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Richard U. Margolis - One of the best experts on this subject based on the ideXlab platform.

  • Expression of phosphacan and Neurocan during early development of mouse retinofugal pathway.
    Brain research. Developmental brain research, 2004
    Co-Authors: K.m Leung, Richard U. Margolis, Sun-on Chan
    Abstract:

    We have investigated whether the two major brain chondroitin sulfate (CS) proteoglycans (PGs), phosphacan and Neurocan, are expressed in patterns that correlate to the axon order changes in the mouse retinofugal pathway. Expression of these proteoglycans was examined by polyclonal antibodies against phosphacan and N- and C-terminal fragments of Neurocan. In E13-E15 mouse embryos, when most optic axons grow in the chiasm and the optic tract, phosphacan and Neurocan were observed in the inner regions of the retina. In the chiasm and the tract, phosphacan but not Neurocan was expressed prominently at the midline and in the deep parts of the tract. Both proteoglycans were observed on the chiasmatic neurons, which have been shown to regulate axon divergence at the chiasmatic midline and the chronotopic fiber ordering in the tract, but phosphacan appeared to be the predominant form that persists to later developmental stages. Intense staining of both proteoglycans was also observed in a strip of glial-like elements in lateral regions of the chiasm, partitioning axons in the stalk from those in the tract. We conclude that phosphacan but not Neurocan is likely the major carrier of the CS glycosaminoglycans that play crucial functions in axon divergence and age-related axon ordering in the mouse optic pathway. Furthermore, localization of these carrier proteins in the optic pathway raises a possibility that these two proteoglycans regulate axon growth and patterning not only through the sulfated sugars but also by interactions of the protein parts with guidance molecules on the optic axons.

  • Developmental changes of aggrecan, versican and Neurocan in the retina and optic nerve
    Experimental eye research, 2004
    Co-Authors: Susanna Popp, Patrice Maurel, Julie S. Andersen, Richard U. Margolis
    Abstract:

    We have used a monoclonal antibody to Neurocan and specific polyclonal antibodies to the non-homologous glycosaminoglycan attachment regions of aggrecan and mRNA splice variants of versican to compare the localization and developmental changes of these structurally related hyaluronan-binding chondroitin sulfate proteoglycans in the rat retina and optic nerve. Staining for aggrecan and versican was first seen at embryonic day 16 in the optic nerve and retina, whereas Neurocan was not detected in the embryonic eye. At postnatal day 0 (P0), beta-versican staining is largely confined to the inner plexiform layer whereas alpha-versican is also apparent in the neuroblastic layer. Both aggrecan and, much more weakly, Neurocan immunoreactivity is present throughout the neonatal retina. At P9, aggrecan and versican immunoreactivity is most intense in the inner and outer plexiform and ganglion cell layers, accompanied by diffuse staining in the inner and outer nuclear layers. Aggrecan and alpha-versican are also present throughout the optic nerve and disk, whereas beta-versican and Neurocan are confined to the laminar beams of the optic nerve. Between P0 and P9 there is a marked increase in beta-versican expression in the inner and outer nuclear layers and in the outer plexiform layer, whereas there is only weak staining of Neurocan in the inner plexiform and ganglion cell layers of P9 retina. By 1 month postnatal the staining pattern of the fully differentiated retinal layers is essentially identical to that seen in the adult, where there is strong aggrecan and alpha-versican immunoreactivity in the retina and optic nerve, whereas beta-versican has essentially disappeared from the adult retina and, similarly to Neurocan, is present only in the laminar beams of the optic nerve. The marked decrease of beta-versican in the retina is consistent with >90% decrease in its concentration in brain during postnatal development, suggesting that the developmental time-course for these proteoglycans in retina parallels that seen in other areas of the central nervous system.

  • The chondroitin sulfate proteoglycans Neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury.
    Experimental neurology, 2003
    Co-Authors: Leonard L. Jones, Richard U. Margolis, Mark H. Tuszynski
    Abstract:

    Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix (ECM) molecules that are widely expressed throughout the developing and adult CNS. In vitro studies demonstrate their potential to restrict neurite outgrowth, and it is believed that CSPGs also inhibit axonal regeneration after CNS injury in vivo. Previous studies demonstrated that CSPGs are generally upregulated after spinal cord injury, and more recent reports have begun to identify individual proteoglycans that may play dominant roles in limiting axonal regeneration. The current study systematically examined the extended deposition patterns after CNS injury of four putatively inhibitory CSPGs that have not been extensively investigated previously in vivo: Neurocan, brevican, phosphacan, and versican. After spinal cord injury, Neurocan, brevican, and versican immunolabeling increased within days in injured spinal cord parenchyma surrounding the lesion site and peaked at 2 weeks. Neurocan and versican were persistently elevated for 4 weeks postinjury, and brevican expression persisted for at least 2 months. On the other hand, phosphacan immunolabeling decreased in the same region immediately following injury but later recovered and then peaked after 2 months. Combined glial fibrillary acidic protein (GFAP) immunohistochemistry and in situ hybridization demonstrated that GFAP astrocytes constituted a source of Neurocan production after spinal cord injury. Thus, the production of several CSPG family members is differentially affected by spinal cord injury, overall establishing a CSPG-rich matrix that persists for up to 2 months following injury. Optimization of strategies to reduce CSPG expression to enhance regeneration may need to target several different family members over an extended period following injury.

  • chondroitin sulfate and chondroitin keratan sulfate proteoglycans of nervous tissue developmental changes of Neurocan and phosphacan
    Journal of Neurochemistry, 2002
    Co-Authors: Birgit Meyerputtlitz, Peter Milev, Richard U. Margolis, Ernst Junker, Irene Zimmer, Renée K. Margolis
    Abstract:

    We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, Neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of Neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the Neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of Neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of Neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of Neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas Neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.

  • Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of Neurocan and phosphacan.
    Journal of neurochemistry, 2002
    Co-Authors: Birgit Meyer-puttlitz, Peter Milev, Richard U. Margolis, Ernst Junker, Irene Zimmer, Renée K. Margolis
    Abstract:

    We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, Neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of Neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the Neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of Neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of Neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of Neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas Neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.

Uwe Rauch - One of the best experts on this subject based on the ideXlab platform.

  • Entorhinal Cortex Lesion in Adult Rats Induces the Expression of the Neuronal Chondroitin Sulfate Proteoglycan Neurocan in Reactive Astrocytes
    2013
    Co-Authors: Carola A. Haas, Uwe Rauch, Niklas Thon, Tobias Merten, Thomas Deller
    Abstract:

    The chondroitin sulfate proteoglycan Neurocan is a major component of brain extracellular matrix during development. Neurocan is primarily synthesized by neurons and has the ability to interact with cell adhesion molecules involved in the regulation of cell migration and axonal growth. Within the first weeks postnatally, Neurocan expression is strongly downregulated. To test whether Neurocan is reexpressed in areas of axonal growth (sprouting) after brain injury, the time course of Neurocan expression was analyzed in the denervated fascia dentata of the rat after entorhinal cortex lesion (12 hr; 1, 2, 4, and 10 d; 2 and 4 weeks; and 6 months after lesion). In the denervated zone, immunohistochemistry revealed Neurocan-positive astrocytes by 2 d after lesion and a diffuse labeling of the extracellular matrix at all later time points. Electron microscopy confirmed the deposition of Neurocan in the extracellular matrix compartment

  • Accumulation of Neurocan, a Brain Chondroitin Sulfate Proteoglycan, in Association with the Retinal Vasculature in RCS Rats
    2013
    Co-Authors: Y. Zhang, Uwe Rauch, Maria-thereza R. Perez
    Abstract:

    PURPOSE. To examine whether and how the retinal distribution of the chondroitin sulfate proteoglycan Neurocan is affected after photoreceptor cell loss and whether it correlates with the multiple secondary cellular changes that accompany the photoreceptor degeneration. METHODS. Retinas from normal rats (Sprague-Dawley; postnatal days [P]0–P70), RCS rats with dystrophic retinas (P0–P300), RCS-rdy � congenic rats with nondystrophic retinas (P0–202), and rhodopsin mutant rats, P23H (P0–P257) and S334ter (P0– P220), were processed for immunohistochemistry using a polyclonal antibody to rat Neurocan. RESULTS. The overall distribution of Neurocan was similar in all retinas examined. Neurocan immunostaining was detected over the nerve fiber layer, the plexiform layers, the photoreceptor outer segments region, and the ciliary epithelium. Wit

  • Detection of Neurocan in cerebrospinal fluid.
    Methods in molecular biology (Clifton N.J.), 2011
    Co-Authors: Uwe Rauch
    Abstract:

    Cerebrospinal fluid (CFS) is the most easily accessible component of the human central nervous system and has been successfully used for the analysis of disease-associated molecular imbalances, particularly for extracellular matrix components. Alterations in the presence of the nervous system-associated chondroitin sulfate proteoglycan Neurocan had been reported from active multiple sclerosis lesions. Neurocan could be detected as a component of human CFS after enrichment of proteoglycans by anion exchange chromatography from pooled liquor as well as individual 300 μL samples by Western blot. However, a general alteration in Neurocan levels in CFS sample with high immunoglobulin content could not be demonstrated. To further reduce the sample size, the development of a PG capturing assay based on polybrene-coated 96-well plates was initiated. This approach could be an interesting alternative option for the analysis of PGs in biological fluid and tissue samples.

  • Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells.
    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2004
    Co-Authors: Hui Zhang, Joachim Kappler, Stephan L. Baader, Michael Sixt, Uwe Rauch
    Abstract:

    Hyaluronan is an unsulfated glycosaminoglycan (GAG) that is ubiquitously expressed in the extracellular matrix (ECM) of all vertebrates, where hyaluronan rich matrices constitute a particular permissive environment for the development of complex biological structures and also for tumor progression. Because of its conserved structure and ubiquitous expression, antibodies for its histochemical detection cannot be produced. We have engineered a fusion protein, Neurocan-GFP, and expressed it as a secreted molecule in mammalian cells. Neurocan-GFP fusion protein specifically binds to hyaluronan and directly visualizes hyaluronan on tissue sections, revealing a very detailed picture of hyaluronan distribution. The fluorescent fusion protein can be used in combination with antibodies and nuclear markers for double or triple staining. In addition, it is suitable to visualize hyaluronan on living cells by time-lapse video microscopy. The successful production and application of the Neurocan-GFP fusion protein opens up new perspectives for using GFP fusion proteins as detection tools in histological and cytological studies complementing conventional antibody and biotin/avidin techniques.

  • Cartilage link protein interacts with Neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6.
    Matrix biology : journal of the International Society for Matrix Biology, 2004
    Co-Authors: Uwe Rauch, Toshitaka Oohashi, Satoshi Hirakawa, Joachim Kappler, Gunnel Roos
    Abstract:

    The interaction of Neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type Neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of Neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of Neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the Neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of Neurocan in developing brain implicates the possibility of a cooperative function of these molecules.

Renée K. Margolis - One of the best experts on this subject based on the ideXlab platform.

  • Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of Neurocan and phosphacan.
    Journal of neurochemistry, 2002
    Co-Authors: Birgit Meyer-puttlitz, Peter Milev, Richard U. Margolis, Ernst Junker, Irene Zimmer, Renée K. Margolis
    Abstract:

    We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, Neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of Neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the Neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of Neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of Neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of Neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas Neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.

  • chondroitin sulfate and chondroitin keratan sulfate proteoglycans of nervous tissue developmental changes of Neurocan and phosphacan
    Journal of Neurochemistry, 2002
    Co-Authors: Birgit Meyerputtlitz, Peter Milev, Richard U. Margolis, Ernst Junker, Irene Zimmer, Renée K. Margolis
    Abstract:

    We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, Neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of Neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the Neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of Neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of Neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of Neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas Neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.

  • high affinity binding and overlapping localization of Neurocan and phosphacan protein tyrosine phosphatase ζ β with tenascin r amphoterin and the heparin binding growth associated molecule
    Journal of Biological Chemistry, 1998
    Co-Authors: Peter Milev, Renée K. Margolis, Atsuro Chiba, Monika Haring, Heikki Rauvala, Melitta Schachner, Barbara Ranscht, Richard U. Margolis
    Abstract:

    Abstract We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans Neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2–7 nmrange. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by ∼60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by ∼75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of Neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (K d = 0.3–8 nm), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on Neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of Neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

  • High Affinity Binding and Overlapping Localization of Neurocan and Phosphacan/Protein-tyrosine Phosphatase-ζ/β with Tenascin-R, Amphoterin, and the Heparin-binding Growth-associated Molecule
    The Journal of biological chemistry, 1998
    Co-Authors: Peter Milev, Renée K. Margolis, Atsuro Chiba, Monika Haring, Heikki Rauvala, Melitta Schachner, Barbara Ranscht, Richard U. Margolis
    Abstract:

    Abstract We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans Neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2–7 nmrange. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by ∼60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by ∼75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of Neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (K d = 0.3–8 nm), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on Neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of Neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

  • TAG-1/axonin-1 is a high-affinity ligand of Neurocan, phosphacan/protein-tyrosine phosphatase-ζ/β, and N-CAM
    The Journal of biological chemistry, 1996
    Co-Authors: Peter Milev, Renée K. Margolis, Patrice Maurel, Monika Haring, Richard U. Margolis
    Abstract:

    Abstract Proteoglycans appear to play an important role in modulating cell-cell and cell-matrix interactions during nervous tissue histogenesis. The nervous tissue-specific chondroitin sulfate proteoglycans Neurocan and phosphacan/protein-tyrosine phosphatase-η/β were found to be high-affinity ligands of the neural cell adhesion molecule TAG-1/axonin-1, with dissociation constants of 0.3 nM and 0.04 nM, respectively. Phosphacan binding was decreased by ∼70% following chondroitinase treatment, whereas binding of Neurocan was not affected. The contribution of chondroitin sulfate chains to the binding of Neurocan and phosphacan to TAG-1/axonin-1 is therefore the opposite of that previously observed for their binding to two other Ig-superfamily neural cell adhesion molecules, Ng-CAM/L1 and N-CAM. Moreover, whereas phosphacan interactions with certain proteins are mediated at least in part by N-linked oligosaccharides on the proteoglycan, N-deglycosylation of phosphacan had no effect on its binding to TAG-1/axonin-1. In addition to the chondroitin sulfate proteoglycans described above, we have demonstrated that N-CAM is a high-affinity ligand of TAG-1/axonin-1 (Kd ∼1 nM), and specific binding of TAG-1/axonin-1 to tenascin-C was also observed (Kd ∼9 nM). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of TAG-1/axonin-1 with all four of these ligands, further supporting the biological significance of their ability to interact in vitro.

Atsuhiko Oohira - One of the best experts on this subject based on the ideXlab platform.

  • Neurocan Is Upregulated in Injured Brain and in
    2016
    Co-Authors: Daniel Morgenstern, Richard A. Asher, Daniel A. Morgenstern, Penny S. Fidler, Kathryn H. Adcock, Joel M. Levine, Cytokine-treated Astrocytes, Atsuhiko Oohira
    Abstract:

    Neurocan is upregulated in injured brain and in cytokine-treated astrocyte

  • Inhibitory Effects of Neurocan and Phosphacan on Neurite Outgrowth from Retinal Ganglion Cells in Culture
    2013
    Co-Authors: Masaru Inatani, Atsuhiko Oohira, Megumi Honjo, Noriaki Kido, Yoshihito Honda, Yasumasa Otori, Yasuo Tano, Hidenobu Tanihara
    Abstract:

    PURPOSE. Neurocan and phosphacan are nervous tissue–specific chondroitin sulfate proteoglycans (CSPGs) that are highly expressed in postnatal rat retina. To elucidate potential roles of Neurocan and phosphacan on neurite outgrowth from retinal ganglion cells (RGCs), in vitro experiments were conducted with purified RGCs. METHODS. Neurocan and phosphacan were purified from postnatal rat brain by DEAE-column chromatography and subsequent gel chromatography. RGCs were obtained from postnatal rat retinas by a two-step immunopanning procedure using an anti-Thy 1,1 antibody and an anti-macrophage antibody. Neurite outgrowth from RGCs was examined on poly-L-lysine (PLL)-conditioned plates, and PLL-conditioned plates treated with Neurocan or phosphacan. RESULTS. Compared with PLL-conditioned plates, Neurocan an

  • Identification of a Nervous Tissue–Specific Chondroitin Sulfate Proteoglycan, Neurocan, in Developing Rat Retina
    2013
    Co-Authors: Masaru Inatani, Atsuhiko Oohira, Hidenobu Tanihara, Megumi Honjo, Yoshihito Honda
    Abstract:

    PURPOSE. To identify the expression of Neurocan, a nervous tissue–specific chondroitin sulfate proteoglycan, in retina and to elucidate its changes during development. METHODS. Expressional changes of Neurocan mRNAs in developing rat retinas were investigated by a semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). The localization and characterization of Neurocan core proteins were also investigated with the use of Western blot analysis and immunohistochemistry. RESULTS. Gene expression of Neurocan was identified in retinas by RT–PCR. Semiquantitative analysis using Southern blot analysis revealed that mRNA expression for Neurocan increased at increasing postnatal stages and that it reached its peak around postnatal day 7 (P7). Immunohistochemical studies demonstrated that in differentiating rat retinal (neuroblast) cells weak Neurocan immunoreactivities were observed throughout the retina on embryonal days 14 (E14) and E16. During the early postnatal period, the immunoreactivities became most conspicuous in the inner and outer plexiform layers on P7 through P14. In adult retinas, only faint immunostaining was detected. Immunoblot analysis showed two positive bands of 220- and 150-kDa core glycoproteins after treatment with chondroitinase ABC. Further immunoblo

  • Expression of Neurocan after transient middle cerebral artery occlusion in adult rat brain.
    Brain research, 2005
    Co-Authors: Kentaro Deguchi, Atsuhiko Oohira, Mikiro Takaishi, Takeshi Hayashi, Shoko Nagotani, Guang Jin, Isao Nagano, Mikio Shoji, Masahiro Miyazaki
    Abstract:

    Neurocan is one of the major chondroitin sulfate proteoglycans in the nervous tissues. The expression and proteolytic cleavage of Neurocan are developmentally regulated in the normal rat brain, and the full-length Neurocan is detected in juvenile brains but not in normal adult brains. Recently, some studies showed that the full-length Neurocan was detectable even in the adult brain when it was exposed to mechanical incision or epileptic stimulation. In the present study, we demonstrated by Western blot analysis that the full-length Neurocan transiently appeared in the peri-ischemic region of transient middle cerebral artery occlusion (tMCAO) in adult rat with a peak level at 4 days after tMCAO. Immunohistochemical analysis showed that a clear positive signal of Neurocan was observed 4 days after tMCAO in the peri-ischemic region of cerebral cortex and caudate, where cells strongly positive in GFAP expression were also distributed. These results indicate that accumulation of the full-length Neurocan produced by reactive astrocytes may be one of the processes for tissue repair and reconstruction of neural networks after focal brain ischemia as well.

  • Aberrant trajectory of thalamocortical axons associated with abnormal localization of Neurocan immunoreactivity in the cerebral neocortex of reeler mutant mice.
    The European journal of neuroscience, 2005
    Co-Authors: Atsuhiko Oohira, Koki Kawamura, Masaharu Ogawa, Hitoshi Kawano
    Abstract:

    We examined the molecular mechanisms underlying the formation of the thalamocortical pathway in the cerebral neocortex of normal and reeler mutant mice. During normal development of the mouse neocortex, thalamic axons immunoreactive for the neural cell adhesion molecule L1 rarely invaded the cortical plate and ran centered in the subplate which is immunoreactive for Neurocan, a brain-specific chondroitin sulfate proteoglycan. On the other hand, in homozygous reeler mutant mice, thalamic axons took an aberrant course to run obliquely through the cortical plate. Injection of bromodeoxyuridine at embryonic day 11 specifically labeled subplate neurons in normal mice, whilst in the reeler neocortex it labeled cells scattered in the cortical plate as well as in the superficial layer (superplate). Neurocan immunoreactivity was associated with the bromodeoxyuridine-positive cells in the superplate, as well as being present in oblique bands within the cortical plate, along which L1-bearing thalamic axons preferentially ran. The present results support our previous hypothesis proposed for normal rats that a heterophilic molecular interaction between L1 and Neurocan is involved in determining the thalamocortical pathway within the neocortical anlage [T. Fukuda et al. (1997) Journal of Comparative Neurology, 382, 141-152].

Peter Milev - One of the best experts on this subject based on the ideXlab platform.

  • Chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of nervous tissue: developmental changes of Neurocan and phosphacan.
    Journal of neurochemistry, 2002
    Co-Authors: Birgit Meyer-puttlitz, Peter Milev, Richard U. Margolis, Ernst Junker, Irene Zimmer, Renée K. Margolis
    Abstract:

    We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, Neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of Neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the Neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of Neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of Neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of Neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas Neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.

  • chondroitin sulfate and chondroitin keratan sulfate proteoglycans of nervous tissue developmental changes of Neurocan and phosphacan
    Journal of Neurochemistry, 2002
    Co-Authors: Birgit Meyerputtlitz, Peter Milev, Richard U. Margolis, Ernst Junker, Irene Zimmer, Renée K. Margolis
    Abstract:

    We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, Neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of Neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the Neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of Neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of Neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of Neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas Neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached approximately 1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-beta-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.

  • high affinity binding and overlapping localization of Neurocan and phosphacan protein tyrosine phosphatase ζ β with tenascin r amphoterin and the heparin binding growth associated molecule
    Journal of Biological Chemistry, 1998
    Co-Authors: Peter Milev, Renée K. Margolis, Atsuro Chiba, Monika Haring, Heikki Rauvala, Melitta Schachner, Barbara Ranscht, Richard U. Margolis
    Abstract:

    Abstract We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans Neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2–7 nmrange. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by ∼60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by ∼75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of Neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (K d = 0.3–8 nm), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on Neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of Neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

  • High Affinity Binding and Overlapping Localization of Neurocan and Phosphacan/Protein-tyrosine Phosphatase-ζ/β with Tenascin-R, Amphoterin, and the Heparin-binding Growth-associated Molecule
    The Journal of biological chemistry, 1998
    Co-Authors: Peter Milev, Renée K. Margolis, Atsuro Chiba, Monika Haring, Heikki Rauvala, Melitta Schachner, Barbara Ranscht, Richard U. Margolis
    Abstract:

    Abstract We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans Neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2–7 nmrange. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by ∼60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by ∼75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of Neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (K d = 0.3–8 nm), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on Neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of Neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

  • TAG-1/axonin-1 is a high-affinity ligand of Neurocan, phosphacan/protein-tyrosine phosphatase-ζ/β, and N-CAM
    The Journal of biological chemistry, 1996
    Co-Authors: Peter Milev, Renée K. Margolis, Patrice Maurel, Monika Haring, Richard U. Margolis
    Abstract:

    Abstract Proteoglycans appear to play an important role in modulating cell-cell and cell-matrix interactions during nervous tissue histogenesis. The nervous tissue-specific chondroitin sulfate proteoglycans Neurocan and phosphacan/protein-tyrosine phosphatase-η/β were found to be high-affinity ligands of the neural cell adhesion molecule TAG-1/axonin-1, with dissociation constants of 0.3 nM and 0.04 nM, respectively. Phosphacan binding was decreased by ∼70% following chondroitinase treatment, whereas binding of Neurocan was not affected. The contribution of chondroitin sulfate chains to the binding of Neurocan and phosphacan to TAG-1/axonin-1 is therefore the opposite of that previously observed for their binding to two other Ig-superfamily neural cell adhesion molecules, Ng-CAM/L1 and N-CAM. Moreover, whereas phosphacan interactions with certain proteins are mediated at least in part by N-linked oligosaccharides on the proteoglycan, N-deglycosylation of phosphacan had no effect on its binding to TAG-1/axonin-1. In addition to the chondroitin sulfate proteoglycans described above, we have demonstrated that N-CAM is a high-affinity ligand of TAG-1/axonin-1 (Kd ∼1 nM), and specific binding of TAG-1/axonin-1 to tenascin-C was also observed (Kd ∼9 nM). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of TAG-1/axonin-1 with all four of these ligands, further supporting the biological significance of their ability to interact in vitro.