Xuesaitong exerts long-term neuroprotection for stroke recovery by inhibiting the ROCKII pathway, in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuesaitong (XST) is a traditional Chinese medicine injection with neuroprotective properties and has been extensively used to treat stroke for many years. The main component of XST is Panax notoginseng saponins (PNS), which is the main extract of the Chinese herbal medicine Panax notoginseng. AIM OF THE STUDY: In this study, we investigated whether XST provided long-term neuroprotection by inhibiting neurite outgrowth inhibitor-A (Nogo-A) and the ROCKII pathway in experimental rats after middle cerebral artery occlusion (MCAO) and in SH-SY5Y cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). MATERIALS AND METHODS: Rats with permanent MCAO were administered XST, Y27632, XST plus Y27632, and nimodipine for 14 and 28 days. Successful MCAO onset was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Neurological deficit score (NDS) was used to assess neurological impairment. Hematoxylin-eosin (HE) staining and immunohistochemical (IHC) analysis of synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) were performed to evaluate cerebral ischemic injury and the neuroprotective capability of XST. Nogo-A levels and the ROCKII pathway were detected by IHC analysis, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) to explore the protective mechanism of XST. OGD/R model was established in SH-SY5Y cells. Cell counting kit 8 (CCK8) was applied to detect the optimum OGD time and XST concentration. The expression levels Nogo-A and ROCKII pathway were determined using western blotting. RESULTS: Our results showed that XST reduced neurological dysfunction and pathological damage, promoted weight gain and synaptic regeneration, reduced Nogo-A mRNA and protein levels, and inhibited the ROCKII pathway in MCAO rats. CCK8 assay displayed that the optimal OGD time and optimal XST concentration were 7 h and 20 µg/mL respectively in SH-SY5Y cells. XST could evidently inhibit OGD/R-induced Nogo-A protein expression and ROCKII pathway activation in SH-SY5Y cells. CONCLUSIONS: The present study suggested that XST exerted long-term neuroprotective effects that assisted in stroke recovery, possibly through inhibition of the ROCKII pathway.

Nogo-A-targeting antibody promotes visual recovery and inhibits neuroinflammation after retinal injury.

N-Methyl-D-aspartate (NMDA)-induced neuronal cell death is involved in a large spectrum of diseases affecting the brain and the retina such as Alzheimer's disease and diabetic retinopathy. Associated neurological impairments may result from the inhibition of neuronal plasticity by Nogo-A. The objective of the current study was to determine the contribution of Nogo-A to NMDA excitotoxicity in the mouse retina. We observed that Nogo-A is upregulated in the mouse vitreous during NMDA-induced inflammation. Intraocular injection of a function-blocking antibody specific to Nogo-A (11C7) was carried out 2 days after NMDA-induced injury. This treatment significantly enhanced visual function recovery in injured animals. Strikingly, the expression of potent pro-inflammatory molecules was downregulated by 11C7, among which TNFα was the most durably decreased cytokine in microglia/macrophages. Additional analyses suggest that TNFα downregulation may stem from cofilin inactivation in microglia/macrophages. 11C7 also limited gliosis presumably via P.Stat3 downregulation. Diabetic retinopathy was associated with increased levels of Nogo-A in the eyes of donors. In summary, our results reveal that Nogo-A-targeting antibody can stimulate visual recovery after retinal injury and that Nogo-A is a potent modulator of excitotoxicity-induced neuroinflammation. These data may be used to design treatments against inflammatory eye diseases.

Anti­Nogo­A antibody promotes brain function recovery after cardiopulmonary resuscitation in rats by reducing apoptosis.

Brain injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is the main cause of neurological dysfunction and death in cardiac arrest. To assess the effect of Nogo­A antibody on brain function in rats following CPR and to explore the underlying mechanisms, CA/CPR (ventricular fibrillation) rats were divided into the CPR+Nogo­A, CPR+saline and sham groups. Hippocampal caspase­3 levels were detected by RT­PCR and immunoblotting. Next, Nogo­A, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cysteinyl aspartate specific proteinase­12 (casapse­12), Bcl­2 and Bax protein levels in the hippocampus were detected by immunoblotting. Coronal brain sections were analyzed by TUNEL assay to detect apoptosis at 72 h, while Nissl staining and electron microscopy were performed to detect Nissl bodies and microstructure at 24 h, respectively. Finally, rats were assessed for neurologic deficits at various times. Nissl staining revealed morphological improvement after Nogo­A antibody treatment. Sub­organelle structure was preserved as assessed by electron microscopy in model animals post­antibody treatment; neurological function was improved as well (P<0.05), while the apoptosis index was decreased (26.2±9.85 vs. 46.6±12.95%; P<0.05). Hippocampal caspase­3 mRNA and protein, Nogo­A protein levels were significantly decreased after antibody treatment (P<0.05). Hippocampal Nogo­A expression was positively correlated with caspase­3 (Pearson's correlation; r=0.790, P=0.000). Hippocampal GRP78 and Bcl­2 protein levels were higher after antibody treatment than these levels noted in the model animals (P<0.05), while CHOP, caspase­12 and Bax levels were reduced (P<0.05). Nogo­A antibody ameliorates neurological function after restoration of spontaneous circulation (ROSC), possibly by suppressing apoptosis induced by endoplasmic reticulum stress.

Nogo-B promotes tumor angiogenesis and provides a potential therapeutic target in hepatocellular carcinoma.

Tumor angiogenesis is one of the hallmarks of cancer as well as an attractive target for cancer therapy. Characterization of novel pathways that act in parallel with the VEGF/VEGFR axis to promote tumor angiogenesis may provide insights into novel anti-angiogenic therapeutic targets. We found that the expression level of Nogo-B is positively correlated with tumor vessel density in hepatocellular carcinoma (HCC). While Nogo-B depletion inhibited tumor angiogenesis, Nogo-B overexpression promoted tumor angiogenesis in a tumor xenograft subcutaneous model of the human HCC cell line. Mechanically, Nogo-B regulates tumor angiogenesis based on its association with integrin αv ß3 and activation of focal adhesion kinase. Moreover, Nogo-B antibody successfully abolished the function of Nogo-B in tumor angiogenesis in vitro and in vivo. Collectively, our results strongly suggest that Nogo-B is an important tumor angiogenic factor and blocking Nogo-B selectively inhibits tumor angiogenesis.

Nogo-A inhibits vascular regeneration in ischemic retinopathy.

Nogo-A is a potent glial-derived inhibitor of axon growth in the injured CNS and acts as a negative regulator of developmental angiogenesis by inhibiting vascular endothelial cell migration. However, its function in pathological angiogenesis has never been studied after ischemic injury in the CNS. Using the mouse model of oxygen-induced retinopathy (OIR) which yields defined zones of retinal ischemia, our goal was to investigate the role of Nogo-A in vascular regeneration. We demonstrate a marked upregulation of the Nogo-A receptor sphingosine 1-phosphate receptor 2 in blood vessels following OIR, while Nogo-A is abundantly expressed in surrounding glial cells. Acute inhibition of Nogo-A with function-blocking antibody 11C7 significantly improved vascular regeneration and consequently prevented pathological pre-retinal angiogenesis. Ultimately, inhibition of Nogo-A led to restoration of retinal function as determined by electrophysiological response of retinal cells to light stimulation. Our data suggest that anti-Nogo-A antibody may protect neuronal cells from ischemic damage by accelerating blood vessel repair in the CNS. Targeting Nogo-A by immunotherapy may improve CNS perfusion after vascular injuries.

NogoA Neutralization Promotes Axonal Restoration After White Matter Injury In Subcortical Stroke.

Blocking axonal growth inhibitor NogoA has been of great interest for promoting axonal recovery from neurological diseases. The present study investigates the therapeutic effects of blocking NogoA, inducing functional recovery and promoting white matter repair in an experimental animal model of stroke. Adult male rats were subjected to white matter injury by subcortical ischemic stroke. Twenty-four hours after surgery, 250 ug of anti-NogoA or anti-IgG-1 were administered through the tail vein. The quantity of NogoA protein was determined by immunohistochemistry in the brain and peripheral organs. In addition, functional status, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers were analyzed. Moreover, an in vitro study was performed in order to strengthen the results obtained in vivo. A lower quantity of NogoA protein was found in the brain and peripheral organs of the animals that received anti-NogoA treatment. The animals receiving anti-NogoA treatment showed significantly better results in terms of functional recovery, fiber tract integrity, axonal sprouting and white matter repair markers compared with the control group at 28 days. White matter integrity was in part restored by antibody-mediated inhibition of NogoA administration in those animals that were subjected to an axonal injury by subcortical stroke. This white matter restoration triggered functional recovery.

Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.

Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.

Nogo-B Facilitates LPS-Mediated Immune Responses by Up-Regulation of TLR4-Signaling in Macrophage RAW264.7.

BACKGROUND/AIMS: Nogo-B, a member of the reticulon family of proteins, is mainly located in the endoplasmic reticulum (ER). Here, we investigate the function and mechanism of Nogo-B in the regulation of TLR4-associated immune responses in the macrophage cell line of RAW264.7. METHODS: Nogo-B was up- and down-regulated through the use of appropriate adenoviral vectors or siRNA, and the effects of Nogo-B on macrophages under liposaccharide (LPS) stimulation were evaluated via western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), flow cytometric analysis, and transwell assay. RESULTS: Our data indicates that the protein of Nogo-B was down-regulated in a time- and dose-dependent manner following LPS administration in the macrophage. Nogo-B overexpression increased the production of inflammatory cytokines (MCP-1, TNF-α, IL-1ß, and TGF-ß), enhanced macrophage migration activities, activated major histocompatibility complex II (MHC II), and elevated the expression of macrophage scavenger receptor 1(MSR1), all of which suggest that Nogo-B is necessary for immune responses and plays an important role in regulating macrophage recruitment. Mechanistically, Nogo-B may enhance TLR4 expression in macrophage surfaces, activate mitogen-activated protein kinase (MAPK) pathways, and initiate inflammatory responses. CONCLUSION: These findings illustrate the key regulatory functions of Nogo-B in facilitating LPS-mediated immune responses through promoting the phosphorylation of MAP kinase.

Nogo-66 Restricts Synaptic Strengthening via Lingo1 and the ROCK2-Cofilin Pathway to Control Actin Dynamics.

Nogo-A restricts long-term potentiation (LTP) at the Schaffer collateral-CA1 pathway in the adult hippocampus via 2 extracellular domains: Nogo-A-Δ20 and Nogo-66. Nogo-66 signals via Nogo Receptor 1 (NgR1) to regulate synaptic function. Whether the NgR1 coreceptors Lingo1 and p75NTR are involved in the signaling in this context is still not known. Moreover, the intracellular cascade mediating the activity of Nogo-66 in restricting LTP is unexplored. We combine electrophysiology and biochemistry in acute hippocampal slices and demonstrate that a loss of function for Lingo1 results in a significant increase in LTP levels at the Schaffer collateral-CA1 pathway, and that Lingo1 is the NgR1 coreceptor mediating the role of Nogo-66 in restricting LTP. Our data show that p75NTR is not involved in mediating the Nogo-66 effect on LTP. Moreover, loss of function for p75NTR and NgR1 equally attenuate LTD, suggesting that p75NTR might mediate the NgR1-dependent regulation of LTD, independently of Nogo-66. Finally, our results indicate that Nogo-66 signaling limits LTP via the ROCK2-Cofilin pathway to control the dynamics of the actin cytoskeleton. The present results elucidate the signaling pathway activated by Nogo-66 to control LTP and contribute to the understanding of how Nogo-A stabilizes the neural circuits to limit activity-dependent plasticity events in the mature hippocampus.

NOGO-A/RTN4A and NOGO-B/RTN4B are simultaneously expressed in epithelial, fibroblast and neuronal cells and maintain ER morphology.

Reticulons (RTNs) are a large family of membrane associated proteins with various functions. NOGO-A/RTN4A has a well-known function in limiting neurite outgrowth and restricting the plasticity of the mammalian central nervous system. On the other hand, Reticulon 4 proteins were shown to be involved in forming and maintaining endoplasmic reticulum (ER) tubules. Using comparative transcriptome analysis and qPCR, we show here that NOGO-B/RTN4B and NOGO-A/RTN4A are simultaneously expressed in cultured epithelial, fibroblast and neuronal cells. Electron tomography combined with immunolabelling reveal that both isoforms localize preferably to curved membranes on ER tubules and sheet edges. Morphological analysis of cells with manipulated levels of NOGO-B/RTN4B revealed that it is required for maintenance of normal ER shape; over-expression changes the sheet/tubule balance strongly towards tubules and causes the deformation of the cell shape while depletion of the protein induces formation of large peripheral ER sheets.

Knockdown of Nogo gene by short hairpin RNA interference promotes functional recovery of spinal cord injury in a rat model.

The specific myelin component Nogo protein is one of the major inhibitory molecules of spinal cord axonal outgrowth following spinal cord injury. The present study aimed to investigate the effects of silencing Nogo protein with shRNA interference on the promotion of functional recovery in a rat model with spinal cord hemisection. Nogo-A short hairpin RNAs (Nogo shRNAs) were constructed and transfected into rats with spinal cord hemisection by adenovirus-mediated transfection. Reverse transcription­polymerase chain reaction and western blotting were performed to analyze the expression of Nogo-A and Growth Associated Protein 43 (GAP-43). In addition, Basso Beattie Bresnahan (BBB) scores were used to assess the functional recovery of rats following spinal cord injury. The results demonstrated that expression of the Nogo­A gene was observed to be downregulated following transfection and GAP­43 expression was observed to increase. The BBB scores were increased following treatment with Nogo shRNAs, indicating functional recovery of the injured nerves. Thus, Nogo-A shRNA interference can knockdown Nogo gene expression and upregulate GAP-43 to promote the functional recovery of spinal cord injury in rats. This finding may advance progress toward assisting the regeneration of injured neurons through the use of Nogo-A shRNA.