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Karl Schilling - One of the best experts on this subject based on the ideXlab platform.

  • The molecular basis of the specificity and cross-reactivity of the NeuN epitope of the neuron-specific splicing regulator, Rbfox3
    Histochemistry and Cell Biology, 2014
    Co-Authors: Stephan Maxeiner, Alexander Glassmann, Karl Schilling
    Abstract:

    Ever since its description and the generation of its defining antibody, some 20 years ago, neural nuclei (NeuN) have been an invaluable tool for developmental neuroscientists and neuropathologists to identify neurons and follow their normal or malignant development. The recent identification of the splicing factor Rbfox3 as the molecule constituting the genuine NeuN epitope has opened up a novel perspective on NeuN immunostaining and its interpretation. Here, we briefly review these recent developments, and we provide a series of data that allow to rationalize the specificity of the NeuN/A60 antibody on aldehyde-fixed tissues on the one hand, and its cross-reactivity with Synapsin I and R3hdm2 on Western blots on the other. We argue that rather than being considered as a mere marker for mature neurons, Rbfox3-mediated NeuN/A60 immunoreactivity may provide a window onto neuronal biology. Specifically, we hypothesize that the phosphorylation-dependent antigenicity of the Rbfox3/NeuN epitope should allow to visualize neuronal physiology realized through Rbfox3, including splicing, on the single-cell level.

  • characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization
    Journal of Neuroscience Research, 2005
    Co-Authors: Daniela Lind, Sebastian Franken, Joachim Kappler, J Jankowski, Karl Schilling
    Abstract:

    NeuN (neuronal nuclei) is an antigen used widely in research and diagnostics to identify postmitotic neurons. The present study aims at an initial understanding of the molecular nature and functional significance of this as yet ill-defined antigen. Using isoelectric focusing, both the 46- and 48-kDa isoforms of NeuN can be separated in multiple spots spanning a pH range of 8-10.5, suggesting that they might be phosphorylated. Enzymatic dephosphorylation abolishes NeuN immunoreactivity, confirming that NeuN is indeed a phosphoprotein, and establishing that binding of the defining antibody depends on its state of phosphorylation. Combined biochemical and immunohistochemical analysis show that both the 46- and the 48-kDa NeuN isoforms can be localized to the cell nucleus as well as in the neuronal cytoplasm. Their relative concentration in these compartments is distinct, however, with the 48-kDa isoform being the predominant isoform in the cytoplasm. Within the nucleus, NeuN is found preferentially in areas of low chromatin density and virtually excluded from areas containing densely packed DNA. The present identification of multiple differentially phosphorylated isoforms of NeuN, together with recent reports on the dependence of NeuN immunoreactivity levels on a variety of physiologic or pathologic signals, suggests a previously unappreciated level of complexity in the regulation of this enigmatic, neuron-specific antigen.

  • developmental and cell type specific expression of the neuronal marker NeuN in the murine cerebellum
    Journal of Neuroscience Research, 2003
    Co-Authors: Anja Weyer, Karl Schilling
    Abstract:

    NeuN is a 46/48-kD nuclear protein antigen used widely to identify postmitotic neurons in both research and diagnostics. It is expressed by neurons throughout the nervous system of a variety of species, including birds, rodents, and man (Mullen et al. [1992] Development 116:201–211). When we sought to use NeuN to follow the developmental progression of murine cerebellar interneurons, we observed that expression of this antigen in the cerebellum was restricted to granule neurons and a small population of cells present in the lower molecular layer of the adult cerebellum. In an attempt to identify these cells, we combined immunostaining for NeuN with a panel of cell type-specific markers to unambiguously identify neurons that express NeuN in the adult and developing cerebellum. In contrast to postmitotic granule neurons, NeuN was not expressed by any other immunocytochemically identified cerebellar interneurons, which comprised basket and stellate cells, Golgi neurons, unipolar brush cells, and Lugaro cells. NeuN-positive cells in the molecular layer failed to express any cell type-specific markers tested. They may represent ectopic granule cells; alternatively, they may represent a hitherto unknown population of cerebellar cells. In vitro experiments suggest that NeuN expression is related closely to granule cell axogenesis. This approach also revealed that the level of NeuN expression could be modulated by chronically depolarizing these cells. Thus, whereas NeuN expression per se is a reliable marker of proliferative capacity, levels of NeuN expression may also be indicative of the physiological status of a postmitotic neuron. © 2003 Wiley-Liss, Inc.

  • developmental and cell type specific expression of the neuronal marker NeuN in the murine cerebellum
    Journal of Neuroscience Research, 2003
    Co-Authors: Anja Weyer, Karl Schilling
    Abstract:

    NeuN is a 46/48-kD nuclear protein antigen used widely to identify postmitotic neurons in both research and diagnostics. It is expressed by neurons throughout the nervous system of a variety of species, including birds, rodents, and man (Mullen et al. [1992] Development 116:201-211). When we sought to use NeuN to follow the developmental progression of murine cerebellar interneurons, we observed that expression of this antigen in the cerebellum was restricted to granule neurons and a small population of cells present in the lower molecular layer of the adult cerebellum. In an attempt to identify these cells, we combined immunostaining for NeuN with a panel of cell type-specific markers to unambiguously identify neurons that express NeuN in the adult and developing cerebellum. In contrast to postmitotic granule neurons, NeuN was not expressed by any other immunocytochemically identified cerebellar interneurons, which comprised basket and stellate cells, Golgi neurons, unipolar brush cells, and Lugaro cells. NeuN-positive cells in the molecular layer failed to express any cell type-specific markers tested. They may represent ectopic granule cells; alternatively, they may represent a hitherto unknown population of cerebellar cells. In vitro experiments suggest that NeuN expression is related closely to granule cell axogenesis. This approach also revealed that the level of NeuN expression could be modulated by chronically depolarizing these cells. Thus, whereas NeuN expression per se is a reliable marker of proliferative capacity, levels of NeuN expression may also be indicative of the physiological status of a postmitotic neuron.

Maria D Ganfornina - One of the best experts on this subject based on the ideXlab platform.

  • myelin extracellular leaflet compaction requires apolipoprotein d membrane management to optimize lysosomal dependent recycling and glycocalyx removal
    Glia, 2018
    Co-Authors: Nadia Garciamateo, Raquel Pascuamaestro, Alberto Perezcastellanos, Concepcion Lillo, Diego Sanchez, Maria D Ganfornina
    Abstract:

    To compact the extracellular sides of myelin, an important transition must take place: from membrane sliding, while building the wraps, to membrane adhesion and water exclusion. Removal of the negatively charged glycocalyx becomes the limiting factor in such transition. What is required to initiate this membrane-zipping process? Knocking-out the Lipocalin Apolipoprotein D (ApoD), essential for lysosomal functional integrity in glial cells, results in a specific defect in myelin extracellular leaflet compaction in peripheral and central nervous system, which results in reduced conduction velocity and suboptimal behavioral outputs: motor learning is compromised. Myelination initiation, growth, intracellular leaflet compaction, myelin thickness or internodal length remain unaltered. Lack of ApoD specifically modifies Plp and P0 protein expression, but not Mbp or Mag. Late in myelin maturation period, ApoD affects lipogenic and growth-related, but not stress-responsive, signaling pathways. Without ApoD, the sialylated glycocalyx is maintained and ganglioside content remains high. In peripheral nervous system, Neu3 membrane sialidase and lysosomal Neu1 are coordinately expressed with ApoD in subsets of Schwann cells. ApoD-KO myelin becomes depleted of Neu3 and enriched in Fyn, a kinase with pivotal roles in transducing axon-derived signals into myelin properties. In the absence of ApoD, partial permeabilization of lysosomes alters Neu1 location as well. Exogenous ApoD rescues ApoD-KO hypersialylated glycocalyx in astrocytes, demonstrating that ApoD is necessary and sufficient to control glycocalyx composition in glial cells. By ensuring lysosomal functional integrity and adequate subcellular location of effector and regulatory proteins, ApoD guarantees the glycolipid recycling and glycocalyx removal required to complete myelin compaction.

Christopher W. Cairo - One of the best experts on this subject based on the ideXlab platform.

  • selective inhibitors of human neuraminidase 1 neu1
    Journal of Medicinal Chemistry, 2018
    Co-Authors: Rachel Heonroberts, Ruixiang Blake Zheng, Alexey V. Pshezhetsky, Christopher W. Cairo
    Abstract:

    Inhibitors of human neuraminidase enzymes (NEU) are recognized as important tools for the study of the biological functions of NEU and will be potent tools for elucidating the role of these enzymes in regulating the repertoire of cellular glycans. Here we report the discovery of selective inhibitors of the human neuraminidase 1 (NEU1) and neuraminidase 2 (NEU2) enzymes with exceptional potency. A library of modified 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) analogues, with variability in the C5- or C9-position, were synthesized and evaluated against four human neuraminidase isoenyzmes (NEU1–4). Hydrophobic groups with an amide linker at the C5 and C9 positions were well accommodated by NEU1, and a hexanamido group was found to give the best potency at both positions. While the C5-hexanamido-C9-hexanamido-DANA analogue did not show synergistic improvements for combined modification, an extended alkylamide at an individual position combined with a smaller group at the second gave increased potenc...

  • molecular dynamics simulations of viral neuraminidase inhibitors with the human neuraminidase enzymes insights into isoenzyme selectivity
    Bioorganic & Medicinal Chemistry, 2018
    Co-Authors: Michele R Richards, Tianlin Guo, Carmanah D Hunter, Christopher W. Cairo
    Abstract:

    Abstract Inhibitors of viral neuraminidase enzymes have been previously developed as therapeutics. Humans can express multiple forms of neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) that share a similar active site and enzymatic mechanism with their viral counterparts. Using a panel of purified human neuraminidase enzymes, we tested the inhibitory activity of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir, oseltamivir, and peramivir against each of the human isoenzymes. We find that, with the exceptions of DANA and zanamivir, these compounds show generally poor activity against the human neuraminidase enzymes. To provide insight into the interactions of viral inhibitors with human neuraminidases, we conducted molecular dynamics simulations using homology models based on coordinates reported for NEU2. Simulations revealed that an organized water is displaced by zanamivir in binding to NEU2 and NEU3 and confirmed the critical importance of engaging the binding pocket of the C7–C9 glycerol sidechain. Our results suggest that compounds designed to target the human neuraminidases should provide more selective tools for interrogating these enzymes. Furthermore, they emphasize a need for additional structural data to enable structure-based drug design in these systems.

  • identification of selective inhibitors for human neuraminidase isoenzymes using c4 c7 modified 2 deoxy 2 3 didehydro n acetylneuraminic acid dana analogues
    Journal of Medicinal Chemistry, 2013
    Co-Authors: Yi Zhang, Alexey V. Pshezhetsky, Amgad Albohy, Yao Zou, Victoria Smutova, Christopher W. Cairo
    Abstract:

    In the past two decades, human neuraminidases (human sialidases, hNEUs) have been found to be involved in numerous pathways in biology. The development of selective and potent inhibitors of these enzymes will provide critical tools for glycobiology, help to avoid undesired side effects of antivirals, and may reveal new small-molecule therapeutic targets for human cancers. However, because of the high active site homology of the hNEU isoenzymes, little progress in the design and synthesis of selective inhibitors has been realized. Guided by our previous studies of human NEU3 inhibitors, we designed a series of C4,C7-modified analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and tested them against the full panel of hNEU isoenzymes (NEU1, NEU2, NEU3, NEU4). We identified inhibitors with up to 38-fold selectivity for NEU3 and 12-fold selectivity for NEU2 over all other isoenzymes. We also identified compounds that targeted NEU2 and NEU3 with similar potency.

  • substrate recognition of the membrane associated sialidase neu3 requires a hydrophobic aglycone
    Biochemistry, 2011
    Co-Authors: Mahendra S Sandbhor, Amgad Albohy, Naoto Soya, Blake R Zheng, Jonathan Cartmell, David R Bundle, John S Klassen, Christopher W. Cairo
    Abstract:

    The human neuraminidases (NEU) consist of a family of four isoforms (NEU1–NEU4). Members of this enzyme family are proposed to have important roles in health and disease through regulation of the composition of cellular sialosides. The NEU3 isoform is a membrane-associated enzyme that cleaves glycolipid substrates. However, few reports have examined the substrate specificity of the enzyme for non-natural substrates. We report here a series of 11 synthetic trisaccharides that feature modifications of the aglycone or the Neu5Ac residue of an octyl β-sialyllactoside. The time course of substrate cleavage by NEU3 was monitored using an electrospray ionization mass spectrometry assay to obtain relative rates (krel). We observed that NEU3 substrate activity was directly dependent upon the hydrophobicity of the aglycone but had no apparent requirement for features of the ceramide headgroup. We also observed that trisaccharides with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac positio...

  • substrate recognition of the membrane associated sialidase neu3 requires a hydrophobic aglycone
    Biochemistry, 2011
    Co-Authors: Mahendra S Sandbhor, Amgad Albohy, Naoto Soya, Blake R Zheng, Jonathan Cartmell, David R Bundle, John S Klassen, Christopher W. Cairo
    Abstract:

    The human neuraminidases (NEU) consist of a family of four isoforms (NEU1–NEU4). Members of this enzyme family are proposed to have important roles in health and disease through regulation of the c...

Pascal Gagneux - One of the best experts on this subject based on the ideXlab platform.

  • sialidases on mammalian sperm mediate deciduous sialylation during capacitation
    Journal of Biological Chemistry, 2012
    Co-Authors: Diana Wu, Liwen Deng, Patrick Secrest, June Zhao, Nissi M Varki, Steven R Lindheim, Pascal Gagneux
    Abstract:

    Abstract Sialic acids mediate many biological functions, including molecular recognition during development and immune response and fertilization. A sialic acid-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their sialic acids. We quantified shed sialic acid monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting Sialidase activity interferes with the sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases from sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.

  • sialidases on mammalian sperm mediate deciduous sialylation during capacitation
    Journal of Biological Chemistry, 2012
    Co-Authors: Liwen Deng, Patrick Secrest, June Zhao, Steven R Lindheim, Nissi Varki, Pascal Gagneux
    Abstract:

    Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.

Taeko Miyagi - One of the best experts on this subject based on the ideXlab platform.

  • lysosomal localization of japanese medaka oryzias latipes neu1 sialidase and its highly conserved enzymatic profiles with human
    Gene, 2016
    Co-Authors: Sena Ryuzono, Petros Kingstone Chigwechokha Chigwechokha, Ryo Takase, Asami Ikeda, Aki Funahashi, Taeko Miyagi, Kazuki Oishi, Masaharu Komatsu, Kazuhiro Shiozaki
    Abstract:

    Abstract Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182 bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase.

  • a crucial role of sialidase neu1 in hyaluronan receptor function of cd44 in t helper type 2 mediated airway inflammation of murine acute asthmatic model
    Clinical and Experimental Immunology, 2010
    Co-Authors: S Katoh, Satoko Senda, Setsuko Moriya, Akio Mori, Kazunori Yamaguchi, S Maeda, H. Fukuoka, T. Wada, Taeko Miyagi
    Abstract:

    Summary CD44 is a highly glycosylated cell adhesion molecule that is involved in lymphocyte infiltration of inflamed tissues. We have demonstrated previously that sialic acid residues of CD44 negatively regulates its receptor function and CD44 plays an important role in the accumulation of T helper type 2 (Th2) cells in the airway of a murine model of acute asthma. Here we evaluated the role of sialidase in the hyaluronic acid (HA) receptor function of CD44 expressed on CD4+ T cells, as well as in the development of a mite antigen-induced murine model of acute asthma. Splenic CD4+ T cell binding of HA was examined with flow cytometry. Expression of sialidases (Neu1, Neu2, Neu3 and Neu4) in spleen cells was evaluated by quantitative real-time reverse transcription–polymerase chain reaction. Airway inflammation and airway hyperresponsiveness (AHR) were evaluated in the asthmatic Neu1-deficient mouse strain SM/J model. Splenic CD4+ T cells from asthmatic model mice displayed increased HA receptor activity of CD44 after culture with the antigen, along with characteristic parallel induction of sialidase (Neu1) expression. This induction of HA binding was suppressed significantly by a sialidase inhibitor and was not observed in SM/J mice. Th2 cytokine concentration and absolute number of Th2 cells in the bronchoalveolar lavage fluid, and AHR were decreased in SM/J mice. In conclusion, HA receptor activity of CD44 and acute asthmatic reactions, including Th2-mediated airway inflammation and AHR, are dependent upon Neu1 enzymatic activity. Our observation suggests that Neu1 may be a target molecule for the treatment of asthma.

  • developmental change of sialidase neu4 expression in murine brain and its involvement in the regulation of neuronal cell differentiation
    Journal of Biological Chemistry, 2009
    Co-Authors: Kazuhiro Shiozaki, Kazunori Yamaguchi, Koichi Koseki, Momo Shiozaki, Hisashi Narimatsu, Taeko Miyagi
    Abstract:

    Abstract Sialidase Neu4 is reported to be dominantly expressed in the mouse brain, but its functional significance is not fully understood. We previously demonstrated that sialidase Neu3, also rich in mouse brain, is up-regulated during neuronal differentiation with involvement in acceleration of neurite formation. To elucidate physiological functions of Neu4, as well as Neu3, we determined expression during mouse brain development by quantitative RT-PCR. Expression was relatively low in the embryonic stage and then rapidly increased at 3–14 days after birth, whereas Neu3 demonstrated high levels in the embryonic stage and down-regulation after birth. Murine Neu4 was found to possess two isoforms differing in expression levels, developmental pattern, and enzymatic character. Distinct from the human isoforms, the murine forms, to a different extent, both catalyzed the removal of sialic acid from gangliosides as well as glycoproteins, and one isoform seemed to act on polysialylated NCAM efficiently, despite the low activity toward ordinary substrates. In situ hybridization demonstrated Neu4 mRNA to be present mainly in the hippocampus in which NCAM is rich and decreases after birth. During retinoic acid-induced differentiation, Neu4 expression was down-regulated in Neuro2a cells. Overexpression of Neu4 resulted in suppression of neurite formation, and its knockdown showed the acceleration. Thin layer chromatography of the glycolipids from Neu4-transfected cells showed ganglioside compositions to be only slightly affected, although lectin blot analysis revealed increased binding to Ricinus communis agglutinin (RCA) lectin of a ∼95-kDa glycoprotein, which decreased with cell differentiation. These results suggest that mouse Neu4 plays an important regulatory role in neurite formation, possibly through desialylation of glycoproteins.

  • design synthesis and biological evaluation of human sialidase inhibitors part 1 selective inhibitors of lysosomal sialidase neu1
    Bioorganic & Medicinal Chemistry Letters, 2008
    Co-Authors: Sadagopan Magesh, Setsuko Moriya, Taeko Miyagi, Hideharu Ishida, Tohru Suzuki, Makoto Kiso
    Abstract:

    We here report the design and synthesis of selective human lysosomal sialidase (NEU1) inhibitors. A series of amide-linked C9 modified DANA (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) analogues were synthesized and their inhibitory activities against all four human sialidases (NEU1–NEU4) were determined. Structure-based approach was used to investigate the basis of selectivity of the compounds with experimentally observed activity. Results from the present study are found to be informative in a qualitative manner for the further design of isoform selective human sialidase inhibitors for therapeutic value.

  • down regulation of sialidase neu4 may contribute to invasive properties of human colon cancers
    Cancer Science, 2007
    Co-Authors: Hideaki Yamanami, Kazuhiro Shiozaki, Kazunori Yamaguchi, Tadashi Wada, Takuji Uemura, Yoichiro Kakugawa, Tsuneaki Hujiya, Taeko Miyagi
    Abstract:

    In mammalian cells, four types of sialidase have been described and found to behave in different ways during carcinogenesis. We previously demonstrated that a human sialidase associated with plasma membranes (NEU3) is up-regulated in human colon cancer and is involved in suppression of apoptosis. Here we document altered expression of another human sialidase, the recently identified NEU4, and evidence of its influence on the malignant phenotype in colon cancers. Human colon mucosa was relatively rich in NEU4, which has been observed to possess short and long isoforms, but hardly contained the latter form. In clear contrast to the NEU3 case, the levels of mRNA for this sialidase were found by quantitative RT-PCR to be markedly decreased in colon cancers. In cultured human colon cancer cells, the enzyme was up-regulated in the early stage of apoptosis induced by either the death ligand TRAIL or serum-depletion, and transfection of NEU4 resulted in acceleration of apoptosis and in decreased invasion and motility. The siRNA-mediated NEU4 targeting, on the other hand, caused a significant inhibition of apoptosis and promotion of invasion and motility. Lectin blot analyses revealed that desialylated forms of nearly 100 kDa glycoproteins were prominently increased with PNA in NEU4 transfectants, whereas only slight changes in glycolipids were detected as assessed by thin layer chromatography. These results suggest that NEU4 plays important roles for maintenance of normal mucosa mostly through desialylation of glycoproteins and that down-regulation may contribute to invasive properties of colon cancers.