenafil, a phosphodiesterase type 5 inhibitor, has been found to produce functional recovery in ischemic rats by increasing the cGMP level and triggering neurogenesis. The aim of this study was to investigate further sildenafil mechanisms. Male Sprague-Dawley rats underwent middle cerebral artery occlusion and reperfusion, followed by intraperitoneal or intravenous treatment of sildenafil starting 2h later. Behavioral tests were performed on day 1 or day 7 after reperfusion, while cerebral infarction, edema, Nissl staining, Fluoro-Jade B staining, and electron microscopy studies were carried out 24h poststroke. The cGMP-dependent Nogo-66 receptor (Nogo-R) pathway, synaptophysin, PSD-95/neuronal nitric oxide synthases (nNOS), brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB), and nerve growth factor (NGF)/tropomyosin-related kinase A (TrkA) were measured. Sildenafil enhanced neurological recovery and inhibited infarction, even following delayed administration 4h after stroke onset. Furthermore, sildenafil reduced the loss of neurons and modulated the expressions of the cGMP-dependent Nogo-R pathway. Moreover, sildenafil protected the structure of synapses and mediated the expressions of synaptophysin, PSD-95/nNOS, BDNF/TrkB, and NGF/TrkA. Sildenafil produces significant neuroprotective effects on injured neurons in acute stroke, and these are mediated by the cGMP-dependent Nogo-R pathway, NGF/TrkA, and BDNF/TrkB.

RNA interference suppression of Nogo-66 receptor prevents Nogo-66-mediated inhibition of invasion and adhesion and simultaneously increases cell apoptosis in C6 cells

Gliomas are the most common primary tumors of the central nervous system (CNS). Nogo-66 is an extracellular domain of Nogo-A, which can block axon regeneration in the CNS after trauma. Some studies have indicated that Nogo-A and its receptor (NgR) are expressed in tumor tissues; however, their roles in tumors are still unknown. We report the impact of Nogo-66 and NgR on the proliferation, apoptosis, adhesion and invasion of C6 glioma cells. Short hairpin RNA (shRNA)-triggered RNA interference was used to inhibit NgR expression in C6cells. Then, an invitro cell adhesion assay was performed to assess the effect of NgR downregulation on the adhesion ability of C6cells. In addition, a chamber assay and a cell scratch assay were conducted to test invasion ability. The spontaneous apoptosis of C6cells was examined by flow cytometry, western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. NgR downregulation resulted in a significant increase of C6 adhesion and invasion activity in the presence of Nogo-66, markedly inhibited proliferation and induced spontaneous apoptosis. In conclusion, knockdown of NgR enhanced invasion and adhesion but increased cell apoptosis in C6cells, suggesting that Nogo-66/NgR might have complex effects on glioma cells.