Schwann cell nerve growth factor receptor expression during initiation of remyelination.

Initiation of remyelination is a promising therapeutic strategy to treat patients with demyelinating diseases, but specific factors that control remyelination are not clear. We first reported that expression of nerve growth factor receptor (NGFR) was increased during initiation of remyelination (Fan and Gelman, Journal of Neuropathology and Experimental Neurology 49: 312, 1990). In this study, we characterized the timing and cellular localization of NGFR expression in a model of segmental demyelination and remyelination using immunohistochemistry and monoclonal antibody 192-IgG, and compared it to an axonal neuropathy. At the onset of demyelination induced by tellurium (Te) poisoning, NGFR antigenicity was selectively expressed within and around demyelinating internodes in rat sciatic nerve. Dual fluorescence staining with myelin-specific antigen showed that NGFR colocalized with demyelinated internodal units with relative specificity; Schwann cell S-100 protein showed a concomitant down-regulation in injured internodes. Peak expression of NGFR occurred during the transition between demyelination and remyelination (day 8 of Te), then declined exponentially. NGFR expression was most prominent in the cytoplasm of daughter Schwann cells as they established contact with denuded axons, and was sharply repressed as compact myelin began to accumulate. Rare colocalization with neurofilament antigens revealed intraxonal deposits of NGFR in segmental demyelination. In the nerve crush model, Schwann cell NGFR expression was not segmentally distributed and was upregulated for a longer period of time. Our data establish that NGFR expression in the peripheral nervous system is not strictly linked to axon elongation, and that it probably functions during the initiation of myelination.     

Sural nerve immunoreactivity for nerve growth factor receptor in a case of localized hypertrophic neuropathy

Summary Immunoreactivity for nerve growth factor receptor (NGFR) was examined using a monoclonal antibody against human NGFR in the sural nerve of a 24-year-old woman, affected by localized hypertrophic neuropathy (LHN). NGFR expression was correlated with electron microscopy and with immunoreactivity for S-100 protein, laminin, HLA-DR, HNK-1, P0 glycoprotein and neurofilament peptides. Our results indicate that in LHN most of whorl-forming cells are NGFR positive and S-100 protein or HLA-DR negative. These data along with the ultrastructural features suggest their origin from perineurium.Supported by funds from Dino Ferrari Center for Neuromuscular and Neurodegenerative Diseases, University of Milan and Italian MS Association     

Synthesis of chimeric mouse nerve growth factor precursor and human beta-nerve growth factor in Escherichia coli: immunological properties

The complete mouse prepro-nerve growth factor (NGF) DNA was fused to the carboxyl terminus of the beta-galactosidase (lac-z) gene of Escherichia coli. Similarly, a genomic fragment encoding the human NGF comprising codons 11 to 106 (from a total of 118) was fused to the fifth codon of the amino terminus of beta-galactosidase. Both bacterial vectors produce high amounts of the chimeric proteins. After cell lysis most of the chimeric mouse preproNGF protein is insoluble and appears in the pellet, whereas the majority of the chimeric human beta-NGF remains in the supernatant. Purification of the fusion proteins from the soluble fraction was achieved by affinity chromatography to p-aminophenyl beta-D-thio-galactoside Sepharose. Yields of the purified chimeric proteins were increased threefold to fourfold by the addition of protease inhibitors in the lysis and chromatography buffers. Their antigenic similarity to the preproNGF and mouse beta-NGF was examined by their interaction to sera raised against synthetic peptides which reproduce sequences of the precursor protein and to sera directed against native and denatured mouse beta-NGF using enzyme-linked immunoabsorbent assay (ELISA) techniques. Antibodies to the peptide N2 (-163 to -139) interacted with high affinity with the chimeric mouse preproNGF protein. Antisera to native and denatured mouse beta-NGF interacted with both chimeric proteins but with a variable degree of affinity. These results provide direct evidence that certain antisera to mouse beta-NGF can cross-react with the human beta-NGF molecule.     

Activation of epidermal growth factor receptor causes astrocytes to form cribriform structures

Epidermal growth factor receptor (EGFR) is expressed in reactive astrocytes following injury in the CNS. However, the effects of activation of the EGFR pathway in astrocytes are not well established. In the present study, we demonstrate that activation of EGFR causes optic nerve astrocytes, as well as brain astrocytes, to form cribriform structures with cavernous spaces. Formation of the cribriform structures is dependent on new protein synthesis and cell proliferation. Platelet-derived growth factor and basic fibroblast growth factor were not effective. Smooth muscle cells and epithelial cells do not form cribriform structures in response to EGFR activation. The formation of the cribriform structures appears to be related to a guided migration of astrocytes and the expression of integrin beta1 and extracellular fibronectin in response to activation of EGFR. The EGFR pathway may be a specific, signal transduction pathway that regulates reactive astrocytes to form cavernous spaces in the glial scars following CNS injury and in the compressed optic nerve in glaucomatous optic nerve neuropathy.     

A soluble brain molecule related to epidermal growth factor receptor is a mitogen inhibitor for astrocytes

The astrocyte mitogenic activity of normal and injured rat brain extracts was greatly enhanced by antibodies to epidermal growth factor receptor (EGFR). The antibodies appear to act by removing from the extracts inhibitory molecules immunologically related to EGFR. Three molecular species recognized by anti-EGFR antibody in brain extracts (molecular weights 41, 52, and 69 kilodaltons) did not seem to originate from EGFR proteolysis. The increase in astrocyte mitogenic activity in brain tissue following injury correlated with a reduction in the levels of soluble EFGR-cross-reacting material and a decrease in mitogen inhibitory activity. The decrease in EGFR-related mitogen inhibitor also correlated with a large increase in astrocyte membrane EGFR immunoreactivity, and intracerebral injection of antibodies to EGFR caused the appearance at the injection site of numerous EGFR-positive reactive astrocytes. Invasion of brain tissue by EGF/EGFR-related blood components may be the signal that initiates astrocyte activation. EGFR-related immunoreactive molecules are also present in extracts of other tissues and may have a general role in the control of cell division.     

Rapid quantitative immunohistochemical assessment of human peripheral neuropathies using a monoclonal antibody against nerve growth factor receptor

Summary An analysis of nerve growth factor (NGF) receptor expression and density in human sural nerve biopsies was performed by immunocytochemistry with a murine monoclonal antibody against the human NGF receptor. Quantitative assessment of immunostaining density was made by histospectrophotometry on frozen sections. Although there was enhanced expression of NGF receptor within endoneurium in all patients with clinical neuropathies, expression was highest in nerves with axonal disease, consistent with the proposal that disruption of axon-Schwann cell interactions triggers the reexpression of the NGF receptor. These results with human nerves, together with previous studies with animal models, suggest that NGF and NGF receptor play important roles in the general response to neuronal injury.     

Effect of nerve activity on transport of nerve growth factor and dopamine β-hydroxylase antibodies in sympathetic neurones

The effect of nerve activity on the uptake and retrograde transport of nerve growth factor (NGF) and dopamine β-hydroxylase (DBH) antibodies was studied by injecting¹²⁵I-labelled NGF and anti-DBH into the anterior eye chamber of guinea-pigs. Decentralization of the ipsilateral superior cervical ganglion (SCG) had no significant effect on the retrograde transport of either NGF or anti-DBH. Phenoxybenzamine produced a 50% increase in anti-DBH but not NGF accumulation and this effect was prevented by prior decentralization. This demonstrates that NGF is taken up independently of the retrieval of synaptic vesicle components.     

Cooperative regulation of nerve growth factor synthesis and secretion in fibroblasts and astrocytes by fibroblast growth factor and other cytokines

Acidic fibroblast growth factor (aFGF) enhances nerve growth factor (NGF) synthesis by astrocytes obtained from various brain regions. NGF secretion by fibrous-shaped astrocytes transformed by dibutyryl-cAMP (db-cAMP) pretreatment was less than that by untreated astrocytes. However, aFGF also enhanced NGF secretion by fibrous-shaped astrocytes. The effects of various kinds of intracellular signaling modulators on NGF synthesis were examined. None of the following second messenger effectors had an effect on NGF synthesis: protein kinase C (PKC) agonist (phorbol myristate acetate (PMA)) or antagonist (sphingosine (SP)). LiCl, and ionomycin (Iono). Further, increases of intracellular cAMP by forskolin (FK) or db-cAMP have no significant effect on NGF synthesis in astrocytes under a standard culture condition. However, NGF synthesis by astrocytes in the presence of aFGF was significantly enhanced by db-cAMP, but not by FK or sodium butyrate. These results indicate that an excessive amount of cAMP enhances the effect of aFGF on NGF synthesis in astrocytes. NGF synthesis in astrocytes was not affected by treatment with anti-aFGF or anti-bFGF neutralizing antibodies, indicating that FGFs are not involved in the autocrine regulation of NGF synthesis in astrocytes. Transforming growth factor-beta 1 (TGF-beta 1), which inhibits some effects of FGFs, increased NGF synthesis in concert with aFGF. Furthermore, the highest NGF synthesis was observed when astrocytes were stimulated by all of the following cytokines: aFGF, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and TGF-beta 1. The mechanism regulating NGF synthesis in fibroblasts obtained from prenatal rat skin was also investigated. Acidic FGF, basic FGF (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha (TGF-alpha), TGF-beta 1, IL-1 beta, and TNF-alpha were found to be regulators of NGF synthesis in skin fibroblasts. Among these cytokines, aFGF is the most potent regulator of NGF synthesis in fibroblasts. NGF synthesis by skin fibroblasts, either in the presence or absence of aFGF, was not modified by any of the following: FK, PMA, SP, LiCl, and Iono. However, db-cAMP significantly enhanced NGF synthesis in both conditions. Sodium butyrate enhanced NGF synthesis in the presence of aFGF, but not in the absence of aFGF. These results suggest that an excessive amount of cAMP and butyrate moiety regulate NGF synthesis in skin fibroblasts in different ways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of estrogen receptor alpha, nerve growth factor, interleukin-2, and androgen receptor in the cerebellum of ovariectomized rats following soybean isoflavone treatment

BACKGROUND: Studies have shown that estrogen receptor alpha (ERα), nerve growth factor(NGF), interleukin-2 (IL-2), and androgen receptor (AR) expression in the cerebellum decreaseswhen estrogen levels decrease in vivo. Soybean isoflavone, a type of non-steroid estrogen withsimilar molecular structure and function to estradiol, exhibits estrogen-like characteristics.OBJECTIVE: To investigate the effects of various doses of soybean isoflavone on expression ofERα, NGF, IL-2, and AR in the cerebellum of ova...     

Aluminum, NO, and nerve growth factor neurotoxicity in cholinergic neurons.

Several neurotoxic compounds, including Al, NO, and beta-amyloid may contribute to the impairment or loss of brain cholinergic neurons in the course of various neurodegenerative diseases. Genotype and phenotypic modifications of cholinergic neurons may determine their variable functional competency and susceptibility to reported neurotoxic insults. Hybrid, immortalized SN56 cholinergic cells from mouse septum may serve as a model for in vitro cholinotoxicity studies. Differentiation by various combinations of cAMP, retinoic acid, and nerve growth factor may provide cells of different morphologic maturity as well as activities of acetylcholine and acetyl-CoA metabolism. In general, differentiated cells appear to be more susceptible to neurotoxic signals than the non-differentiated ones, as evidenced by loss of sprouting and connectivity, decreases in choline acetyltransferase and pyruvate dehydrogenase activities, disturbances in acetyl-CoA compartmentation and metabolism, insufficient or excessive acetylcholine release, as well as increased expression of apoptosis markers. Each neurotoxin impaired both acetylcholine and acetyl-CoA metabolism of these cells. Activation of p75 or trkA receptors made either acetyl-CoA or cholinergic metabolism more susceptible to neurotoxic influences, respectively. Neurotoxins aggravated detrimental effects of each other, particularly in differentiated cells. Thus brain cholinergic neurons might display a differential susceptibility to Al and other neurotoxins depending on their genotype or phenotype-dependent variability of the cholinergic and acetyl-CoA metabolism.     

Vasopressin induces human mesangial cell growth via induction of vascular endothelial growth factor secretion

Vasoactive hormones, growth factors, and cytokines are important in promoting mesangial cell growth, a characteristic feature of many glomerular diseases. Vascular endothelial growth factor (VEGF) is an endothelial mitogen and promoter of vascular permeability that is constitutively expressed in human glomeruli, but its role in the kidney is still unclear. In the present study, we investigated the ability of vasopressin (AVP) to stimulate VEGF secretion by and correlation with AVP-induced cell growth in human mesangial cells. AVP caused time- and concentration-dependent increases in VEGF secretion from human mesangial cells, which was in turn potently inhibited by a V_1A receptor-selective antagonist, confirming that this secretion is a V_1A receptor-mediated event. VEGF also induced mesangial cell growth which was completely inhibited on administration of an anti-VEGF neutralizing antibody. Further, AVP-induced mesangial cell growth was completely abolished by the V_1A receptor-selective antagonist and partially inhibited by an anti-VEGF neutralizing antibody. These results suggest that AVP stimulates VEGF secretion by human mesangial cells via V_1A receptors. This secreted VEGF may function as an autocrine hormone to regulate mesangial cell growth, a mechanism by which AVP might contribute to progressive glomerular diseases such as diabetic nephropathy.     

Express,on of estrogen receptor alpha,nerve growth factor,interleukin-2, and androgen receptor in the cerebellum of ovariectomized rats following soybean isoflavone treatment

BACKGROUND： Studies have shown that estrogen receptor alpha （ERα）, nerve growth factor （NGF）, interleukin-2 （IL-2）, and androgen receptor （AR） expression in the cerebellum decreases when estrogen levels decrease in vivo. Soybean isoflavone, a type of non-steroid estrogen with similar molecular structure and function to estradiol, exhibits estrogen-like characteristics. OBJECTIVE： To investigate the effects of various doses of soybean isoflavone on expression of ERa, NGF, IL-2, and AR in the cerebellum of ovariectomized rat, and to determine whether there is a dose-dependent effect.DESIGN, TIME AND SETTING： Controlled trial at the cellular and molecular level. The study was performed at the Experimental Animal Engineering Center, College of Veterinary Medicine, Sichuan Agricultural University from July 2006 to May 2008. MATERIALS： Soybean isoflavone, comprised of daidzin, genistein and isoflavone, was provided by Taiyuan Yuantai Biochemical Industry, China. The ERα, NGF, IL-2, and AR in situ hybridization kit, rabbit anti-rat ERa, NGF, IL-2, and AR monoclonal antibodies, and SABC kit were purchased from Wuhan Boster Biological Technology, China. METHODS： A total of 50 female, Sprague Dawley rats, aged 3 months, were randomly assigned to 5 groups, with 10 animals in each group. With the exception of the sham-operation group （abdominal cavity opening alone）, all rats underwent bilateral ovariectomy. At 14 days after surgery, rats in the high-, middle-, and low-dose soybean isoflavone groups were subcutaneously injected with 1.5, 1.0, and 0.5 mg/kg soybean isoflavone, respectively, every 2 days for 6 consecutive weeks. Rats in the sham-operation and ovariectomized groups were subcutaneously injected with absolute alcohol （0.5 mL/kg）. MAIN OUTCOME MEASURES： Expression levels and distribution of ERα, NGF, IL-2, and AR in the cerebellum were detected by immunohistochemistry and in situ hybridization. RESULTS： Compared with the sham-operation group, immunoreactive products and hybridization signals of ERa, NGF, IL-2, and AR were significantly decreased in the cerebellar cortex and nuclei of ovariectomized rats （P 〈 0.05 or P 〈 0.01）, but increased following soybean isoflavone treatment. In particular, levels of the high-dose soybean isoflavone group were almost restored to levels of the sham-operation group （P 〉 0.05）. The immunoreactive products were primarily located in the cytoplasm and neurites, and rarely in the cell membrane and nuclei. However, the hybridization signals were predominantly located in the nuclei, but rarely in the cytoplasm, cell membrane, or neurites. CONCLUSION： Soybean isoflavone upregulated ERα, NGF, IL-2, and AR protein and gene expression in a dose-dependent manner, and played an important role in sustaining and protecting structure and function of cerebellar neurons. Moreover, the similarity of expression patterns of these molecules indicated that they were mutually interactive during the regulation of soybean isoflavone to the cerebellum.     

Distribution and relative density of p75 nerve growth factor receptors in the rat brain as a function of age and treatment with antibodies to nerve growth factor.

It is clear that nerve growth factor (NGF) has a role in the central nervous system. In order to begin to determine the possible roles of NGF in the CNS, neonatal rats were given daily subcutaneous injections of antibodies to NGF (ANTI-NGF) beginning at birth for a period of one month. By utilizing the monoclonal antibody, 192-IgG, which recognizes the p75 NGF receptor (NGFR), and standard immunohistochemical techniques we have localized p75 NGFR in variously aged ANTI-NGF-treated animals and compared the anatomic localization and relative density of the p75 NGFR immunoreactive (p75 NGFR-I) regions to same age untreated and preimmune sera-treated littermates. We confirm previously reported localizations of p75 NGFR-I in the rat brain. In addition, we demonstrate that p75 NGFR-I levels of ANTI-NGF-treated rats found in the molecular, the granular and the Purkinje cell layers of the cerebellum, the vestibular nuclei, the spinal tract of V and the cochlear nuclei remain at lower concentrations compared to same-age control animals. We also demonstrate that p75 NGFR-I levels in the basal nucleus approaches background levels after ANTI-NGF treatment. We hypothesize that ANTI-NGF biologically inactivates NGF, which over a period of 30 days results in decreased p75 NGFR-I. These results are consistent with neuronal loss in these regions following ANTI-NGF treatment. Furthermore, the immunological methods used to produce the specific deficits in the present study may have broader implications with respect to usefulness as a method for determining the dependency of CNS neuronal populations for a putative neurotrophic factor and as a method for the development of models of neurodegenerative diseases.     

Transient expression of full-length and truncated forms of the human nerve growth factor receptor

To facilitate the characterization of nerve growth factor (NGF) receptor and mutated forms of the receptor, we have set up a rapid, efficient transient expression system utilizing COS cells. The human NGF receptor is a 427 amino acid protein with a hydrophobic signal sequence, a 222 amino acid extracellular domain, a single transmembrane domain and a 155 amino acid intracellular domain. The NGF receptor and a truncated form lacking the cytoplasmic domain were expressed in a COS cell expression system. Both recombinant proteins were detected on the cell surface and at a perinuclear site. Specific binding of 125I-NGF to the recombinant proteins was detected by chemical cross-linking. The extracellular domain of the NGF receptor was also expressed in the same system and detected in the COS cell endoplasmic reticulum and in the culture supernatant. This recombinant protein also specifically binds NGF.     

Phenytoin enhances the phosphorylation of epidermal growth factor receptor and fibroblast growth factor receptor in the subventricular zone and promotes the proliferation of neural precursor cells and oligodendrocyte differentiation

Phenytoin is a widely used antiepileptic drug that induces cell proliferation in several tissues, such as heart, bone, skin, oral mucosa and neural precursors. Some of these effects are mediated via fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (EGFR). These receptors are strongly expressed in the adult ventricular–subventricular zone (V‐SVZ), the main neurogenic niche in the adult brain. The aim of this study was to determine the cell lineage and cell fate of V‐SVZ neural progenitors expanded by phenytoin, as well as the effects of this drug on EGFR/FGFR phosphorylation. Male BALB/C mice received 10 mg/kg phenytoin by oral cannula for 30 days. We analysed the proliferation of V‐SVZ neural progenitors by immunohistochemistry and western blot. Our findings indicate that phenytoin enhanced twofold the phosphorylation of EGFR and FGFR in the V‐SVZ, increased the number of bromodeoxyuridine (BrdU)+/Sox2+ and BrdU+/doublecortin+ cells in the V‐SVZ, and expanded the population of Olig2‐expressing cells around the lateral ventricles. After phenytoin removal, a large number of BrdU+/Receptor interacting protein (RIP)+ cells were observed in the olfactory bulb. In conclusion, phenytoin enhanced the phosphorylation of FGFR and EGFR, and promoted the expression of neural precursor markers in the V‐SVZ. In parallel, the number of oligodendrocytes increased significantly after phenytoin removal.     

Distribution and relative density of p75 nerve growth factor receptors in the rat spinal cord as a function of age and treatment with antibodies to nerve growth factor.

It has been postulated that nerve growth factor (NGF) binding to the low-affinity fast-dissociating NGF receptor (p75 NGFR) on Schwann cells and growing neurites is involved with the molecular feedback necessary for continued neurite extension during development and regeneration. Since central projections of somatosensory fibers sprout into the spinal cord after daily neonatal injections of antibodies to NGF (ANTI-NGF) for a one month period, it is of interest to determine if the distribution of p75 NGFR correlates with the occurrence of sprouting. Spinal cords from three groups of rats: untreated, preimmune sera treated and ANTI-NGF treated were examined on postnatal days (PD) 0, 14 and 30. The p75 NGFR distribution was determined using the monoclonal antibody 192 with standard immunohistochemical techniques and the optical density of the immunoreaction product was quantified using an Amersham image analysis system. The 192 immunoreaction product was localized to laminae I-IV, the dorsal columns, the dorsolateral funiculus, Lissauer's tract (LT) and the ventral horn on PD 0; to laminae I-III and medial IV and LT on PD 14; and laminae I-II and LT on PD 30. The untreated and preimmune sera treated groups show no difference in distribution. In the ANTI-NGF treated group, the 192 immunoreaction product was localized to laminae I-V and LT on PD 14 and to laminae I-III and medial IV and LT on PD 30. Similarly, the optical density of the ANTI-NGF treated group was significantly greater than same aged untreated and preimmune sera treated groups, but was not statistically different from these two groups examined 14 days earlier. Thus, ANTI-NGF treatment interferes with the postnatal downregulation of p75 NGFR in the dorsal horn and may provide for continued neurite growth.     

Distribution of beta-adrenergic receptor subtypes in human post-mortem brain: alterations in limbic regions of schizophrenics.

The distribution of the beta 1 (beta 1) and beta 2 (beta 2) subtypes of the beta-adrenergic receptor was examined in rat and nondiseased control human tissue. The distribution of the beta 1 and beta 2 receptors was also examined in schizophrenic cases, with additional studies in schizophrenic suicide and nonschizophrenic suicide cases. Scatchard analysis of the binding of [125I]iodopindolol (IPIN) to cortical membranes showed a similar Kd in human (177 pM) and rat (161 pM), but a lower maximum binding site (Bmax) in the human tissue (18.7 fmol/mg protein and 55.6 fmol/mg protein). For the autoradiographic studies [125I]IPIN was used to visualize both subtypes (total) or was displaced with the selective beta 1-receptor antagonist ICI-89,406 to visualize beta 2 sites, or with the selective beta 2-receptor antagonist ICI-118,551 to visualize beta 1 sites. Important differences in the regional distribution of the two subtypes of the beta-adrenergic receptors were noted between rat and human. In the nucleus accumbens and ventral putamen (ventral striatum), a patchy distribution of beta 1 receptors was observed that was not evident in the rat. These patches were aligned with markers of the matrix compartment of the striatum. The schizophrenic cases showed significant increases in the labeling of the beta 1-receptor patches with [125I]IPIN. In contrast to the frontal cortex of the nondisease controls, the parietal and temporal cortex showed a high ratio of beta 1 to beta 2 receptors and a highly laminar organization of the subtypes. [125I]IPIN binding to beta 1 receptors was highest in the external laminae with the reverse gradient for the beta 2 subtype. The medial temporal cortex displayed an alteration in the ratio of the 2 subtypes of the beta-adrenergic receptor, with the parahippocampus and hippocampus of the human, in contrast to the rat brain, predominantly expressing the beta 2 receptor. Moreover, there were consistently higher densities of beta 2 receptors in the hippocampus of the right hemisphere than the left hemisphere of the nondisease controls. There was not a left and right hemispheric asymmetry of beta 2 receptors in the hippocampus of elderly schizophrenics or in young schizophrenics who committed suicide. The asymmetry was evident in nonschizophrenic suicides, suggesting that the lack of asymmetry in the hippocampus of schizophrenics is evident early in the disease process. Thus limbic structures show alterations in the patterning of beta 1 and beta 2 receptors in the schizophrenic cases.