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.

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.

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.

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.

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.

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.

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.

Astrocytes as a target for Nogo-A and implications for synapse formation in vitro and in a model of acute demyelination.

Central nervous system (CNS) function depends on precise synaptogenesis, which is shaped by environmental cues and cellular interactions. Astrocytes are outstanding regulators of synapse development and plasticity through contact-dependent signals and through the release of pro- and antisynaptogenic factors. Conversely, myelin and its associated proteins, including Nogo-A, affect synapses in a inhibitory fashion and contribute to neural circuitry stabilization. However, the roles of Nogo-A-astrocyte interactions and their implications in synapse development and plasticity have not been characterized. Therefore, we aimed to investigate whether Nogo-A affects the capacity of astrocytes to induce synaptogenesis. Additionally, we assessed whether downregulation of Nogo-A signaling in an in vivo demyelination model impacts the synaptogenic potential of astrocytes. Our in vitro data show that cortical astrocytes respond to Nogo-A through RhoA pathway activation, exhibiting stress fiber formation and decreased ramified morphology. This phenotype was associated with reduced levels of GLAST protein and aspartate uptake, decreased mRNA levels of the synaptogenesis-associated genes Hevin, glypican-4, TGF-ß1 and BDNF, and decreased and increased protein levels of Hevin and SPARC, respectively. Corroborating these findings, conditioned medium from Nogo-A-treated astrocytes suppressed the formation of structurally and functionally mature synapses in cortical neuronal cultures. After cuprizone-induced acute demyelination, we observed reduced immunostaining for Nogo-A in the visual cortex accompanied by higher levels of Hevin expression in astrocytes and an increase in excitatory synapse density. Hence, we suggest that interactions between Nogo-A and astrocytes might represent an important pathway of plasticity regulation and could be a target for therapeutic intervention in demyelinating diseases in the future.

The Regulatory Role of Reticulons in Neurodegeneration: Insights Underpinning Therapeutic Potential for Neurodegenerative Diseases.

In the last few decades, cytoplasmic organellar dysfunction, such as that of the endoplasmic reticulum (ER), has created a new area of research interest towards the development of serious health maladies including neurodegenerative diseases. In this context, the extensively dispersed family of ER-localized proteins, i.e. reticulons (RTNs), is gaining interest because of its regulative control over neural regeneration. As most neurodegenerative diseases are pathologically manifested with the accretion of misfolded proteins with subsequent induction of ER stress, the regulatory role of RTNs in neural dysfunction cannot be ignored. With the limited information available in the literature, delineation of the functional connection between rising consequences of neurodegenerative diseases and RTNs need to be elucidated. In this review, we provide a broad overview on the recently revealed regulatory roles of reticulons in the pathophysiology of several health maladies, with special emphasis on neurodegeneration. Additionally, we have also recapitulated the decisive role of RTN4 in neurite regeneration and highlighted how neurodegeneration and proteinopathies are mechanistically linked with each other through specific RTN paralogues. With the recent findings advocating zebrafish Rtn4b (a mammalian Nogo-A homologue) downregulation following central nervous system (CNS) lesion, RTNs provides new insight into the CNS regeneration. However, there are controversies with respect to the role of Rtn4b in zebrafish CNS regeneration. Given these controversies, the connection between the unique regenerative capabilities of zebrafish CNS by distinct compensatory mechanisms and Rtn4b signalling pathway could shed light on the development of new therapeutic strategies against serious neurodegenerative diseases.

Identification of a new functional domain of Nogo-A that promotes inflammatory pain and inhibits neurite growth through binding to NgR1.

Nogo-A is a key inhibitory molecule to axon regeneration, and plays diverse roles in other pathological conditions, such as stroke, schizophrenia, and neurodegenerative diseases. Nogo-66 and Nogo-Δ20 fragments are two known functional domains of Nogo-A, which act through the Nogo-66 receptor (NgR1) and sphingosine-1-phosphate receptor 2 (S1PR2), respectively. Here, we reported a new functional domain of Nogo-A, Nogo-A aa 846-861, was identified in the Nogo-A-specific segment that promotes complete Freund's adjuvant (CFA)-induced inflammatory pain. Intrathecal injection of its antagonist peptide 846-861PE or the specific antibody attenuated the CFA-induced inflammatory heat hyperalgesia. The 846-861 PE reduced the content of transient receptor potential vanilloid subfamily member 1 (TRPV1) in dorsal root ganglia (DRG) and decreased the response of DRG neurons to capsaicin. These effects were accompanied by a reduction in LIMK/cofilin phosphorylation and actin polymerization. GST pull-down and fluorescence resonance energy transfer (FRET) assays both showed that Nogo-A aa 846-861 bound to NgR1. Moreover, we demonstrated that Nogo-A aa 846-861 inhibited neurite outgrowth from cortical neurons and DRG explants. We concluded that Nogo-A aa 846-861 is a novel ligand of NgR1, which activates the downstream signaling pathways that inhibit axon growth and promote inflammatory pain.

Nogo-A Induced Polymerization of Microtubule Is Involved in the Inflammatory Heat Hyperalgesia in Rat Dorsal Root Ganglion Neurons.

The microtubule, a major constituent of cytoskeletons, was shown to bind and interact with transient receptor potential vanilloid subfamily member 1 (TRPV1), and serves a pivotal role to produce thermal hyperalgesia in inflammatory pain. Nogo-A is a modulator of microtubule assembly and plays a key role in maintaining the function of TRPV1 in inflammatory heat pain. However, whether the microtubule dynamics modulated by Nogo-A in dorsal root ganglion (DRG) neurons participate in the inflammatory pain is not elucidated. Here we reported that the polymerization of microtubules in the DRG neurons, as indicated by the acetylated α-tubulin, tubulin polymerization-promoting protein 3 (TPPP3), and microtubule numbers, was significantly elevated in the complete Freund's adjuvant (CFA) induced inflammatory pain. Consistent with our previous results, knock-out (KO) of Nogo-A protein significantly attenuated the heat hyperalgesia 72 h after CFA injection and decreased the microtubule polymerization via up-regulation of phosphorylation of collapsin response mediator protein 2 (CRMP2) in DRG. The colocalization of acetylated α-tubulin and TRPV1 in DRG neurons was also reduced dramatically in Nogo-A KO rats under inflammatory pain. Moreover, the down-regulation of TRPV1 in DRG of Nogo-A KO rats after injection of CFA was reversed by intrathecal injection of paclitaxel, a microtubule stabilizer. Furthermore, intrathecal injection of nocodazole (a microtubule disruptor) attenuated significantly the CFA-induced inflammatory heat hyperalgesia and the mechanical pain in a rat model of spared nerve injury (SNI). In these SNI cases, the Nogo-A and acetylated α-tubulin in DRG were also significantly up-regulated. We conclude that the polymerization of microtubules promoted by Nogo-A in DRG contributes to the development of inflammatory heat hyperalgesia mediated by TRPV1.

Plasticity-Enhancing Effects of Levodopa Treatment after Stroke.

Dopaminergic treatment in combination with rehabilitative training enhances long-term recovery after stroke. However, the underlying mechanisms on structural plasticity are unknown. Here, we show an increased dopaminergic innervation of the ischemic territory during the first week after stroke induced in Wistar rats subjected to transient occlusion of the middle cerebral artery (tMCAO) for 120 min. This response was also found in rats subjected to permanent focal ischemia induced by photothrombosis (PT) and mice subjected to PT or tMCAO. Dopaminergic branches were detected in the infarct core of mice and rats in both stroke models. In addition, the Nogo A pathway was significantly downregulated in rats treated with levodopa (LD) compared to vehicle-treated animals subjected to tMCAO. Specifically, the number of Nogo A positive oligodendrocytes as well as the levels of Nogo A and the Nogo A receptor were significantly downregulated in the peri-infarct area of LD-treated animals, while the number of Oligodendrocyte transcription factor 2 positive cells increased in this region after treatment. In addition, we observed lower protein levels of Growth Associated Protein 43 in the peri-infarct area compared to sham-operated animals without treatment effect. The results provide the first evidence of the plasticity-promoting actions of dopaminergic treatment following stroke.

Anti-Nogo-A Antibodies As a Potential Causal Therapy for Lower Urinary Tract Dysfunction after Spinal Cord Injury.

Loss of bladder control is common after spinal cord injury (SCI) and no causal therapies are available. Here we investigated whether function-blocking antibodies against the nerve-fiber growth inhibitory protein Nogo-A applied to rats with severe SCI could prevent development of neurogenic lower urinary tract dysfunction. Bladder function of rats with SCI was repeatedly assessed by urodynamic examination in fully awake animals. Four weeks after SCI, detrusor sphincter dyssynergia had developed in all untreated or control antibody-infused animals. In contrast, 2 weeks of intrathecal anti-Nogo-A antibody treatment led to significantly reduced aberrant maximum detrusor pressure during voiding and a reduction of the abnormal EMG high-frequency activity in the external urethral sphincter. Anatomically, we found higher densities of fibers originating from the pontine micturition center in the lumbosacral gray matter in the anti-Nogo-A antibody-treated animals, as well as a reduced number of inhibitory interneurons in lamina X. These results suggest that anti-Nogo-A therapy could also have positive effects on bladder function clinically.SIGNIFICANCE STATEMENT After spinal cord injury, loss of bladder control is common. Detrusor sphincter dyssynergia is a potentially life-threatening consequence. Currently, only symptomatic treatment options are available. First causal treatment options are urgently needed in humans. In this work, we show that function-blocking antibodies against the nerve-fiber growth inhibitory protein Nogo-A applied to rats with severe spinal cord injury could prevent development of neurogenic lower urinary tract dysfunction, in particular detrusor sphincter dyssynergia. Anti-Nogo-A therapy has entered phase II clinical trial in humans and might therefore soon be the first causal treatment option for neurogenic lower urinary tract dysfunction.

Nogo receptor antagonist LOTUS exerts suppression on axonal growth-inhibiting receptor PIR-B.

Damaged axons in the adult mammalian central nervous system have a restricted regenerative capacity mainly because of Nogo protein, which is a major myelin-associated axonal growth inhibitor with binding to both receptors of Nogo receptor-1 (NgR1) and paired immunoglobulin-like receptor (PIR)-B. Lateral olfactory tract usher substance (LOTUS) exerts complete suppression of NgR1-mediated axonal growth inhibition by antagonizing NgR1. However, the regulation of PIR-B functions in neurons remains unknown. In this study, protein-protein interactions analyses found that LOTUS binds to PIR-B and abolishes Nogo-binding to PIR-B completely. Reverse transcription-polymerase chain reaction and immunocytochemistry revealed that PIR-B is expressed in dorsal root ganglions (DRGs) from wild-type and Ngr1-deficient mice (male and female). In these DRG neurons, Nogo induced growth cone collapse and neurite outgrowth inhibition, but treatment with the soluble form of LOTUS completely suppressed them. Moreover, Nogo-induced growth cone collapse and neurite outgrowth inhibition in Ngr1-deficient DRG neurons were neutralized by PIR-B function-blocking antibodies, indicating that these Nogo-induced phenomena were mediated by PIR-B. Our data show that LOTUS negatively regulates a PIR-B function. LOTUS thus exerts an antagonistic action on both receptors of NgR1 and PIR-B. This may lead to an improvement in the defective regeneration of axons following injury.

Expression of Nogo-A in dorsal root ganglion in rats with cauda equina injury.

OBJECTIVE: To investigate the expression of Nogo-A in dorsal root ganglion (DRG) in rats with cauda equina injury and the therapeutic effects of blocking Nogo-A and its receptor. METHODS AND MATERIALS: Fifty-eight male Sprague-Dawley rats were divided randomly into either the sham operation group (n = 24) or the cauda equina compression (CEC) control group (n = 34). Behavioral, histological, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were conducted to assess the establishment of the model. The dynamic expression change of Nogo-A was evaluated using real time-qPCR. Immunofluorescence was used to evaluate the expression of Nogo-A in the DRG and cauda equina. Furthermore, 20 male Sprague-Dawley rats were equally divided into 4 groups, including the sham group, the CEC group, the NEP1-40 (the NgR antagonist peptide) treatment group, and the JTE-013 (the S1PR2 antagonist) treatment group. Behavioral assessments and western blotting were used to evaluate the therapeutic effect of cauda equina injury via blocking Nogo-A and its receptor. RESULTS: Tactile allodynia and heat hyperalgesia in the CEC model developed as soon as 1 day after surgery and recovered to normal at 7 days, which was followed by the downregulation of Nogo-A in DRG neurons. However, the locomotor function impairment in the CEC model showed a different prognosis from the sensory function, which was consistent with the expression change of Nogo-A in the spinal cord. Immunofluorescence results also demonstrated that Nogo A-positive/NF200-negative neurons and axons increased in the DRG and cauda equina 7 days after surgery. Surprisingly, Schwann cells, which myelinate axons in the PNS, also expressed considerable amounts of Nogo-A. Then, after blocking the Nogo-A/NgR signaling pathway by NEP1-40, significant improvement of mechanical allodynia was identified in the first 2 days after the surgery. Western blotting suggested the NEP1-40 treatment group had lower expression of cleaved caspase-3 than the CEC and JTE-013 treatment group. CONCLUSION: Neuronal Nogo-A in the DRG may be involved in regeneration and play a protective role in the CEC model. Whereas Nogo-A, released from the injured axons or expressed by Schwann cells, may act as an inhibiting factor in the process of CEC repairment. Thus, blocking the Nogo-A/NgR signaling pathway can alleviate mechanical allodynia by apoptosis inhibition.

Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone.

The promotion of axonal regeneration is required for functional recovery from stroke and various neuronal injuries. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. Nogo-66, a C-terminal domain of Nogo-A, binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibits neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors, suggesting it is an important target for the prevention of axonal growth inhibition. By using the indirect immunofluorescence method, we show for the first time that a cell-permeable cAMP analog (dibutyryl-cAMP) induced a rapid decrease in the cell surface expression of NgR1 in Neuroscreen-1 (NS-1) cells. The biotinylation method revealed that cAMP indeed induced internalization of NgR1 within minutes. Other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, also induced this process. This internalization was found to be reversible and influenced by intracellular levels of cAMP. Using selective activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA, but dependent on Epac. The decrease in cell surface expression of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, it is likely that besides axonal growth inhibitors affecting neurons, neurons themselves also self-regulate their sensitivity to axonal growth inhibitors, as influenced by intracellular cAMP/Epac. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors, and enhance functional recovery after stroke and neuronal injuries.

Fasudil reduces ß-amyloid levels and neuronal apoptosis in APP/PS1 transgenic mice via inhibition of the Nogo-A/NgR/RhoA signaling axis.

Recent studies have shown that Nogo-A and the Nogo-A receptor affect ß-amyloid metabolism and the downstream Rho GTP enzyme signaling pathway, which may affect the levels of ß-amyloid and tau. Nogo-A may play a key role in the pathogenesis of Alzheimer's disease. However, the underlying molecular mechanisms of Fasudil treatment in Alzheimer's disease are not yet clear. Our results have found that Fasudil treatment for two months substantially ameliorated behavioral deficits, diminished ß-amyloid plaque and tau protein pathology, and alleviated neuronal apoptosis in APP/PS1 transgenic mice. More importantly, two well-established markers for synaptic function, growth-associated protein 43 and synaptophysin, were upregulated after Fasudil treatment. Finally, the levels of Nogo-A, Nogo-A receptor complex NgR/p75NTR/LINGO-1 and the downstream Rho/Rho kinase signaling pathway were significantly reduced. These findings suggest that Fasudil exerts its neuroprotective function in Alzheimer's disease by inhibiting the Nogo-A/NgR1/RhoA signaling pathway.

Application of Antibodies to Neuronally Expressed Nogo-A Increases Neuronal Survival and Neurite Outgrowth.

Nogo-A, a glycoprotein expressed in oligodendrocytes and central nervous system myelin, inhibits regeneration after injury. Antibodies against Nogo-A neutralize this inhibitory activity, improve locomotor recovery in spinal cord-injured adult mammals, and promote regrowth/sprouting/saving of damaged axons beyond the lesion site. Nogo-A is also expressed by neurons. Complete ablation of Nogo-A in all cell types expressing it has been found to lead to recovery in some studies but not in others. Neuronal ablation of Nogo-A reduces axonal regrowth after injury. In view of these findings, we hypothesized that, in addition to neutralizing Nogo-A in oligodendrocytes and myelin, Nogo-A antibodies may act directly on neuronal Nogo-A to trigger neurite outgrowth and neuronal survival. Here, we show that polyclonal and monoclonal antibodies against Nogo-A enhance neurite growth and survival of cultured cerebellar granule neurons and increase expression of the neurite outgrowth-promoting L1 cell adhesion molecule and polysialic acid. Application of inhibitors of signal transducing molecules, such as c-src, c-fyn, protein kinase A, and casein kinase II reduce antibody-triggered neurite outgrowth. These observations indicate that the recovery-promoting functions of antibodies against Nogo-A may not only be due to neutralizing Nogo-A in oligodendrocytes and myelin, but also to their interactions with Nogo-A on neurons.

Inactivation of sphingosine-1-phosphate receptor 2 (S1PR2) decreases demyelination and enhances remyelination in animal models of multiple sclerosis.

Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) in which multiple sites of blood-brain barrier (BBB) disruption, focal inflammation, demyelination and tissue destruction are the hallmarks. Here we show that sphingosine-1-phosphate receptor 2 (S1PR2) has a negative role in myelin repair as well as an important role in demyelination by modulating BBB permeability. In lysolecithin-induced demyelination of adult mouse spinal cord, S1PR2 inactivation by either the pharmacological inhibitor JTE-013 or S1PR2 gene knockout led to enhanced myelin repair as determined by higher numbers of differentiated oligodendrocytes and increased numbers of remyelinated axons at the lesion sites. S1PR2 inactivation in lysolecithin-induced demyelination of the optic chiasm, enhanced oligodendrogenesis and improved the behavioral outcome in an optokinetic reflex test. In order to see the effect of S1PR2 inactivation on demyelination, experimental autoimmune encephalitis (EAE) was induced by MOG-peptide. S1PR2 inhibition or knockout decreased the extent of demyelinated areas as well as the clinical disability in this EAE model. Both toxin induced and EAE models showed decreased BBB leakage and reduced numbers of Iba1+ macrophages following S1PR2 inactivation. Our results suggest that S1PR2 activity impairs remyelination and also enhances BBB leakage and demyelination. The former effect could be mediated by Nogo-A, as antagonism of this factor enhances remyelination and S1PR2 can act as a Nogo-A receptor.

RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.

Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.