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Expression of a Second Ecto-5′-Nucleotidase Variant Besides the Usual Protein in Symptomatic Phase of Experimental Autoimmune Encephalomyelitis

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Abstract

Ecto-5′-nucleotidase/cluster of differentiation 73 (CD73) (eN) is a 70-kDa glycoprotein expressed in several different mammalian tissues and cell types. It is the rate-limiting enzyme of the purine catabolic pathway, which catalyzes the hydrolysis of AMP to produce adenosine with known anti-inflammatory and immunosuppressive actions. There is strong evidence for lymphocyte and endothelial cell eN having a role in experimental autoimmune encephalomyelitis (EAE), but the role of eN in cell types within the central nervous system is less clear. We have previously shown that eN activity significantly increased in the lumbar spinal cord during EAE. The present study is aimed to explore molecular pattern of the eN upregulation over the course of the disease and cell type(s) accountable for the induction. EAE was induced in Dark Agouti (DA) rats by immunization with the spinal cord tissue homogenate and adjuvant. Animals were sacrificed 8, 15, and 28 days following immunization (D8, D15, and D28), i.e., at time points which corresponded to the presymptomatic, symptomatic, and postsymptomatic phases of the disease, respectively. Significant increase in eN activity and its upregulation at the gene and the protein levels were demonstrated at D15 and less prominently at D28 in comparison to control. Additionally, reactive astrocytes abundantly present in the lumbar spinal cord parenchyma were identified as principal cell type with significantly elevated eN expression. In all experimental groups, eN was expressed as a 71-kDa protein band of uniform abundance, whereas the overexpression of eN at D15 and D28 was associated with the expression of a second 75-kDa eN variant. The possible outcome of eN upregulation during EAE as a part of protective astrocyte repertoire contributing to the resolution of the disease is discussed.

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Abbreviations

ATP:

Adenosine triphosphate

CNS:

Central nervous system

D:

Days after immunization

EAE:

Experimental autoimmune encephalomyelitis

eN:

Ecto-5′-nucleotidase

ENT:

Equilibrative nucleoside transporter

MS:

Multiple sclerosis

NTPDase:

Nucleoside triphosphate diphosphohydrolase

Panx1:

pannexin 1

References

  • Airas L, Niemela J, Yegutkin GG, Jalkanen S (2007) Mechanism of action of IFN-β in the treatment of multiple sclerosis: a special reference to CD73 and adenosine. Ann N Y Acad Sci 1110:641–648

    Article  CAS  PubMed  Google Scholar 

  • Aloisi F, Penna G, Cerase J, Menendez IB, Adorini L (1997) IL-12 production by central nervous system microglia is inhibited by astrocytes. J Immunol 159:1604–1612

    CAS  PubMed  Google Scholar 

  • Ascherio A, Munger KL (2007) Environmental risk factors for multiple sclerosis. II. Noninfectious factors. Ann Neurol 61:504–513

    Article  CAS  PubMed  Google Scholar 

  • Bjelobaba I, Parabucki A, Lavrnja I, Stojkov D, Dacic S, Pekovic S, Rakic L, Stojiljkovic M, Nedeljkovic N (2011) Dynamic changes in the expression pattern of ecto-5′-nucleotidase in the rat model of cortical stab injury. J Neurosci Res 89:862–873

    Article  CAS  PubMed  Google Scholar 

  • Blazevski J, Petkovic F, Momcilovic M, Jevtic B, Miljkovic D, Mostarica-Stojkovic M (2013) High interleukin-10 expression within the central nervous system may be important for initiation of recovery of Dark Agouti rats from experimental autoimmune encephalomyelitis. Immunobiology 218:1192–1199

    Article  CAS  PubMed  Google Scholar 

  • Bours MJ (2006) Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol Ther 112:358–404

    Article  CAS  PubMed  Google Scholar 

  • Braun N, Zhu Y, Krieglstein J, Culmsee C, Zimmermann H (1998) Upregulation of the enzyme chain hydrolyzing extracellular ATP after transient forebrain ischemia in the rat. J Neurosci 18:4891–4900

    CAS  PubMed  Google Scholar 

  • Brisevac D, Bjelobaba I, Bajic A, Clarner T, Stojiljkovic M, Beyer M, Andjus P, Kipp M, Nedeljkovic N (2012) Regulation of ecto-5′-nucleotidase (CD73) in cultured cortical astrocytes by different inflammatory factors. Neurochem Int 61:681–688

    Article  CAS  PubMed  Google Scholar 

  • Brück W (2005) The pathology of multiple sclerosis is the result of focal inflammatory demyelination with axonal damage. J Neurol 252S:v3–v9

    Article  Google Scholar 

  • Chen Y, Swanson RA (2003) Astrocytes and brain injury. J Cereb Blood Flow Metab 23:137–149

    Article  PubMed  Google Scholar 

  • Christensen LD (1996) No correlation between CD73 expression and ecto-5′-nucleotidase activity on blood mononuclear cells in vitro. Evidence of CD73 (ecto-5′-nucleotidase) on blood mononuclear cells with distinct antigenic properties. APMIS 10:126–134

    Article  Google Scholar 

  • Cuhna RA, Ribeiro JA (2000) ATP as a presynaptic modulator. Life Sci 68:119–137

    Article  Google Scholar 

  • Cunha RA, Brendel P, Zimmermann H, Ribeiro JA (2000) Immunologically distinct isoforms of ecto-5′-nucleotidase in nerve terminals of different areas of the rat hippocampus. J Neurochem 74:334–338

    Article  CAS  PubMed  Google Scholar 

  • Dong Y, Benveniste EN (2001) Immune function of astrocytes. Glia 36:180–190

    Article  CAS  PubMed  Google Scholar 

  • Eltzschig HK, Sitkovsky MV, Robson SC (2012) Purinergic signaling during inflammation. N Engl J Med 367:2322–2333

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Eng LF, Gerstl B, Vanderhaeghen JJ (1970) A study of proteins in old multiple sclerosis plaques. Trans Am Soc Neurochem 1:42

    Google Scholar 

  • Farina C, Aloisi F, Meinl E (2007) Astrocytes are active players in cerebral innate immunity. Trends Immunol 28:138–145

    Article  CAS  PubMed  Google Scholar 

  • Fiebich BL, Akter S, Akundi RS (2014) The two-hit hypothesis for neuroinflammation: role of exogenous ATP in modulating inflammation in the brain. Front Cell Neurosci 8(260):1–11. doi:10.3389/fncel.2014.00260

    Google Scholar 

  • Fini C, Amoresano A, Andolfo A, D’Auria S, Floridi A, Paolini S, Pucci P (2000) Mass spectrometry study of ecto-5′-nucleotidase from bull seminal plasma. Eur J Biochem 267:4978–4987

    Article  CAS  PubMed  Google Scholar 

  • Franke H, Verkhratsky A, Burnstock G, Illes P (2012) Pathophysiology of astroglial purinergic signalling. Purinergic Signal 8:629–657

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Garcia Redondo PA, Nakamura CV, de Souza W, Morgado–Díaz JA (2004) Differential expression of sialic acid and N-acetyl galactosamine residues on the cell surface of intestinal epithelial cells according to normal or metastatic potential. J Histochem Cytochem 52:629–640

    Article  Google Scholar 

  • Garcıa-Ayllon MS, Campoy FJ, Vidal CJ, Munoz-Delgado E (2001) Identification of inactive ecto-5′-nucleotidase in normal mouse muscle and its increased activity in dystrophic Lama2 dy mice. J Neurosci Res 66:656–665

    Article  PubMed  Google Scholar 

  • Gray EG, Whittaker VP (1962) The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation. J Anat 96:79–88

    PubMed Central  CAS  PubMed  Google Scholar 

  • Grkovic I, Bjelobaba I, Nedeljkovic N, Mitrovic N, Drakulic D, Stanojlovic M, Horvat A (2014) Developmental increase in ecto-5′-nucleotidase activity overlaps with appearance of two immunologically distinct enzyme isoforms in rat hippocampal synaptic plasma membranes. J Mol Neurosci 54:109–118

    Article  CAS  PubMed  Google Scholar 

  • Henttinen T, Jalkanen S, Yegutkin GG (2003) Adherent leukocytes prevent adenosine formation and impair endothelial barrier function by ecto-5′-nucleotidase/CD73-dependent mechanism. J Biol Chem 278:24888–24895

    Article  CAS  PubMed  Google Scholar 

  • Hide I, Tanaka M, Inoue A, Nakajima K, Kohsaka S, Inoue K, Nakata Y (2000) Extracellular ATP triggers tumor necrosis factor-alpha release from rat microglia. J Neurochem 75:965–972

    Article  CAS  PubMed  Google Scholar 

  • Hostenbach S, Cambron M, D’haeseleer M, Kooijman R, De Keyser J (2014) Astrocyte loss and astrogliosis in neuroinflammatory disorders. Neurosci Lett 565:39–41

    Article  CAS  PubMed  Google Scholar 

  • Ingram G, Loveless S, Howell OW, Hakobyan S, Dancey B, Harris CL, Robertson NP, Neal JW, Morgan BP (2014) Complement activation in multiple sclerosis plaques: an immunohistochemical analysis. Acta Neuropathol Commun 2:53

    Article  PubMed Central  PubMed  Google Scholar 

  • John GR, Lee SC, Song X, Rivieccio M, Brosnan CF (2005) IL-1-regulated responses in astrocytes: relevance to injury and recovery. Glia 49:161–176

    Article  PubMed  Google Scholar 

  • Jun S, Ochoa-Reparaz J, Zlotkowska D, Hoyt T, Pascual DW (2012) Bystander-mediated stimulation of proteolipid protein-specific regulatory T (Treg) cells confers protection against experimental autoimmune encephalomyelitis (EAE) via TFG-β. J Neuroimmunol 245:39–47

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Keegan BM, Noseworthy JH (2002) Multiple sclerosis. Annu Rev Med 53:285–302

    Article  CAS  PubMed  Google Scholar 

  • Kobie JJ, Shah PR, Yang L, Rebhahn JA, Fowell DJ, Mosmann TR (2006) T regulatory and primed uncommitted CD4 T cells express CD73, which suppresses effector CD4 T cells by converting 5′-adenosine monophosphate to adenosine. J Immunol 177:6780–6786

    Article  CAS  PubMed  Google Scholar 

  • Lassmann H, Bruck W, Lucchinetti CF (2007) The immunopathology of multiple sclerosis: an overview. Brain Pathol 17:210–218

    Article  PubMed  Google Scholar 

  • Lavrnja I, Bjelobaba I, Stojiljkovic M, Pekovic S, Mostatica-Stojkovic M, Stosic-Grujicic S, Nedeljkovic N (2009) Time-course changes in ectonucleotidase activities during experimental autoimmune encephalomyelitis. Neurochem Int 55:193–198

    Article  CAS  PubMed  Google Scholar 

  • Lavrnja I, Savic D, Bjelobaba I, Dacic S, Bozic I, Parabucki A, Nedeljkovic N, Pekovic S, Rakic L, Stojiljkovic M (2012) The effect of ribavirin on reactive astrogliosis in experimental autoimmune encephalomyelitis. J Pharmacol Sci 119:221–232

    Article  CAS  PubMed  Google Scholar 

  • Liedtke W, Edelmann W, Chiu FC, Kucherlapati R, Raine CS (1998) Experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion. Am J Pathol 152:251–259

    PubMed Central  CAS  PubMed  Google Scholar 

  • Matute C (2011) Glutamate and ATP signalling in white matter pathology. J Anat 219:53–64

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mills JH, Thompson LF, Mueller C, Waickman AT, Jalkanen S, Niemela J, Airas L, Bynoe MS (2008) CD73 is required for efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalomyelitis. PNAS 105:9325–9330

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mills JH, Alabanza LM, Mahamed DA, Bynoe MS (2012) Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis. J Neuroinflammation 9:193

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Misumi Y, Ogata S, Hirose S, Ikehara Y (1990) Primary structure of rat liver 5′-nucleotidase deduced from the cDNA. Presence of the COOH-terminal hydrophobic domain for possible posttranslational modification by glycophospholipid. J Biol Chem 265:2178–2183

    CAS  PubMed  Google Scholar 

  • Morote-Garcia JC, Sanchez Del Campo LF, Campoy FJ, Vidal CJ, Munoz-Delgado E (2006) The increased ecto-5′-nucleotidase activity in muscle, heart and liver of laminin alpha2-deficient mice is not caused by an elevation in the mRNA content. Int J Biochem Cell Biol 38:1092–1101

    Article  CAS  PubMed  Google Scholar 

  • Nair A, Frederick TJ, Miller SD (2008) Astrocytes in multiple sclerosis: a product of their environment. Cell Mol Life Sci 65:2702–2720

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Navarro JM, Olmo N, Lopez-Conejo MT, Lizarbe MA (1998) Ecto-5′-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells. Mol Cell Biochem 187:121–131

    Article  CAS  PubMed  Google Scholar 

  • Nedeljkovic N, Bjelobaba I, Subasic S, Lavrnja I, Pekovic S, Stojkov D, Vjestica A, Rakic L, Stojiljkovic M (2006) Upregulation of ectonucleotidase activity after cortical stab injury in rat. Cell Biol Int 30:541–546

    Article  CAS  PubMed  Google Scholar 

  • Niemela J, Ifergan I, Yegutkin GG, Jalkanen S, Prat A, Airas L (2008) IFN-b regulates CD73 and adenosine expression at the blood–brain barrier. Eur J Immunol 38:2718–2726

    Article  CAS  PubMed  Google Scholar 

  • Niino M, Fukazawa T, Kikuchi S, Sasaki H (2007) Recent advances in genetic analysis of multiple sclerosis: genetic associations and therapeutic implications. Expert Rev Neurother 7:1175–1188

    Article  CAS  PubMed  Google Scholar 

  • Roth J, Zuber C, Park S, Jang I, Lee Y, Kysela KG, Le Fourn V, Santimaria R, Guhl B, Cho JW (2010) Protein N-glycosylation, protein folding, and protein quality control. Mol Cells 30:497–506

    Article  CAS  PubMed  Google Scholar 

  • Serhan CN, Savill J (2005) Resolution of inflammation: the beginning programs the end. Nat Immunol 6:1191–1197

    Article  CAS  PubMed  Google Scholar 

  • Thompson LF, Takedachi M, Ebisuno Y, Tanaka T, Miyasaka M, Mills JH, Bynoe MS (2008) Regulation of leukocyte migration across endothelial barriers by ecto-5′-nucleotidase-generated adenosine. Nucleosides Nucleotides Nucleic Acids 27:755–760

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Virgilio FD, Ceruti S, Bramanti P, Abbracchio MP (2009) Purinergic signalling in inflammation of the central nervous system. Trends Neurosci 32:79–87

    Article  PubMed  Google Scholar 

  • Vogel M, Kowalewski HJ, Zimmermann H, Janetzko A, Margolis RU, Wollny HE (1991) Association of the HNK-1 epitope with 5′-nucleotidase from Torpedo marmorata (electric ray) electric organ. Biochem J 278:199–202

    PubMed Central  CAS  PubMed  Google Scholar 

  • Voskuhl RR, Peterson S, Song B, Ao Y, Morales LBJ, Tiwari-Woodruff S, Sofroniew MV (2009) Reactive astrocytes form scar-like perivascular barriers to leukocytes during adaptive immune inflammation of the CNS. J Neurosci 29:11511–11522

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wada I, Himeno M, Furuno K, Kato K (1986) Biosynthesis and intracellular transport of rat liver 5′-nucleotidase. J Biol Chem 261:2222–2227

    CAS  PubMed  Google Scholar 

  • Warford J, Jones QR, Nichols M, Sullivan V, Rupasinghe HP, Robertson GS (2014) The flavonoid-enriched fraction AF4 suppresses neuroinflammation and promotes restorative gene expression in a mouse model of experimental autoimmune encephalomyelitis. J Neuroimmunol 268:71–83

    Article  CAS  PubMed  Google Scholar 

  • Zhang Q, Cui F, Fang L, Hong J, Zheng B, Zhang JZ (2013) TNF-α impairs differentiation and function of TGF-β-induced Treg cells in autoimmune diseases through Akt and Smad3 signaling pathway. J Mol Cell Biol 5:85–98

    Article  CAS  PubMed  Google Scholar 

  • Zimmermann H (1992) 5′-Nucleotidase—molecular structure and functional aspects. Biochem J 285:345–365

    PubMed Central  CAS  PubMed  Google Scholar 

  • Zimmermann H (2000) Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362:299–309

    Article  CAS  PubMed  Google Scholar 

  • Zimmermann H, Braun N (1999) Ecto-nucleotidases: molecular structures, catalytic properties, and functional roles in the nervous system. Prog Brain Res 120:371–385

    Article  CAS  PubMed  Google Scholar 

  • Zimmermann H, Zebisch M, Strater N (2012) Cellular functions and molecular structure of ecto-nucleotidases. Purinergic Signal 8:437–502

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

This study is supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Project No. III41014.

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Authors and Affiliations

  1. Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Boulevard Despot Stefan 142, Belgrade, 11060, Serbia

    Irena Lavrnja, Danijela Savic, Iva Bozic, Ivana Bjelobaba & Sanja Pekovic

  2. Institute for Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 3, Belgrade, 11001, Serbia

    Danijela Laketa & Nadezda Nedeljkovic

Authors
  1. Irena Lavrnja
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  2. Danijela Laketa
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  3. Danijela Savic
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  4. Iva Bozic
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  5. Ivana Bjelobaba
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  6. Sanja Pekovic
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  7. Nadezda Nedeljkovic
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Corresponding author

Correspondence to Nadezda Nedeljkovic.

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Irena Lavrnja and Danijela Laketa contributed equally to this work.

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Lavrnja, I., Laketa, D., Savic, D. et al. Expression of a Second Ecto-5′-Nucleotidase Variant Besides the Usual Protein in Symptomatic Phase of Experimental Autoimmune Encephalomyelitis. J Mol Neurosci 55, 898–911 (2015). https://doi.org/10.1007/s12031-014-0445-x

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