The adhesion and migration of microglia to ß-amyloid (Aß) is decreased with aging and inhibited by Nogo/NgR pathway.

BACKGROUND: Alzheimer's disease is characterized by progressive accumulation of ß-amyloid (Aß)-containing amyloid plaques, and microglia play a critical role in internalization and degradation of Aß. Our previous research confirmed that Nogo-66 binding to Nogo receptors (NgR) expressed on microglia inhibits cell adhesion and migration in vitro. METHODS: The adhesion and migration of microglia isolated from WT and APP/PS1 mice from different ages were measured by adhesion assays and transwells. After NEP1-40 (a competitive antagonist of Nogo/NgR pathway) was intracerebroventricularly administered via mini-osmotic pumps for 2 months in APP/PS1 transgenic mice, microglial recruitment toward Aß deposits and CD36 expression were determined. RESULTS: In this paper, we found that aging led to a reduction of microglia adhesion and migration to fAß1-42 in WT and APP/PS1 mice. The adhesion and migration of microglia to fAß1-42 were downregulated by the Nogo, which was mediated by NgR, and the increased inhibitory effects of the Nogo could be observed in aged mice. Moreover, Rho GTPases contributed to the effects of the Nogo on adhesion and migration of microglia to fAß1-42 by regulating cytoskeleton arrangement. Furthermore, blocking the Nogo/NgR pathway enhanced recruitment of microglia toward Aß deposits and expression of CD36 in APP/PS1 mice. CONCLUSION: Taken together, Nogo/NgR pathway could take part in Aß pathology in AD by modulating microglial adhesion and migration to Aß and the Nogo/NgR pathway might be an important target for treating AD.

Cetuximab and Taxol co-modified collagen scaffolds show combination effects for the repair of acute spinal cord injury.

Injury-activated endogenous neural stem cells (NSCs) in the spinal cord have promising therapeutic applications for rebuilding the neuronal relays after spinal cord injury (SCI) because of their lack of immune-rejection following exogenous cell transplantation. However, these NSCs rarely differentiate into neurons and the damaged axonal regenerative ability is drastically reduced due to the adverse SCI microenvironment. Cetuximab, an EGFR signaling antagonist, has demonstrated the ability of promoting NSC differentiation into neurons. Taxol, in addition to stabilizing microtubules, has shown potential for enhancing axonal regeneration and reducing scar formation after SCI. In this study, we further verified the combined therapeutic effects of Cetuximab and Taxol on inhibition of scar deposition and promotion of neuronal differentiation, axonal outgrowth and functional recovery in a rat severe SCI model. A linear orderly collagen scaffold modified with Cetuximab and Taxol was grafted into the SCI site after the complete removal of 4 mm of spinal tissue. The results showed that the combined functional scaffold implantation significantly increased neural regeneration to reconnect the neural network. Moreover, scaffold transplantation decreases the deposition of varied scar-related inhibitors within the lesion center, further reflecting the need for a combination dedicated to increasing motor function following SCI. Collagen scaffold based-combined therapy provides a potential strategy for improving functional restoration of the injured spinal cord.

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.

The blockage of the Nogo/NgR signal pathway in microglia alleviates the formation of Aß plaques and tau phosphorylation in APP/PS1 transgenic mice.

BACKGROUND: Alzheimer's disease (AD) is characterized by extracellular ß-amyloid (Aß) plaques, neurofibrillary tangles (NFTs), and microglia-dominated neuroinflammation. The Nogo/NgR signal pathway is involved in AD pathological features, but the detailed mechanism needs further investigation. Our previous studies have confirmed that the activation of NgR on microglia by Nogo promotes the expression of proinflammatory cytokines and inhibits cell adhesion and migration behaviors. In the present study, we investigated the effects of Nogo/NgR signaling pathway on the pathological features of AD and possible mechanisms. METHODS: After NEP1-40 (a competitive antagonist of Nogo/NgR pathway) was intracerebroventricularly administered via mini-osmotic pumps for 2 months in amyloid precursor protein (APP)/PS1 transgenic mice, plaque load, tau phosphorylation, and inflammatory responses were determined. After primary mouse neurons were exposed to the conditioned medium from BV-2 microglia stimulated by Nogo, the production of Aß and phosphorylation of tau was quantified by ELISA and western blot. RESULTS: Inhibition of the Nogo/NgR signaling pathway ameliorated pathological features including amyloid plaques and phosphorylated levels of tau in APP/PS1 mice. In addition, after treatment with the conditioned medium from BV-2 microglia stimulated by Nogo, Aß production and tau phosphorylation in cultured neurons were increased. The conditioned medium also increased the expression of APP, its amyloidogenic processing, and the activity of GSK3ß in neurons. The conditioned medium was also proinflammatory medium, and the blockage of the Nogo/NgR pathway improved the neuroinflammatory environment in APP/PS1 mice. CONCLUSIONS: Taken together, the neuroinflammation mediated by Nogo/NgR pathway in microglia could directly take part in the pathological process of AD by influencing the amyloidogenesis and tau phosphorylation. These results contribute to a better understanding of AD pathogenesis and could offer a new therapeutic option for delaying the progression of AD.

Asynchronous therapy targeting Nogo-A enhances neurobehavioral recovery by reducing neuronal loss and promoting neurite outgrowth after cerebral ischemia in mice.

Therapeutics targeting the Nogo-A signal pathway hold promise to promote recovery following brain injury. Based on the temporal characteristics of Nogo-A expression in the process of cerebral ischemia and reperfusion, we tested a novel asynchronous treatment, in which TAT-M9 was used in the early stage to decrease neuronal loss, and TAT-NEP1-40 was used in the delayed stage to promote neurite outgrowth after bilateral common carotid artery occlusion (BCCAO) in mice. Both TAT-M9 and TAT-NEP1-40 were efficiently delivered into the brains of mice by intraperitoneal injection. TAT-M9 treatment promoted neuron survival and inhibited neuronal apoptosis. Asynchronous therapy with TAT-M9 and TAT-NEP1-40 increased the expression of Tau, GAP43 and MAP-2 proteins, and enhanced short-term and long-term cognitive functions. In conclusion, the asynchronous treatment had a long-term neuroprotective effect, which reduced neurologic injury and apoptosis, promoted neurite outgrowth and enhanced functional recovery after ischemia. It suggests that this asynchronous treatment could be a promising therapy for cerebral ischemia in humans.

Effect of oral administration of pig spinal cord hydrolysate on clinical and histopathological symptoms of experimental allergic encephalomyelitis in rats.

Oral tolerance is the natural occurring phenomenon of a decreased immune response to previously fed antigens, which prevents induction of a response to dietary antigens. One of the mechanisms is deletion of T lymphocytes reactive to the fed antigen. Knowing that phenomenon, it seems appropriate to engage this mechanism for treatment of autoimmune diseases. Multiple sclerosis (MS) is an autoimmunological disease which causes neurological impairment in humans. Autoreactive T lymphocytes migrate through the open blood-brain barrier into the central nervous system (CNS), where they recognize myelin antigens as foreign, and induce an inflammatory response against the myelin sheath, which causes demyelination and even axonal loss. Experimental allergic encephalomyelitis (EAE), an animal model of MS, resembles the autoimmunological aspect of the disease. We used a broad spectrum of myelin antigens to induce EAE, and also to induce oral tolerance by giving myelin epitopes intragastrically to rats. The aim of our study was to evaluate whether pig spinal cord hydrolysate given intragastrically is able to evoke oral tolerance in rats with an animal model of MS - EAE. In our experiments we fed female Lewis rats with pig spinal cord hydrolysate at doses of 5, 20 and 100 mg per kg of body weight. We observed diminished clinical symptoms of ongoing EAE in rats fed with all doses of pig spinal cord hydrolysate. In the histopathological study, intensity of the inflammatory process in spinal cord was similar in rats not fed with EAE and in rats fed with lower doses of pig spinal cord hydrolysate. In animals fed with the highest dose of pig spinal cord hydrolysate, intensification of the inflammatory response was observed. These results were confirmed by morphometric evaluations. We found that feeding animals with preparations containing myelin antigens can reduce EAE symptoms, which may indicate oral tolerance induction, but the obtained results also underline the importance of dose of the orally given antigens, because of the possibility of enhancement of the inflammatory process in the CNS.

Amygdala NRG1-ErbB4 is critical for the modulation of anxiety-like behaviors.

Anxiety disorder is related to the pathophysiology of psychiatric diseases, including major depression, substance abuse, and schizophrenia. The amygdala is important for manifestation and modulation of anxiety. However, relatively little is known regarding the mechanisms that control the amygdala inhibitory activity that is involved in anxiety. We found that almost all ErbB4, which is the only autonomous receptor of neuregulin 1 (NRG1) in the basolateral amygdala (BLA), was expressed in GABAergic neurons. Endogenous NRG1-ErbB4 signaling pathway in the BLA could modulate anxiety-like behaviors and GABA release, whereas it had no effect on glutamatergic transmission. The administration of NRG1 into the BLA of high-anxiety mice alleviated their anxiety and enhanced GABAergic neurotransmission. Moreover, exogenous NRG1 also produced an anxiolytic effect in the stressed mice. Together, these observations indicated that NRG1-ErbB4 signaling is critical to maintaining GABAergic activity in the amygdala and thus to modulating anxiety-like behaviors. Because NRG1 and ErbB4 are susceptibility genes of schizophrenia, our findings might also help to explain the potential mechanism of emotional abnormality in schizophrenia.

Polyphenols from green tea prevent antineuritogenic action of Nogo-A via 67-kDa laminin receptor and hydrogen peroxide.

Axonal regeneration after injury to the CNS is hampered by myelin-derived inhibitors, such as Nogo-A. Natural products, such as green tea, which are neuroprotective and safe for long-term therapy, would complement ongoing various pharmacological approaches. In this study, using nerve growth factor-differentiated neuronal-like Neuroscreen-1 cells, we show that extremely low concentrations of unfractionated green tea polyphenol mixture (GTPP) and its active ingredient, epigallocatechin-3-gallate (EGCG), prevent both the neurite outgrowth-inhibiting activity and growth cone-collapsing activity of Nogo-66 (C-terminal domain of Nogo-A). Furthermore, a synergistic interaction was observed among GTPP constituents. This preventive effect was dependent on 67-kDa laminin receptor (67LR) to which EGCG binds with high affinity. The antioxidants N-acetylcysteine and cell-permeable catalase abolished this preventive effect of GTPP and EGCG, suggesting the involvement of sublethal levels of H2 O2 in this process. Accordingly, exogenous sublethal concentrations of H2 O2 , added as a bolus dose (5 µM) or more effectively through a steady-state generation (1-2 µM), mimicked GTPP in counteracting the action of Nogo-66. Exogenous H2 O2 mediated this action by bypassing the requirement of 67LR. Taken together, these results show for the first time that GTPP and EGCG, acting through 67LR and elevating intracellular sublethal levels of H2 O2 , inhibit the antineuritogenic action of Nogo-A. Currently, several agents are being evaluated for overcoming axonal growth inhibitors to promote functional recovery after stroke and spinal cord injury. Epigallocatechin-3-gallate (EGCG), present in green tea polyphenol mixture (GTPP), prevents antineuritogenic activity of Nogo-A, a myelin-derived axonal growth inhibitor. The preventive action of EGCG involves the cell-surface-associated 67-kDa laminin receptor and H2 O2 . GTPP may complement ongoing efforts to treat neuronal injuries.>

CNS axon regeneration inhibitors stimulate an immediate early gene response via MAP kinase-SRF signaling.

BACKGROUND: CNS axon regeneration inhibitors such as Nogo and CSPGs (Chondroitin Sulfate Proteoglycans) are major extrinsic factors limiting outgrowth of severed nerve fibers. However, knowledge on intracellular signaling cascades and gene expression programs activated by these inhibitors in neurons is sparse. Herein we studied intracellular signaling cascades activated by total myelin, Nogo and CSPGs in primary mouse CNS neurons. RESULTS: Total myelin, Nogo and CSPGs stimulated gene expression activity of the serum response factor (SRF), a central gene regulator of immediate early (IEG) and actin cytoskeletal gene transcription. As demonstrated by pharmacological interference, SRF-mediated IEG activation by myelin, Nogo or CSPGs depended on MAP kinase, to a lesser extent on Rho-GTPase but not on PKA signaling. Stimulation of neurons with all three axon growth inhibitors activated the MAP kinase ERK. In addition to ERK activation, myelin activated the IEG c-Fos, an important checkpoint of neuronal survival vs. apoptosis. Employing Srf deficient neurons revealed that myelin-induced IEG activation requires SRF. This suggests an SRF function in mediating neuronal signaling evoked by axon regeneration associated inhibitors. Besides being a signaling target of axon growth inhibitors, we show that constitutively-active SRF-VP16 can be employed to circumvent neurite growth inhibition imposed by myelin, Nogo and CSPGs. CONCLUSION: In sum, our data demonstrate that axon regeneration inhibitors such as Nogo trigger gene expression programs including an SRF-dependent IEG response via MAP kinases and Rho-GTPases.

Protein synthesis dependence of growth cone collapse induced by different Nogo-A-domains.

BACKGROUND: The protein Nogo-A regulates axon growth in the developing and mature nervous system, and this is carried out by two distinct domains in the protein, Nogo-A-Δ20 and Nogo-66. The differences in the signalling pathways engaged in axon growth cones by these domains are not well characterized, and have been investigated in this study. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed growth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 using explanted chick dorsal root ganglion neurons growing on laminin/poly-lysine substratum. Collapse induced by purified Nogo-A-Δ20 peptide is dependent on protein synthesis whereas that induced by Nogo-66 peptide is not. Nogo-A-Δ20-induced collapse is accompanied by a protein synthesis-dependent rise in RhoA expression in the growth cone, but is unaffected by proteasomal catalytic site inhibition. Conversely Nogo-66-induced collapse is inhibited ∼ 50% by proteasomal catalytic site inhibition. CONCLUSION/SIGNIFICANCE: Growth cone collapse induced by the Nogo-A domains Nogo-A-Δ20 and Nogo-66 is mediated by signalling pathways with distinguishable characteristics concerning their dependence on protein synthesis and proteasomal function.

NogoR1 and PirB signaling stimulates neural stem cell survival and proliferation.

Neural stem cells (NSCs) and neural progenitors (NPs) in the mammalian neocortex give rise to the main cell types of the nervous system. The biological behavior of these NSCs and NPs is regulated by extracellular niche derived autocrine-paracrine signaling factors on a developmental timeline. Our previous reports [Plos One 2010;5:e15341; J Neurochem 2011;117:565-578] have shown that chondroitin sulfate proteoglycan and ApolipoproteinE are autocrine-paracrine survival factors for NSCs. NogoA, a myelin related protein, is expressed in the cortical ventricular zones where NSCs reside. However, the functional role of Nogo signaling proteins in NSC behavior is not completely understood. In this study, we show that NogoA receptors, NogoR1 and PirB, are expressed in the ventricular zone where NSCs reside between E10.5 and 14.5 but not at E15.5. Nogo ligands stimulate NSC survival and proliferation in a dosage-dependent manner in vitro. NogoR1 and PirB are low and high affinity Nogo receptors, respectively and are responsible for the effects of Nogo ligands on NSC behavior. Inhibition of autocrine-paracrine Nogo signaling blocks NSC survival and proliferation. In NSCs, NogoR1 functions through Rho whereas PirB uses Shp1/2 signaling pathways to control NSC behavior. Taken together, this work suggests that Nogo signaling is an important pathway for survival of NSCs.

Spinal motor neurite outgrowth over glial scar inhibitors is enhanced by coculture with bone marrow stromal cells.

BACKGROUND CONTEXT: Transplantation of bone marrow cells into spinal cord lesions promotes functional recovery in animal models, and recent clinical trials suggest possible recovery also in humans. The mechanisms responsible for these improvements are still unclear. PURPOSE: To characterize spinal cord motor neurite interactions with human bone marrow stromal cells (MSCs) in an in vitro model of spinal cord injury (SCI). STUDY DESIGN/SETTING: Previously, we have reported that human MSCs promote the growth of extending sensory neurites from dorsal root ganglia (DRG), in the presence of some of the molecules present in the glial scar, which are attributed with inhibiting axonal regeneration after SCI. We have adapted and optimized this system replacing the DRG with a spinal cord culture to produce a central nervous system (CNS) model, which is more relevant to the SCI situation. METHODS: We have developed and characterized a novel spinal cord culture system. Human MSCs were cocultured with spinal motor neurites in substrate choice assays containing glial scar-associated inhibitors of nerve growth. In separate experiments, MSC-conditioned media were analyzed and added to spinal motor neurites in substrate choice assays. RESULTS: As has been reported previously with DRG, substrate-bound neurocan and Nogo-A repelled spinal neuronal adhesion and neurite outgrowth, but these inhibitory effects were abrogated in MSC/spinal cord cocultures. However, unlike DRG, spinal neuronal bodies and neurites showed no inhibition to substrates of myelin-associated glycoprotein. In addition, the MSC secretome contained numerous neurotrophic factors that stimulated spinal neurite outgrowth, but these were not sufficient stimuli to promote spinal neurite extension over inhibitory concentrations of neurocan or Nogo-A. CONCLUSIONS: These findings provide novel insight into how MSC transplantation may promote regeneration and functional recovery in animal models of SCI and in the clinic, especially in the chronic situation in which glial scars (and associated neural inhibitors) are well established. In addition, we have confirmed that this CNS model predominantly comprises motor neurons via immunocytochemical characterization. We hope that this model may be used in future research to test various other potential interventions for spinal injury or disease states.

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.

Effects of Nogo-A receptor antagonist on the regulation of the Wnt signaling pathway and neural cell proliferation in newborn rats with hypoxic ischemic encephalopathy.

Hypoxic ischemic encephalopathy is a serious condition due to inadequate oxygen supply to the brain. Regeneration of neural cells is a critical process for repairing the damaged brain. Nogo has been identified as an inhibitor of neurite outgrowth that is specific to the brain. In the present study, the Nogo-A receptor (NgR) antagonist NEP1-40 was used to study the effects of inhibition of NgR on the regeneration of neural cells and the related Wnt signaling pathway in newborn rats. The investigation focused on the transcription factors regulated in the Wnt signaling pathway during the repair process, together with the proliferation of neural cells. The results indicated that c-Jun and c-Myc were the main transcription factors involved in the Wnt signaling pathway, while neural cell proliferation in the subventricular zone was increased during this process.

Autoantigen conformation influences both B- and T-cell responses and encephalitogenicity.

It has become increasingly clear that only antibodies recognizing conformation-dependent epitopes of myelin oligodendrocyte glycoprotein (MOG) have a demyelinating potential in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Nevertheless, for the induction of EAE, most studies to date have used MOG peptides or bacterially expressed MOG, neither of which contain the tertiary structure of the native antigen. Non-refolded recombinant human MOG does not induce EAE in DA rats. Therefore, we refolded this protein in order to assess the influence of MOG conformation on its pathogenicity in DA rats. DA rats immunized with refolded human MOG developed severe acute EAE. As expected, rats immunized with the refolded protein had a higher amount of conformational MOG antibodies present in serum. But in addition, a striking effect of MOG refolding on the generation of T-cell responses was found. Indeed, T-cell responses against the encephalitogenic MOG 91-108 epitope were greatly enhanced after refolding. Therefore, we conclude that refolding of MOG increases its pathogenicity both by generating conformation-dependent MOG antibodies and by enhancing its processing or/and presentation on MHC molecules. These data are important in regard to investigations of the pathogenic potential of many (auto)antigens.

The effect of growth factors and soluble Nogo-66 receptor protein on transplanted neural stem/progenitor survival and axonal regeneration after complete transection of rat spinal cord.

Adult central mammalian axons show minimal regeneration after spinal cord injury due to loss of oligodendrocytes, demyelination of surviving axons, absence of growth-promoting molecules, and inhibitors of axonal outgrowth. In the present study, we attempted to address these impediments to regeneration by using a combinatory strategy to enhance cell survival and regeneration after complete spinal cord transection (SCT) in adult rats. The strategy comprised: 1) adult rat brain-derived neural stem/progenitor cells (NSPCs) preseeded on laminin-coated chitosan channels; 2) extramedullary chitosan channels to promote axonal regrowth and reduce the barrier caused by scarring; 3) local delivery of a novel rat soluble Nogo-66 receptor protein [NgR(310)ecto-Fc, referred to as NgR] to block the inhibitory effect of myelin-based inhibitors; and 4) local delivery of basic fibroblast growth factor, epidermal growth factor, and platelet-derived growth factor to enhance survival and promote differentiation of transplanted cells. Compared with our previous studies where brain-derived NSPCs preseeded in extramedullary chitosan channels were implanted in the same SCT model but without growth factors and NgR, the present channel-growth factor combination produced greater numbers of surviving NSPCs after SCT. Also, the growth factors promoted preferential differentiation of NSPCs toward oligodendrocytes, while NgR significantly decreased astrocytic differentiation of NSPCs. NgR alone or in combination with NSPCs significantly enhanced the total number of myelinated fibers in the bridge and increased the area of the bridging tissue between the cord stumps. The combination of NgR, growth factors, and NSPCs had synergistic effect on bridge formation. However, only a small number of descending corticospinal tract axons grew into the central portions of the bridges as shown by anterograde tracing of the corticospinal tract with BDA. The majority of the regenerated axons in the channels originated from local host neurons adjacent to the tissue bridges. In conclusion, we showed that growth factors increased survival of transplanted NSPCs whereas NgR enhanced axonal regeneration, but the combination did not have additive effects on functional recovery or regeneration.

Nogo-66 inhibits adhesion and migration of microglia via GTPase Rho pathway in vitro.

Nogo-66 is a 66-amino-acid-residue extracellular domain of Nogo-A, which plays a key role in inhibition neurite outgrowth of central nervous system through binding to the Nogo-66 receptor (NgR) expressed on the neuron. Recent studies have confirmed that NgR is also expressed on the surface of macrophages/microglia in multiple sclerosis, but its biological effects remain unknown. In the present study, our results demonstrated that Nogo-66 triggered microglia anti-adhesion and inhibited their migration in vitro, which was mediated by NgR. We also assessed the roles of small GTP (glycosyl phosphatidylinositol)-binding proteins of the Rho family as the downstream signal transducers on the microglia adhesion and mobility induced by Nogo-66. The results showed that Nogo-66 activated RhoA and reduced the activity of Cdc42 in the meanwhile, which further triggered the anti-adhesion and migration inhibition effects to microglia. Nogo-66 inhibited microglia polarization and membrane protrusion formation, thus might eventually contribute to the decreasing capability of cell mobility. Taken together, the Nogo-66/NgR pathway may modulate neuroinflammation via mediating microglia adhesion and migration in addition to its role in neurons. Better understanding the relationship between Nogo-66/NgR and neuroinflammation may help targeting NgR for treating central nervous system diseases related with inflammation.

Nogo-66 receptor activation inhibits neurite outgrowth and increases ß-amyloid protein secretion of cortical neurons.

A Nogo-A to Nogo-66 receptor (NgR) pathway is well known to contribute to the inhibition of the neurite regeneration of adult central nervous system neurons after traumatic injuries. Recent evidence suggests that Nogo-A and NgR are involved in the pathology of Alzheimer's disease (AD), as evidenced by the fact that Nogo-A is overexpressed by hippocampal neurons in patients with AD and is associated with ß-amyloid protein (Aß) deposits in senile plaques. In the present experiments, we investigated the potential role of Nogo-A in both neurite outgrowth and Aß generation in cortical neurons. Our results showed that activation of NgR not only inhibited neurite outgrowth in cortical neurons by activating the rho-associated coiled coil-containing protein kinase (ROCK) and protein kinase C, but also promoted their Aß secretion, which was at least in part activated by ROCK. These findings suggest that the overexpression of Nogo-A and the activation of NgR inhibit neurite outgrowth and alter neuronal metabolism, resulting in overproduction and/or release of Aß, which in turn may trigger the onset and development of AD. Inhibition of ROCK can promote neurite outgrowth and reduce Aß production of cortical neuron, which suggests that ROCK appears to be a good target for AD therapy.

Nogo-66 inhibits the dye-coupling of astrocytic gap junctions in vitro.

Communication between astrocytes via the gap junction is crucial for maintaining homeostasis of the extra-neuronal microenvironment of the central nervous system. Dysfunction of astrocytic gap junctions is involved in many brain disorders. Our previous studies demonstrated a novel co-localization of Nogo-66 receptor at glial gap junctions in rat cerebellum and posterior pituitary. The present study was aimed at exploring whether Nogo-66 can modulate glial gap junctions in vitro. We confirmed the co-localization of Nogo-66 receptor with Cx43 in cultured astrocytes, and stimulated astrocytes with myelin extracts, or Nogo-66-Fc conditioned medium. Finally, we expressed and purified a functionally effective GST-Nogo-66 peptide. Lucifer yellow transfer assay was adopted to measure the gap junction permeability. The results showed that the spreading of Lucifer yellow was inhibited significantly by all three treatments as compared with their corresponding controls. Therefore, this study shows a novel inhibitory effect of Nogo-66 on the permeability of astrocytic gap junctions, suggesting a presumable role of Nogo-66 receptor in modulating the glial gap junction.

Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice.

BACKGROUND AND PURPOSE: Cannabis extracts and several cannabinoids have been shown to exert broad anti-inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration. EXPERIMENTAL APPROACH: We used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG-immunized animals and on MOG-specific T-cell proliferation. KEY RESULTS: Treatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T-cell recruitment in the spinal cord of MOG-injected mice. Moreover, CBD inhibited MOG-induced T-cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB(1) and CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: CBD, a non-psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T-cell proliferation by CBD appeared to contribute to these beneficial effects.