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Examination of Gelatinase Isoforms in Rodent Models of Acute Neurodegenerative Diseases Using Two-Dimensional Zymography

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Zymography

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1626))

Abstract

Pathological activation of gelatinases (matrix metalloproteinase-2 and -9; MMP-2/-9) has been shown to cause a number of detrimental outcomes in neurodegenerative diseases. In gel gelatin zymography is a highly sensitive methodology commonly used in revealing levels of gelatinase activity and in separating the proform and active form of gelatinases, based on their different molecular weights. However, this methodology is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity can be regulated at transcriptional and/or post-translational levels under in vivo conditions resulting in alternation of their isoelectric focusing (IEF) points. In this chapter, we describe an advanced methodology, termed two-dimensional zymography, combining IEF with zymographic electrophoresis under non-reducing conditions to achieve significant improvement in separation of the gelatinase isoforms in both cell-based and in vivo models for acute brain injuries and neuroinflammation.

Fanjun Meng (Deceased)

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References

  1. Kessenbrock K, Plaks V, Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141(1):52–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Parks WC, Wilson CL, Lopez-Boado YS (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 4(8):617–629

    Article  CAS  PubMed  Google Scholar 

  3. Mott JD, Werb Z (2004) Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 16(5):558–564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yong VW (2005) Metalloproteinases: mediators of pathology and regeneration in the CNS. Nat Rev Neurosci 6(12):931–944

    Article  CAS  PubMed  Google Scholar 

  5. Rosenberg GA (2009) Matrix metalloproteinases and their multiple roles in neurodegenerative diseases. Lancet Neurol 8(2):205–216

    Article  CAS  PubMed  Google Scholar 

  6. Kaplan A, Spiller KJ, Towne C, Kanning KC, Choe GT, Geber A, Akay T, Aebischer P, Henderson CE (2014) Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration. Neuron 81(2):333–348

    Article  CAS  PubMed  Google Scholar 

  7. Agrawal SM, Lau L, Yong VW (2008) MMPs in the central nervous system: where the good guys go bad. Semin Cell Dev Biol 19(1):42–51

    Article  CAS  PubMed  Google Scholar 

  8. Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA (2002) S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science 297(5584):1186–1190

    Article  CAS  PubMed  Google Scholar 

  9. Chaturvedi M, Kaczmarek L (2014) Mmp-9 inhibition: a therapeutic strategy in ischemic stroke. Mol Neurobiol 49(1):563–573

    Article  CAS  PubMed  Google Scholar 

  10. Hadass O, Tomlinson BN, Gooyit M, Chen S, Purdy JJ, Walker JM, Zhang C, Giritharan AB, Purnell W, Robinson CR 2nd, Shin D, Schroeder VA, Suckow MA, Simonyi A, Sun GY, Mobashery S, Cui J, Chang M, Gu Z (2013) Selective inhibition of matrix metalloproteinase-9 attenuates secondary damage resulting from severe traumatic brain injury. PLoS One 8(10):e76904

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chen ZL, Strickland S (1997) Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 91(7):917–925

    Article  CAS  PubMed  Google Scholar 

  12. Montaner J, Alvarez-Sabin J, Molina CA, Angles A, Abilleira S, Arenillas J, Monasterio J (2001) Matrix metalloproteinase expression is related to hemorrhagic transformation after cardioembolic stroke. Stroke 32(12):2762–2767

    Article  CAS  PubMed  Google Scholar 

  13. Horstmann S, Kalb P, Koziol J, Gardner H, Wagner S (2003) Profiles of matrix metalloproteinases, their inhibitors, and laminin in stroke patients: influence of different therapies. Stroke 34(9):2165–2170

    Article  PubMed  Google Scholar 

  14. Switzer JA, Hess DC, Ergul A, Waller JL, Machado LS, Portik-Dobos V, Pettigrew LC, Clark WM, Fagan SC (2011) Matrix metalloproteinase-9 in an exploratory trial of intravenous minocycline for acute ischemic stroke. Stroke 42(9):2633–2635

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Castellanos M, Leira R, Serena J, Pumar JM, Lizasoain I, Castillo J, Davalos A (2003) Plasma metalloproteinase-9 concentration predicts hemorrhagic transformation in acute ischemic stroke. Stroke 34(1):40–46

    Article  CAS  PubMed  Google Scholar 

  16. Gasche Y, Fujimura M, Morita-Fujimura Y, Copin JC, Kawase M, Massengale J, Chan PH (1999) Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: a possible role in blood-brain barrier dysfunction. J Cereb Blood Flow Metab 19(9):1020–1028

    Article  CAS  PubMed  Google Scholar 

  17. Lapchak PA, Chapman DF, Zivin JA (2000) Metalloproteinase inhibition reduces thrombolytic (tissue plasminogen activator)-induced hemorrhage after thromboembolic stroke. Stroke 31(12):3034–3040

    Article  CAS  PubMed  Google Scholar 

  18. Wang J, Tsirka SE (2005) Neuroprotection by inhibition of matrix metalloproteinases in a mouse model of intracerebral haemorrhage. Brain 128(Pt 7):1622–1633

    Article  PubMed  Google Scholar 

  19. Van Wart HE, Birkedal-Hansen H (1990) The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc Natl Acad Sci U S A 87(14):5578–5582

    Article  PubMed  PubMed Central  Google Scholar 

  20. Park AJ, Matrisian LM, Kells AF, Pearson R, Yuan ZY, Navre M (1991) Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the “cysteine switch”. J Biol Chem 266(3):1584–1590

    CAS  PubMed  Google Scholar 

  21. Vandooren J, Van den Steen PE, Opdenakker G (2013) Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9): the next decade. Crit Rev Biochem Mol Biol 48(3):222–272

    Article  CAS  PubMed  Google Scholar 

  22. Rossano R, Larocca M, Riviello L, Coniglio MG, Vandooren J, Liuzzi GM, Opdenakker G, Riccio P (2014) Heterogeneity of serum gelatinases MMP-2 and MMP-9 isoforms and charge variants. J Cell Mol Med 18(2):242–252

    Article  CAS  PubMed  Google Scholar 

  23. Okamoto T, Akaike T, Nagano T, Miyajima S, Suga M, Ando M, Ichimori K, Maeda H (1997) Activation of human neutrophil procollagenase by nitrogen dioxide and peroxynitrite: a novel mechanism for procollagenase activation involving nitric oxide. Arch Biochem Biophys 342(2):261–274

    Article  CAS  PubMed  Google Scholar 

  24. Okamoto T, Akaike T, Sawa T, Miyamoto Y, van der Vliet A, Maeda H (2001) Activation of matrix metalloproteinases by peroxynitrite-induced protein S-glutathiolation via disulfide S-oxide formation. J Biol Chem 276(31):29596–29602

    Article  CAS  PubMed  Google Scholar 

  25. Viappiani S, Nicolescu AC, Holt A, Sawicki G, Crawford BD, Leon H, van Mulligen T, Schulz R (2009) Activation and modulation of 72 kDa matrix metalloproteinase-2 by peroxynitrite and glutathione. Biochem Pharmacol 77(5):826–834

    Article  CAS  PubMed  Google Scholar 

  26. Sariahmetoglu M, Crawford BD, Leon H, Sawicka J, Li L, Ballermann BJ, Holmes C, Berthiaume LG, Holt A, Sawicki G, Schulz R (2007) Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation. FASEB J 21(10):2486–2495

    Article  CAS  PubMed  Google Scholar 

  27. Granelli-Piperno A, Reich E (1978) A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med 148(1):223–234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Vandooren J, Geurts N, Martens E, Van den Steen PE, Opdenakker G (2013) Zymography methods for visualizing hydrolytic enzymes. Nat Methods 10(3):211–220

    Article  CAS  PubMed  Google Scholar 

  29. Schulz R (2007) Intracellular targets of matrix metalloproteinase-2 in cardiac disease: rationale and therapeutic approaches. Annu Rev Pharmacol Toxicol 47:211–242

    Article  CAS  PubMed  Google Scholar 

  30. Chen S, Meng F, Chen Z, Tomlinson BN, Wesley JM, Sun GY, Whaley-Connell AT, Sowers JR, Cui J, Gu Z (2015) Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries. PLoS One 10(4):e0123852

    Article  PubMed  PubMed Central  Google Scholar 

  31. Gu Z, Cui J, Brown S, Fridman R, Mobashery S, Strongin AY, Lipton SA (2005) A highly specific inhibitor of matrix metalloproteinase-9 rescues laminin from proteolysis and neurons from apoptosis in transient focal cerebral ischemia. J Neurosci 25(27):6401–6408

    Article  CAS  PubMed  Google Scholar 

  32. Cui J, Chen S, Zhang C, Meng F, Wu W, Hu R, Hadass O, Lehmidi T, Blair GJ, Lee M, Chang M, Mobashery S, Sun GY, Gu Z (2012) Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia. Mol Neurodegener 7:21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Shen S, Yu S, Binek J, Chalimoniuk M, Zhang X, Lo SC, Hannink M, Wu J, Fritsche K, Donato R, Sun GY (2005) Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells. Neurochem Int 47(4):298–307

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by the American Heart Association National Scientist Development award (09SDG2260983), The Dana Foundation, The National Football Leagues (NFL) Charities Foundation, The University of Missouri Mizzou Advantage One Health One Medicine Program and the Department of Pathology Research funds to Z.G.

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Authors and Affiliations

  1. Department of Pathology and Anatomical Sciences, University of Missouri School of Medicine, Columbia, MO, USA

    Shanyan Chen, Fanjun Meng, Zhenzhou Chen, Zhe Qu, Jiankun Cui & Zezong Gu

  2. Interdisciplinary Neuroscience Program, University of Missouri, Columbia, MO, USA

    Shanyan Chen

  3. Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO, USA

    Jiankun Cui & Zezong Gu

Authors
  1. Shanyan Chen
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  2. Fanjun Meng
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  3. Zhenzhou Chen
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  4. Zhe Qu
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  5. Jiankun Cui
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  6. Zezong Gu
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Correspondence to Zezong Gu .

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Editors and Affiliations

  1. Centre for Environmental, Biology and Chemistry Research, Facultad de Ciencias y Tecnología, University of Carabobo, Valencia, Venezuela

    Jeff Wilkesman

  2. Centre for Environmental, Biology and Chemistry Research Facultad de Ciencias y Tecnología, University of Carabobo, Valencia, Venezuela

    Liliana Kurz

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Chen, S., Meng, F., Chen, Z., Qu, Z., Cui, J., Gu, Z. (2017). Examination of Gelatinase Isoforms in Rodent Models of Acute Neurodegenerative Diseases Using Two-Dimensional Zymography. In: Wilkesman, J., Kurz, L. (eds) Zymography. Methods in Molecular Biology, vol 1626. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7111-4_13

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  • DOI: https://doi.org/10.1007/978-1-4939-7111-4_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7109-1

  • Online ISBN: 978-1-4939-7111-4

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