Nogo-A-mediated constraints on activity-dependent synaptic plasticity and associativity in rat hippocampal CA2 synapses.

Hippocampal area CA2 has garnered attention in recent times owing to its significant involvement in social memory and distinctive plasticity characteristics. Research has revealed that the CA2 region demonstrates a remarkable resistance to plasticity, particularly in the Schaffer Collateral (SC)-CA2 pathway. In this study we investigated the role of Nogo-A, a well-known axon growth inhibitor and more recently discovered plasticity regulator, in modulating plasticity within the CA2 region. The findings demonstrate that blocking Nogo-A in male rat hippocampal slices facilitates the establishment of both short-term and long-term plasticity in the SC-CA2 pathway, while having no impact on the Entorhinal Cortical (EC)-CA2 pathway. Additionally, the study reveals that inhibiting Nogo-A enables association between the SC and EC pathways. Mechanistically, we confirm that Nogo-A operates through its well-known co-receptor, p75 neurotrophin receptor (p75NTR), and its downstream signaling factor such as Rho-associated protein kinase (ROCK), as their inhibition also allows plasticity induction in the SC-CA2 pathway. Additionally, the induction of long-term depression (LTD) in both the EC and SC-CA2 pathways led to persistent LTD, which was not affected by Nogo-A inhibition. Our study demonstrates the involvement of Nogo-A mediated signaling mechanisms in limiting synaptic plasticity within the CA2 region.

Nogo-A neutralization in the central nervous system with a blood-brain barrier-penetrating antibody.

The poor penetration of monoclonal antibodies (mAb) across the blood-brain barrier (BBB) impedes the development of regenerative therapies for neurological diseases. For example, Nogo-A is a myelin-associated protein highly expressed in the central nervous system (CNS) whose inhibitory effects on neuronal plasticity can be neutralized with direct administration of 11C7 mAb in CNS tissues/fluids, but not with peripheral administrations such as intravenous injections. Therefore, in the present study, we engineered a CNS-penetrating antibody against Nogo-A by combining 11C7 mAb and the single-chain variable fragment (scFv) of 8D3, a rat antibody binding transferrin receptor 1 (TfR) and mediating BBB transcytosis (11C7-scFv8D3). The binding of 11C7-scFv8D3 to Nogo-A and to TfR/CD71 was validated by capture ELISA and Biolayer Interferometry. After intravenous injection in mice, capture ELISA measurements revealed fast plasma clearance of 11C7-scFv8D3 concomitantly with brain and spinal cord accumulation at levels up to 19 fold as high as those of original 11C7 mAb. 11C7-scFv8D3 detection in the parenchyma indicated effective blood-to-CNS transfer. A single dose of 11C7-scFv8D3 induced stronger activation of the growth-promoting AkT/mTOR/S6 signaling pathway than 11C7 mAb or control antibody. Taken together, our results show that BBB-crossing 11C7-scFv8D3 engages Nogo-A in the mouse CNS and stimulates neuronal growth mechanisms.

Nogo-A is secreted in extracellular vesicles, occurs in blood and can influence vascular permeability.

Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.

Anti-Nogo-A Antibody Therapy Improves Functional Outcome Following Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) can cause sensorimotor deficits, and recovery is slow and incomplete. There are no effective pharmacological treatments for recovery from TBI, but research indicates potential for anti-Nogo-A antibody (Ab) therapy. This Ab neutralizes Nogo-A, an endogenous transmembrane protein that inhibits neuronal plasticity and regeneration. OBJECTIVE: We hypothesized that anti-Nogo-A Ab treatment following TBI results in disinhibited axonal growth from the contralesional cortex, the establishment of new compensatory neuronal connections, and improved function. METHODS: We modeled TBI in rats using the controlled cortical impact method, resulting in focal brain damage and motor deficits like those observed in humans with a moderate cortical TBI. Rats were trained on the skilled forelimb reaching task and the horizontal ladder rung walking task. They were then given a TBI, targeting the caudal forelimb motor cortex, and randomly divided into 3 groups: TBI-only, TBI + Anti-Nogo-A Ab, and TBI + Control Ab. Testing resumed 3 days after TBI and continued for 8 weeks, when rats received an injection of the anterograde neuronal tracer, biotinylated dextran amine (BDA), into the corresponding area contralateral to the TBI. RESULTS: We observed significant improvement in rats that received anti-Nogo-A Ab treatment post-TBI compared to controls. Analysis of BDA-positive axons revealed that anti-Nogo-A Ab treatment resulted in cortico-rubral plasticity to the deafferented red nucleus. Conclusions. Anti-Nogo-A Ab treatment may improve functional recovery via neuronal plasticity to brain areas important for skilled movements, and this treatment shows promise to improve outcomes in humans who have suffered a TBI.

Novel Function of Nogo-A as Negative Regulator of Endothelial Progenitor Cell Angiogenic Activity: Impact in Oxygen-Induced Retinopathy.

Endothelial Progenitor Cells (EPCs) can actively participate in revascularization in oxygen-induced retinopathy (OIR). Yet the mechanisms responsible for their dysfunction is unclear. Nogo-A, whose function is traditionally related to the inhibition of neurite function in the central nervous system, has recently been documented to display anti-angiogenic pro-repellent properties. Based on the significant impact of EPCs in retinal vascularization, we surmised that Nogo-A affects EPC function, and proceeded to investigate the role of Nogo-A on EPC function in OIR. The expression of Nogo-A and its specific receptor NgR1 was significantly increased in isolated EPCs exposed to hyperoxia, as well as in EPCs isolated from rats subjected to OIR compared with respective controls (EPCs exposed to normoxia). EPCs exposed to hyperoxia displayed reduced migratory and tubulogenic activity, associated with the suppressed expression of prominent EPC-recruitment factors SDF-1/CXCR4. The inhibition of Nogo-A (using a Nogo-66 neutralizing antagonist peptide) or siRNA-NGR1 in hyperoxia-exposed EPCs restored SDF-1/CXCR4 expression and, in turn, rescued the curtailed neovascular functions of EPCs in hyperoxia. The in vivo intraperitoneal injection of engineered EPCs (Nogo-A-inhibited or NgR1-suppressed) in OIR rats at P5 (prior to exposure to hyperoxia) prevented retinal and choroidal vaso-obliteration upon localization adjacent to vasculature; coherently, the inhibition of Nogo-A/NgR1 in EPCs enhanced the expression of key angiogenic factors VEGF, SDF-1, PDGF, and EPO in retina; CXCR4 knock-down abrogated suppressed NgR1 pro-angiogenic effects. The findings revealed that hyperoxia-induced EPC malfunction is mediated to a significant extent by Nogo-A/NgR1 signaling via CXCR4 suppression; the inhibition of Nogo-A in EPCs restores specific angiogenic growth factors in retina and the ensuing vascularization of the retina in an OIR model.

Constraint therapy promotes motor cortex remodeling and functional improvement by regulating c-Jun/miR-182-5p/Nogo - A signals in hemiplegic cerebral palsy mice.

BACKGROUND: Our previous study has confirmed that constraint-induced movement therapy (CIMT) could promote neural remodeling in hemiplegic cerebral palsy (HCP) mice through Nogo-A/NgR/RhoA/ROCK signaling, however, the upstream mechanism was still unclear. Therefore, the present study aimed to further explore the mechanism of CIMT regulating the expression of Nogo-A in HCP mice. METHOD: HCP mice were well established through ligating the left common carotid artery of 7-day-old pups and being placed in a hypoxic box which was filled with a mixture of 8% oxygen and 92% nitrogen. CIMT intervention was conducted by taping to fix the entire arm of the contralateral side (left) to force the mice to use the affected limb (right). Bioinformatics prediction and luciferase experiment were performed to confirm that miR-182-5p was targeted with Nogo-A. The beam test and grip test were applied to examine the behavioral performance under the intervention of c-Jun and CIMT. Also, immunofluorescence, Golgi staining, and transmission electron microscopy were conducted to show that the lenti-expression of c-Jun could increases the expression of myelin, and downregulates the expression of Nogo-A under the CIMT on HCP mice. RESULT: (1) The beam walking test and grip test experiment results showed that compared with the control group, the HCP + nCIMT group's forelimb grasping ability and balance coordination ability were decreased (P < 0.05). (2) The results of Golgi staining, and transmission electron microscopy showed that the thickness of myelin sheath and the density of dendritic spines in the HCP + nCIMT group were lower than those in the control group (P < 0.05). Compared with the HCP + nCIMT group, the cerebral cortex myelin sheath thickness, dendrite spine density and nerve filament expression were increased in HCP + CIMT group (P < 0.05). (3) Immunofluorescence staining showed that the expression of Nogo-A in the cerebral cortex of the HCP + nCIMT group was higher than that of the HCP + CIMT group (P < 0.05). Compared with the HCP + CIMT group, the expression of Nogo-A in the HCP + LC + CIMT group was decreased and, in the HCP, + SC + CIMT group was significantly increased (P < 0.05). Compared with the HCP + nCIMT group, the expression of c-Jun in the control, HCP + CIMT, HCP + LC + nCIMT and HCP + LC + CIMT groups was significantly increased, and in the HCP + SC + CIMT was decreased (P < 0.05). (4) Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that the expression level of miR-182-5p in the HCP + LC + CIMT group was more increased than that in the HCP + nCIMT group (P < 0.05). The expression level of miR-182-5p in the HCP + LC + CIMT group was higher than that in the HCP + LC + nCIMT group and the HCP + SC + CIMT group (P < 0.05). CONCLUSION: These data identified that CIMT might stimulate the remodeling of neurons and myelin in the motor cortex by partially inhibiting the c-Jun/miR-182-5p/Nogo-A pathway, thereby facilitating the grasping performance and balance function of HCP mice.

How does Nogo receptor influence demyelination and remyelination in the context of multiple sclerosis?

Multiple sclerosis (MS) can progress with neurodegeneration as a consequence of chronic inflammatory mechanisms that drive neural cell loss and/or neuroaxonal dystrophy in the central nervous system. Immune-mediated mechanisms can accumulate myelin debris in the disease extracellular milieu during chronic-active demyelination that can limit neurorepair/plasticity and experimental evidence suggests that potentiated removal of myelin debris can promote neurorepair in models of MS. The myelin-associated inhibitory factors (MAIFs) are integral contributors to neurodegenerative processes in models of trauma and experimental MS-like disease that can be targeted to promote neurorepair. This review highlights the molecular and cellular mechanisms that drive neurodegeneration as a consequence of chronic-active inflammation and outlines plausible therapeutic approaches to antagonize the MAIFs during the evolution of neuroinflammatory lesions. Moreover, investigative lines for translation of targeted therapies against these myelin inhibitors are defined with an emphasis on the chief MAIF, Nogo-A, that may demonstrate clinical efficacy of neurorepair during progressive MS.

Nogo-A and LINGO-1: Two Important Targets for Remyelination and Regeneration.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that causes progressive neurological disability in most patients due to neurodegeneration. Activated immune cells infiltrate the CNS, triggering an inflammatory cascade that leads to demyelination and axonal injury. Non-inflammatory mechanisms are also involved in axonal degeneration, although they are not fully elucidated yet. Current therapies focus on immunosuppression; however, no therapies to promote regeneration, myelin repair, or maintenance are currently available. Two different negative regulators of myelination have been proposed as promising targets to induce remyelination and regeneration, namely the Nogo-A and LINGO-1 proteins. Although Nogo-A was first discovered as a potent neurite outgrowth inhibitor in the CNS, it has emerged as a multifunctional protein. It is involved in numerous developmental processes and is necessary for shaping and later maintaining CNS structure and functionality. However, the growth-restricting properties of Nogo-A have negative effects on CNS injury or disease. LINGO-1 is also an inhibitor of neurite outgrowth, axonal regeneration, oligodendrocyte differentiation, and myelin production. Inhibiting the actions of Nogo-A or LINGO-1 promotes remyelination both in vitro and in vivo, while Nogo-A or LINGO-1 antagonists have been suggested as promising therapeutic approaches for demyelinating diseases. In this review, we focus on these two negative regulators of myelination while also providing an overview of the available data on the effects of Nogo-A and LINGO-1 inhibition on oligodendrocyte differentiation and remyelination.

Intranasal delivery of full-length anti-Nogo-A antibody: A potential alternative route for therapeutic antibodies to central nervous system targets.

Antibody delivery to the CNS remains a huge hurdle for the clinical application of antibodies targeting a CNS antigen. The blood-brain barrier and blood-CSF barrier restrict access of therapeutic antibodies to their CNS targets in a major way. The very high amounts of therapeutic antibodies that are administered systemically in recent clinical trials to reach CNS targets are barely viable cost-wise for broad, routine applications. Though global CNS delivery of antibodies can be achieved by intrathecal application, these procedures are invasive. A non-invasive method to bring antibodies into the CNS reliably and reproducibly remains an important unmet need in neurology. In the present study, we show that intranasal application of a mouse monoclonal antibody against the neurite growth-inhibiting and plasticity-restricting membrane protein Nogo-A leads to a rapid transfer of significant amounts of antibody to the brain and spinal cord in intact adult rats. Daily intranasal application for 2 wk of anti-Nogo-A antibody enhanced growth and compensatory sprouting of corticofugal projections and functional recovery in rats after large unilateral cortical strokes. These findings are a starting point for clinical translation for a less invasive route of application of therapeutic antibodies to CNS targets for many neurological indications.

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

Subsequently to the publication of this paper, an interested reader drew to the authors' attention that, in Fig. 4A on p. 6 showing the effects of NEP1­40 on MBP expression as determined via immunohistochemical analysis, certain of the data panels appeared to be overlapping, such that they may have been derived from the same original source. After having examined their original data, the authors have realized that these data panels were inadvertently assembled incorrectly. A corrected version of Fig. 4 is shown below, incorporating data from one of the alternative experiments in Fig. 4A. Note that these errors did not significantly affect the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 24: 844, 2021; DOI: 10.3892/mmr.2021.12484].

Inhibition of Nogo-A rescues synaptic plasticity and associativity in APP/PS1 animal model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss and cognitive decline. Synaptic impairment is one of the first events to occur in the progression of this disease. Synaptic plasticity and cellular association of various plastic events have been shown to be affected in AD models. Nogo-A, a well-known axonal growth inhibitor with a recently discovered role as a plasticity suppressor, and its main receptor Nogo-66 receptor 1 (NGR1) have been found to be overexpressed in the hippocampus of Alzheimer's patients. However, the role of Nogo-A and its receptor in the pathology of AD is still widely unknown. In this work we set out to investigate whether Nogo-A is working as a plasticity suppressor in AD. Our results show that inhibition of the Nogo-A pathway via the Nogo-R antibody in an Alzheimer's mouse model, APP/PS1, leads to the restoration of both synaptic plasticity and associativity in a protein synthesis and NMDR-dependent manner. We also show that inhibition of the p75NTR pathway, which is strongly associated with NGR1, restores synaptic plasticity as well. Mechanistically, we propose that the restoration of synaptic plasticity in APP/PS1 via inhibition of the Nogo-A pathway is due to the modulation of the RhoA-ROCK2 pathway and increase in plasticity related proteins. Our study identifies Nogo-A as a plasticity suppressor in AD models hence targeting Nogo-A could be a promising strategy to understanding AD pathology.

Association of Nogo-A Gene Polymorphisms with Cerebral Palsy in Southern China: A Case-Control Study.

Cerebral palsy (CP) is a motor and postural disorder syndrome caused by the nonprogressive dysfunction of the developing brain. Previous studies strongly indicated that the Nogo-A gene might be related to the pathogenesis of CP. The objective of this research was to explore the relationship between Nogo-A polymorphisms (rs1012603, rs12464595, and rs2864052) and CP in Southern China. The Hardy-Weinberg equilibrium (HWE) testing, allele and genotype frequencies analysis, and haplotype association analysis were applied to the genotyping of 592 CP children and 600 controls. The results showed that the allele and genotype frequencies of rs1012603 of CP group were significantly different from the control group. The haplotype "TTGGG" was significantly associated with an increased risk of CP. The allele frequencies of rs1012603 were significant differences between CP with spastic diplegia, female CP cases, and controls. Furthermore, significant differences in allele and genotype frequencies were also noticed between GMFCS I of CP and controls for rs1012603, and significant differences in allele and genotype frequencies were observed between the ADL (>9) of CP and controls for rs1012603 and rs12464595. This study showed that the SNPs rs1012603 of Nogo-A were significantly correlated with CP, and the correlations were also found in spastic diplegia, GMFCS I of CP, ADL (>9) of CP, and female subgroups, indicating that Nogo-A might mainly affect mild types of CP and there might be sex-related differences.

Zinc Ameliorates Nogo-A Receptor and Osteocalcin Gene Expression in Memory-Sensitive Rat Hippocampus Impaired by Intracerebroventricular Injection of Streptozotocin.

Metabolic dysfunction is a critical step in the etiopathogenesis of Alzheimer's disease. In this progressive neurological disorder, impaired zinc homeostasis has a key role that needs to be clarified. The aim of this study was to investigate the effect of zinc deficiency and administration on hippocampal Nogo-A receptor and osteocalcin gene expression in rats injected with intracerebroventricular streptozotocin (icv-STZ). Forty male Wistar rats were divided into 5 groups in equal numbers: Sham 1 group received icv artificial cerebrospinal fluid (aCSF); Sham 2 group received icv a CSF and i.p. saline; STZ group received 3 mg/kg icv STZ; STZ-Zn-deficient group received 3 mg/kg icv STZ and fed a zinc-deprived diet; STZ-Zn-supplemented group received 3 mg/kg icv STZ and i.p. zinc sulfate (5 mg/kg/day). Hippocampus tissue samples were taken following the cervical dislocation of the animals under general anesthesia. Nogo-A receptor and osteocalcin gene expression levels were determined by real-time-PCR method. Zinc supplementation attenuated the increase in hippocampal Nogo-A receptor gene expression, which was significantly increased in zinc deficiency. Again, zinc supplementation upregulated the intrinsic protective mechanisms of the brain by activating osteocalcin-expressing cells in the brain. The results of the study show that zinc has critical effects on Nogo-A receptor gene expression and hippocampal osteocalcin gene expression levels in the memory-sensitive rat hippocampus that is impaired by icv-STZ injection. These results are the first to examine the effect of zinc deficiency and supplementation on hippocampal Nogo-A receptor and osteocalcin gene expression in icv-STZ injection in rats.

Modulation of the Microglial Nogo-A/NgR Signaling Pathway as a Therapeutic Target for Multiple Sclerosis.

Current therapeutics targeting chronic phases of multiple sclerosis (MS) are considerably limited in reversing the neural damage resulting from repeated inflammation and demyelination insults in the multi-focal lesions. This inflammation is propagated by the activation of microglia, the endogenous immune cell aiding in the central nervous system homeostasis. Activated microglia may transition into polarized phenotypes; namely, the classically activated proinflammatory phenotype (previously categorized as M1) and the alternatively activated anti-inflammatory phenotype (previously, M2). These transitional microglial phenotypes are dynamic states, existing as a continuum. Shifting microglial polarization to an anti-inflammatory status may be a potential therapeutic strategy that can be harnessed to limit neuroinflammation and further neurodegeneration in MS. Our research has observed that the obstruction of signaling by inhibitory myelin proteins such as myelin-associated inhibitory factor, Nogo-A, with its receptor (NgR), can regulate microglial cell function and activity in pre-clinical animal studies. Our review explores the microglial role and polarization in MS pathology. Additionally, the potential therapeutics of targeting Nogo-A/NgR cellular mechanisms on microglia migration, polarization and phagocytosis for neurorepair in MS and other demyelination diseases will be discussed.

Nogo-A Regulates the Fate of Human Dental Pulp Stem Cells toward Osteogenic, Adipogenic, and Neurogenic Differentiation.

Human teeth are highly innervated organs that contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. Specific molecules are often used in these treatments to favorably modulate the function and fate of stem cells. Nogo-A, a key regulator of neuronal growth and differentiation, is already used in clinical tissue regeneration trials. While the functions of Nogo-A in neuronal tissues are extensively explored, its role in teeth still remains unknown. In this work, we first immunohistochemically analyzed the distribution of Nogo-A protein in the dental pulp of human teeth. Nogo-A is localized in a variety of cellular and structural components of the dental pulp, including odontoblasts, fibroblasts, neurons and vessels. We also cross-examined Nogo expression in the various pulp cell clusters in a single cell RNA sequencing dataset of human dental pulp, which showed high levels of expression in all cell clusters, including that of stem cells. We then assessed the role of Nogo-A on the fate of human dental pulp stem cells and their differentiation capacity in vitro. Using immunostaining, Alizarin Red S, Nile Red and Oil Red O staining we showed that Nogo-A delayed the differentiation of cultured dental pulp stem cells toward the osteogenic, adipogenic and neurogenic lineages, while addition of the blocking anti-Nogo-A antibody had opposite effects. These results were further confirmed by qRT-PCR, which demonstrated overexpression of genes involved in osteogenic (RUNX2, ALP, SP7/OSX), adipogenic (PPAR-γ2, LPL) and neurogenic (DCX, TUBB3, NEFL) differentiation in the presence of the anti-Nogo-A antibody. Conversely, the osteogenic and adipogenic genes were downregulated by Nogo-A. Taken together, our results show that the functions of Nogo-A are not restricted to neuronal cells but are extended to other cell populations, including dental pulp stem cells. We show that Nogo-A regulates their fates toward osteogenic, adipogenic and neurogenic differentiation, thus indicating its potential use in clinics.

Nogo-A/NgR signaling regulates stemness in cancer stem-like cells derived from U87MG glioblastoma cells.

Neurite outgrowth inhibitor A (Nogo-A), a member of the reticulon 4 family, is an axon regeneration inhibitor that is negatively associated with the malignancy of oligodendroglial tumors. It has been suggested that the Nogo-A/Nogo Receptor (NgR) pathway plays a promoting effect in regulating cancer stem-like cells (CSCs) derived from glioblastoma, indicating that Nogo-A could exert different roles in CSCs than those in parental cancer cells. In the present study, CSCs were generated from the human Uppsala 87 malignant glioma (U87MG) cell line. These U87MG-CSCs were characterized by the upregulation of CD44 and CD133, which are two markers of stemness. The expression levels of Nogo-A and the differentiation of U87MG-CSCs were investigated. In addition, the proliferation, invasion and colony formation U87MG-CSCs were examined. Using culture in serum-containing medium, U87MG-CSCs were differentiated into neuron-like cells specifically expressing MAP2, ß-III-tubulin and nestin. Nogo-A was upregulated in U87MG-CSCs compared with parental cells. Knockdown of Nogo-A and inhibition of the Nogo-A/NgR signaling pathway in U87MG-CSCs markedly decreased cell viability, cell cycle entry, invasion and tumor formation, indicating that Nogo-A could regulate U87MG-CSC function. Moreover, Nogo-A was involved in intracellular ATP synthesis and scavenging of accumulated reactive oxygen species. Nogo-A/NgR pathway exerted protective effects against hypoxia-induced non-apoptotic and apoptotic cell death. These results suggest that Nogo-A plays an important role in regulating U87MG-CSCs via the Nogo-A/NgR signaling pathway. Nogo-A may also different roles in U87MG-CSCs compared with their parental cells.

miR-182-5p Regulates Nogo-A Expression and Promotes Neurite Outgrowth of Hippocampal Neurons In Vitro.

Nogo-A protein is a key myelin-associated inhibitor of axonal growth, regeneration, and plasticity in the central nervous system (CNS). Regulation of the Nogo-A/NgR1 pathway facilitates functional recovery and neural repair after spinal cord trauma and ischemic stroke. MicroRNAs are described as effective tools for the regulation of important processes in the CNS, such as neuronal differentiation, neuritogenesis, and plasticity. Our results show that miR-182-5p mimic specifically downregulates the expression of the luciferase reporter gene fused to the mouse Nogo-A 3'UTR, and Nogo-A protein expression in Neuro-2a and C6 cells. Finally, we observed that when rat primary hippocampal neurons are co-cultured with C6 cells transfected with miR-182-5p mimic, there is a promotion of the outgrowth of neuronal neurites in length. From all these data, we suggest that miR-182-5p may be a potential therapeutic tool for the promotion of axonal regeneration in different diseases of the CNS.

The Effect of Mild Hypothermia on Nogo-A and Neurological Function in the Brain after Cardiopulmonary Resuscitation in Rats.

ObjectiveWe investigated the dynamic changes of Nogo-A protein in brain and the effects of mild therapeutic hypothermia (MTH) on its expression after cardiopulmonary resuscitation (CPR). Methods: Western-blotting and neurological scoring of 45 rats subjected to cardiac arrest and CPR with and without MTR were performed to investigate the changes in the expression of Nogo-A protein in the hippocampus and cortex over a period of time ranging from 6 h to 72 h after restoration of spontaneous circulation (ROSC). Results: Nogo-A expression levels were increased at 6 h after CPR in the hippocampus and cortex, peaked at 24 h in the cortex, and at 48 h in the hippocampus. The expression of Nogo-A in the MTR group was significantly lower at 12 h (p < 0.05) compared to those with no MTR after ROSC. Conclusions: MTR blunts the expression of Nogo-A protein in the hippocampus and cortex after cardiac arrest and resuscitation, and MTR may provide cerebral protection after ischemia.

Co-Expression of Nogo-A in Dopaminergic Neurons of the Human Substantia Nigra Pars Compacta Is Reduced in Parkinson's Disease.

Parkinson's disease is mainly characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Together with the small number, the high vulnerability of the dopaminergic neurons is a major pathogenic culprit of Parkinson's disease. Our previous findings of a higher survival of dopaminergic neurons in the substantia nigra co-expressing Nogo-A in an animal model of Parkinson's disease suggested that Nogo-A may be associated with dopaminergic neurons resilience against Parkinson's disease neurodegeneration. In the present study, we have addressed the expression of Nogo-A in the dopaminergic neurons in the substantia nigra in postmortem specimens of diseased and non-diseased subjects of different ages. For this purpose, in a collaborative effort we developed a tissue micro array (TMA) that allows for simultaneous staining of many samples in a single run. Interestingly, and in contrast to the observations gathered during normal aging and in the animal model of Parkinson's disease, increasing age was significantly associated with a lower co-expression of Nogo-A in nigral dopaminergic neurons of patients with Parkinson's disease. In sum, while Nogo-A expression in dopaminergic neurons is higher with increasing age, the opposite is the case in Parkinson's disease. These observations suggest that Nogo-A might play a substantial role in the vulnerability of dopaminergic neurons in Parkinson's disease.

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

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