Function of Nogo‐A/Nogo‐A Receptor in Alzheimer's Disease

Nogo‐A is a protein inhibiting axonal regeneration, which is considered a major obstacle to nerve regeneration after injury in mammals. Rapid progress has been achieved in new physiopathological function of Nogo‐A in Alzheimer's disease in the past decade. Recent research shows that through binding to Nogo‐A receptor, Nogo‐A plays an important role in Alzheimer's disease (AD) pathogenesis. Particularly, Nogo‐A/Nogo‐A receptors modulate the generation of amyloid β‐protein (Aβ), which is thought to be a major cause of AD. This review describes the recent development of Nogo‐A, Nogo‐A receptor, and downstream signaling involved in AD and pharmacological basis of therapeutic drugs. We concluded the Nogo‐A/Nogo‐A receptor provide new insight into potential mechanisms and promising therapy strategies in AD.     

Protein microarray analysis identifies human cellular prion protein interactors

Aims: To obtain an insight into the function of cellular prion protein (PrPC), we studied PrPC‐interacting proteins (PrPIPs) by analysing a protein microarray. Methods: We identified 47 novel PrPIPs by probing an array of 5000 human proteins with recombinant human PrPC spanning amino acid residues 23–231 named PR209. Results: The great majority of 47 PrPIPs were annotated as proteins involved in the recognition of nucleic acids. Coimmunoprecipitation and cell imaging in a transient expression system validated the interaction of PR209 with neuronal PrPIPs, such as FAM64A, HOXA1, PLK3 and MPG. However, the interaction did not generate proteinase K‐resistant proteins. KeyMolnet, a bioinformatics tool for analysing molecular interaction on the curated knowledge database, revealed that the complex molecular network of PrPC and PrPIPs has a significant relationship with AKT, JNK and MAPK signalling pathways. Conclusions: Protein microarray is a useful tool for systematic screening and comprehensive profiling of the human PrPC interactome. Because the network of PrPC and interactors involves signalling pathways essential for regulation of cell survival, differentiation, proliferation and apoptosis, these observations suggest a logical hypothesis that dysregulation of the PrPC interactome might induce extensive neurodegeneration in prion diseases.     

Nogo‐A expression in oligodendroglial tumors

Nogo‐A belongs to the reticulon protein family and is expressed in the inner and outer loops of myelin sheaths of oligodendrocytes. We analyzed the patterns of Nogo‐A expression in human gliomas in an effort to identify a useful marker for the characterization of oligodendroglial tumors. We determined the expression of Nogo‐A in a panel of 58 astrocytic and oligodendroglial tumors using immunohistochemistry and compared the expression of Nogo‐A with Olig‐2, a recently identified marker for oligodendrogliomas. To localize Nogo‐A expression, immunofluorescent staining was performed using other glial markers (MAP‐2 and GFAP). We also confirmed the overexpression of the Nogo‐A protein in 53 astrocytic and oligodendroglial tumors using Western blot analysis. Based on immunohistochemical analysis, Nogo‐A and Olig‐2 had specificity in the detection of oligodendroglial tumors from astrocytic tumors (P = 0.001). The level of Nogo‐A staining was highly correlated with Olig‐2 (P = 0.001). The sensitivity and specificity of Nogo‐A for oligodendroglial tumors was 86.9% and 57.1%, respectively. Nogo‐A expression overlapped that of other oligodendroglial markers, but with different patterns of expression. Western blot analysis revealed that Nogo‐A is predominantly expressed in 85.7% of oligodendroglioma cells and 93.7% of anaplastic oligodendroglioma cells. Like other oligodendroglial markers, Nogo‐A is highly expressed in oligodendroglial tumors; however, it does not serve as a definite marker specific for oligodendroglial tumors.     

Combination treatment with anti‐Nogo‐A and chondroitinase ABC is more effective than single treatments at enhancing functional recovery after spinal cord injury

Anti‐Nogo‐A antibody and chondroitinase ABC (ChABC) enzyme are two promising treatments that promote functional recovery after spinal cord injury (SCI). Treatment with them has encouraged axon regeneration, sprouting and functional recovery in a variety of spinal cord and central nervous system injury models. The two compounds work, in part, through different mechanisms, so it is possible that their effects will be additive. In this study, we used a rat cervical partial SCI model to explore the effectiveness of a combination of anti‐Nogo‐A, ChABC, and rehabilitation. We found that spontaneous recovery of forelimb functions reflects the extent of the lesion on the ipsilateral side. We applied a combination treatment with acutely applied anti‐Nogo‐A antibody followed by delayed ChABC treatment starting at 3 weeks after injury, and rehabilitation starting at 4 weeks, to accommodate the requirement that anti‐Nogo‐A be applied acutely, and that rehabilitation be given after the cessation of anti‐Nogo‐A treatment. We found that single treatment with either anti‐Nogo‐A or ChABC, combined with rehabilitation, produced functional recovery of similar magnitude. The combination treatment, however, was more effective. Both single treatments produced increases in sprouting and axon regeneration, but the combination treatment produced greater increases. Anti‐Nogo‐A stimulated growth of a greater number of axons with a diameter of > 3 μm, whereas ChABC treatment stimulated increased growth of finer axons with varicosities. These results point to different functions of Nogo‐A and chondroitin sulfate proteoglycans in axonal regeneration. The combination of anti‐Nogo‐A, ChABC and rehabilitation shows promise for enhancing functional recovery after SCI.     

Upregulation of axon guidance molecules in the adult central nervous system of Nogo‐A knockout mice restricts neuronal growth and regeneration

Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo‐A/Nogo receptor (NgR1) signaling pathway. Nogo‐A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody‐mediated neutralization of Nogo‐A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo‐A KO vs. wild‐type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4‐positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth‐inhibitory than WT but more than Nogo‐A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo‐A KO myelin as compared with WT neurons, supporting increased EphA4‐mediated growth inhibition in Nogo‐A KO mice. Consistently, in vivo, Nogo‐A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo‐A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo‐A.     

2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase: A novel RNA‐binding protein that inhibits protein synthesis

2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP) is one of the earliest myelin‐related proteins to be specifically expressed in differentiating oligodendrocytes (ODCs) in the central nervous system (CNS) and is implicated in myelin biogenesis. CNP possesses an in vitro enzymatic activity, whose in vivo relevance remains to be defined, because substrates with 2′,3,‐cyclic termini have not yet been identified. To characterize CNP function better, we previously determined the structure of the CNP catalytic domain by NMR. Interestingly, the structure is remarkably similar to the plant cyclic nucleotide phosphodiesterase (CPDase) from A. thaliana and the bacterial 2′‐5′ RNA ligase from T. thermophilus, which are known to play roles in RNA metabolism. Here we show that CNP is an RNA‐binding protein. Furthermore, by using precipitation analyses, we demonstrate that CNP associates with poly(A)⁺ mRNAs in vivo and suppresses translation in vitro in a dose‐dependent manner. With SELEX, we isolated RNA aptamers that can suppress the inhibitory effect of CNP on translation. We also demonstrate that CNP1 can bridge an association between tubulin and RNA. These results suggest that CNP1 may regulate expression of mRNAs in ODCs of the CNS. © 2008 Wiley‐Liss, Inc.     

Combined with anti‐Nogo‐A antibody treatment,BDNF did not compensate the extra deleterious motor effect caused by large size cervical cord hemisection in adult macaques

In spinal cord injured adult mammals, neutralizing the neurite growth inhibitor Nogo‐A with antibodies promotes axonal regeneration and functional recovery, although axonal regeneration is limited in length. Neurotrophic factors such as BDNF stimulate neurite outgrowth and protect axotomized neurons. Can the effects obtained by neutralizing Nogo‐A, inducing an environment favorable for axonal sprouting, be strengthened by adding BDNF? A unilateral incomplete hemicord lesion at C7 level interrupted the main corticospinal component in three groups of adult macaque monkeys: control monkeys (n = 6), anti‐Nogo‐A antibody‐treated monkeys (n = 7), and anti‐Nogo‐A antibody and BDNF‐treated monkeys (n = 5). The functional recovery of manual dexterity was significantly different between the 3 groups of monkeys, the lowest in the control group. Whereas the anti‐Nogo‐A antibody‐treated animals returned to manual dexterity performances close to prelesion ones, irrespective of lesion size, both the control and the anti‐Nogo‐A/BDNF animals presented a limited functional recovery. In the control group, the limited spontaneous functional recovery depended on lesion size, a dependence absent in the combined treatment group (anti‐Nogo‐A antibody and BDNF). The functional recovery in the latter group was significantly lower than in anti‐Nogo‐A antibody‐treated monkeys, although the lesion was larger in three out of the five monkeys in the combined treatment group.     

Behavioral analysis of the huntingtin‐associated protein 1 ortholog trak‐1 in Caenorhabditis elegans

The precise role of huntingtin‐associated protein 1 (HAP1) is not known, but studies have shown that it is important for early development and survival. A Caenorhabditis elegans ortholog of HAP1, T27A3.1 (also called trak‐1), has been found and is expressed in a subset of neurons. Potential behavioral functions of three knockout lines of T27A3.1 were examined. From its suspected role in mice we hypothesize that T27A3.1 might be involved in egg hatching and early growth, mechanosensation, chemosensation, sensitivity to osmolarity, and synaptic transmission. Our studies show that the knockout worms are significantly different from the wild‐type (WT) worms only in the synaptic transmission test, which was measured by adding aldicarb, an acetylcholinesterase inhibitor. The change in function was determined by measuring the number of worms paralyzed. However, when the T27A3.1 worms were tested for egg hatching and early growth, mechanosensation, chemosensation, and sensitivity to osmolarity, there were no significant differences between the knockout and WT worms. © 2016 Wiley Periodicals, Inc.     

Isoform‐specific localization of Nogo protein in the optic pathway of mouse embryos

Expression of Nogo protein was investigated in the optic pathway of embryonic mice by using isoform‐specific antibodies Bianca and 11C7, which recognize Nogo‐A/B and Nogo‐A, respectively. Our previous reports from using antibody N18 have shown that Nogo is localized on the radial glia in the retina and at the midline of the ventral diencephalon in mouse embryos during the ingrowth of retinal ganglion cells (RGCs) axons. This glial‐specific localization is markedly different from findings in other studies. This study showed Nogo‐A/B primarily on radial glia in the retina at E13 and then later on retinal ganglion cells and axons at E14 and E15, whereas Nogo‐A was expressed preferentially by RGCs and their axons. In the ventral diencephalon, Nogo‐A/B was expressed strongly on radial glia, particularly in those located in the midline region of the chiasm but also on RGC axons. In Nogo‐A knockout embryos, the isoform Nogo‐B (revealed by Bianca) was observed on radial glia in the ventral diencephalon and on RGCs and their axons. We concluded that Nogo‐A is localized on the ganglion cells and retinal axons, whereas Nogo‐B is expressed by the radial glia in the optic pathway. Nogo‐B may play an important role in guiding axon growth in decisive regions of the visual pathway, which include the optic disc and the optic chiasm. J. Comp. Neurol. 524:2322–2334, 2016. © 2016 Wiley Periodicals, Inc.     

Further evaluation of [11C]MP‐10 as a radiotracer for phosphodiesterase 10A: PET imaging study in rhesus monkeys and brain tissue metabolite analysis

[¹¹C]MP‐10 is a potent and specific PET tracer previously shown to be suitable for imaging the phosphodiesterase 10A (PDE10A) in baboons with reversible kinetics and high specific binding. However, another report indicated that [¹¹C]MP‐10 displayed seemingly irreversible kinetics in rhesus monkeys, potentially due to the presence of a radiolabeled metabolite capable of penetrating the blood‐brain‐barrier (BBB) into the brain. This study was designed to address the discrepancies between the species by re‐evaluating [¹¹C]MP‐10 in vivo in rhesus monkey with baseline scans to assess tissue uptake kinetics and self‐blocking scans with unlabeled MP‐10 to determine binding specificity. Ex vivo studies with one rhesus monkey and 4 Sprague‐Dawley rats were also performed to investigate the presence of radiolabeled metabolites in the brain. Our results indicated that [¹¹C]MP‐10 displayed reversible uptake kinetics in rhesus monkeys, albeit slower than in baboons. Administration of unlabeled MP‐10 reduced the binding of [¹¹C]MP‐10 in a dose‐dependent manner in all brain regions including the cerebellum. Consequently, the cerebellum appeared not to be a suitable reference tissue in rhesus monkeys. Regional volume of distribution (V_T) was mostly reliably derived with the multilinear analysis (MA1) method. In ex vivo studies in the monkey and rats only negligible amount of radiometabolites was seen in the brain of either species. In summary, results from the present study strongly support the suitability of [¹¹C]MP‐10 as a radiotracer for PET imaging and quantification of PDE10A in nonhuman primates. Synapse 69:86–95, 2015. © 2014 Wiley Periodicals, Inc.     

Fluorescent Visualisation of the Hypothalamic Oxytocin Neurones Activated by Cholecystokinin‐8 in Rats Expressing c‐fos‐Enhanced Green Fluorescent Protein and Oxytocin‐Monomeric Red Fluorescent Protein 1 Fusion Transgenes

The up‐regulation of c‐fos gene expression is widely used as a marker of neuronal activation elicited by various stimuli. Anatomically precise observation of c‐fos gene products can be achieved at the RNA level by in situ hybridisation or at the protein level by immunocytochemistry. Both of these methods are time and labour intensive. We have developed a novel transgenic rat system that enables the trivial visualisation of c‐fos expression using an enhanced green fluorescent protein (eGFP) tag. These rats express a transgene consisting of c‐fos gene regulatory sequences that drive the expression of a c‐fos‐eGFP fusion protein. In c‐fos‐eGFP transgenic rats, robust nuclear eGFP fluorescence was observed in osmosensitive brain regions 90 min after i.p. administration of hypertonic saline. Nuclear eGFP fluorescence was also observed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) 90 min after i.p. administration of cholecystokinin (CCK)‐8, which selectively activates oxytocin (OXT)‐secreting neurones in the hypothalamus. In double transgenic rats that express c‐fos‐eGFP and an OXT‐monomeric red fluorescent protein 1 (mRFP1) fusion gene, almost all mRFP1‐positive neurones in the SON and PVN expressed nuclear eGFP fluorescence 90 min after i.p. administration of CCK‐8. It is possible that not only a plane image, but also three‐dimensional reconstruction image may identify cytoplasmic vesicles in an activated neurone at the same time.     

Proinflammatory cytokine regulation of cyclic AMP‐phosphodiesterase 4 signaling in microglia in vitro and following CNS injury

Cyclic AMP suppresses immune cell activation and inflammation. The positive feedback loop of proinflammatory cytokine production and immune activation implies that cytokines may not only be regulated by cyclic AMP but also conversely regulate cyclic AMP. This study examined the effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β on cyclic AMP‐phosphodiesterase (PDE) signaling in microglia in vitro and after spinal cord injury (SCI) or traumatic brain injury (TBI). TNF‐α or IL‐1β stimulation produced a profound reduction (>90%) of cyclic AMP within EOC2 microglia from 30 min that then recovered after IL‐1β but remained suppressed with TNF‐α through 24 h. Cyclic AMP was also reduced in TNF‐α‐stimulated primary microglia, albeit to a lesser extent. Accompanying TNF‐α‐induced cyclic AMP reductions, but not IL‐1β, was increased cyclic AMP‐PDE activity. The role of PDE4 activity in cyclic AMP reductions was confirmed by using Rolipram. Examination of pde4 mRNA revealed an immediate, persistent increase in pde4b with TNF‐α; IL‐1β increased all pde4 mRNAs. Immunoblotting for PDE4 showed that both cytokines increased PDE4A1, but only TNF‐α increased PDE4B2. Immunocytochemistry revealed PDE4B nuclear translocation with TNF‐α but not IL‐1β. Acutely after SCI/TBI, where cyclic AMP levels are reduced, PDE4B was localized to activated OX‐42⁺ microglia; PDE4B was absent in OX‐42⁺ cells in uninjured spinal cord/cortex or inactive microglia. Immunoblotting showed PDE4B2 up‐regulation from 24 h to 1 wk post‐SCI, the peak of microglia activation. These studies show that TNF‐α and IL‐1β differentially affect cyclic AMP‐PDE signaling in microglia. Targeting PDE4B2 may be a putative therapeutic direction for reducing microglia activation in CNS injury and neurodegenerative diseases. © 2012 Wiley Periodicals, Inc.     

The Uncoupling Protein 2 ‐866G > A Polymorphism is Associated with the Risk of Ischemic Stroke in Chinese Type 2 Diabetic Patients

Aims: To determine genetic predispsitions for diabetic cerebral ischemia, we investigated the relationship between the ‐866G>A polymorphism of uncoupling protein (UCP) 2 and the risk of ischemic stroke in two cohorts of type 2 diabetic patients. Methods: A total of 844 type 2 diabetic patients with 4‐year prospective study were examined using a case‐control methodology. And 404 cases with ischemical stroke, 440 cases without ischemical stroke. The ‐866G>A polymorphism in UCP2 was genotyped by TaqMan MGB probe method. Results: The ‐866G>A SNP in UCP2 was significantly associated with diabetic ischemical stroke (odds ratio [OR]= 1.94; 95% confidence interval [CI]= 0.68 to1.31; P < 0.037). Similar results were observed for baseline cases of IS. Stratification by sex confirmed an allelic association with IS in women, whereas no association was observed in men. Conclusions: The A allele of the ‐866G>A variant of UCP2 was associated with increased risk of IS in Chinese diabetic women with type 2 diabetes in a 4‐year prospective study. This association was independent of other common IS risk factors.