1887

Abstract

We established abnormal isoform of prion protein (PrP)-specific double immunostaining using mAb 132, which recognizes aa 119–127 of the PrP molecule, and novel PrP-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrP-specific double immunostaining, we analysed PrP in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrP-specific double immunostaining showed the existence of PrP positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrP species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrP staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrP stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrP stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrP in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrP in N2a-3 lack the N-terminal portion. In contrast, nearly half PrP detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrP that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrP-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000514
2016-08-01
2019-10-16
Loading full text...

Full text loading...

/deliver/fulltext/jgv/97/8/2030.html?itemId=/content/journal/jgv/10.1099/jgv.0.000514&mimeType=html&fmt=ahah

References

  1. Arnold J. E. , Tipler C. , Laszlo L. , Hope J. , Landon M. , Mayer R. J. . ( 1995;). The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain. . J Pathol 176: 403–411. [CrossRef] [PubMed]
    [Google Scholar]
  2. Béranger F. , Mangé A. , Goud B. , Lehmann S. . ( 2002;). Stimulation of PrP(C) retrograde transport toward the endoplasmic reticulum increases accumulation of PrP(Sc) in prion-infected cells. . J Biol Chem 277: 38972–38977. [CrossRef] [PubMed]
    [Google Scholar]
  3. Borchelt D. R. , Taraboulos A. , Prusiner S. B. . ( 1992;). Evidence for synthesis of scrapie prion proteins in the endocytic pathway. . J Biol Chem 267: 16188–16199.[PubMed]
    [Google Scholar]
  4. Carimalo J. , Cronier S. , Petit G. , Peyrin J. M. , Boukhtouche F. , Arbez N. , Lemaigre-Dubreuil Y. , Brugg B. , Miquel M. C. . ( 2005;). Activation of the JNK–c-Jun pathway during the early phase of neuronal apoptosis induced by PrP106-126 and prion infection. . Eur J Neurosci 21: 2311–2319. [CrossRef] [PubMed]
    [Google Scholar]
  5. Caughey B. , Raymond G. J. . ( 1991;). The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. . J Biol Chem 266: 18217–18223.[PubMed]
    [Google Scholar]
  6. Caughey B. , Raymond G. J. , Ernst D. , Race R. E. . ( 1991;). N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state. . J Virol 65: 6597–6603.[PubMed]
    [Google Scholar]
  7. Chen S. G. , Teplow D. B. , Parchi P. , Teller J. K. , Gambetti P. , Autilio-Gambetti L. . ( 1995;). Truncated forms of the human prion protein in normal brain and in prion diseases. . J Biol Chem 270: 19173–19180.[PubMed] [CrossRef]
    [Google Scholar]
  8. Cronier S. , Laude H. , Peyrin J. M. . ( 2004;). Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death. . Proc Natl Acad Sci U S A 101: 12271–12276. [CrossRef] [PubMed]
    [Google Scholar]
  9. Cronier S. , Beringue V. , Bellon A. , Peyrin J. M. , Laude H. . ( 2007;). Prion strain- and species-dependent effects of antiprion molecules in primary neuronal cultures. . J Virol 81: 13794–13800. [CrossRef] [PubMed]
    [Google Scholar]
  10. Curin Serbec V. , Bresjanac M. , Popovic M. , Pretnar Hartman K. , Galvani V. , Rupreht R. , Cernilec M. , Vranac T. , Hafner I. et al. ( 2004;). Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt-Jacob's disease-affected and normal brain tissue. . J Biol Chem 279: 3694–3698. [CrossRef] [PubMed]
    [Google Scholar]
  11. Dron M. , Moudjou M. , Chapuis J. , Salamat M. K. , Bernard J. , Cronier S. , Langevin C. , Laude H. . ( 2010;). Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly cell- and tissue-dependent. . J Biol Chem 285: 10252–10264. [CrossRef] [PubMed]
    [Google Scholar]
  12. Fournier J. G. , Escaig-Haye F. , Grigoriev V. . ( 2000;). Ultrastructural localization of prion proteins: physiological and pathological implications. . Microsc Res Tech 50: 76–88. [CrossRef] [PubMed]
    [Google Scholar]
  13. Fuhrmann M. , Mitteregger G. , Kretzschmar H. , Herms J. . ( 2007;). Dendritic pathology in prion disease starts at the synaptic spine. . J Neurosci 27: 6224–6233. [CrossRef] [PubMed]
    [Google Scholar]
  14. Godsave S. F. , Wille H. , Kujala P. , Latawiec D. , DeArmond S. J. , Serban A. , Prusiner S. B. , Peters P. J. . ( 2008;). Cryo-immunogold electron microscopy for prions: toward identification of a conversion site. . J Neurosci 28: 12489–12499. [CrossRef] [PubMed]
    [Google Scholar]
  15. Godsave S. F. , Wille H. , Pierson J. , Prusiner S. B. , Peters P. J. . ( 2013;). Plasma membrane invaginations containing clusters of full-length PrPSc are an early form of prion-associated neuropathology in vivo. . Neurobiol Aging 34: 1621–1631. [CrossRef] [PubMed]
    [Google Scholar]
  16. Goold R. , Rabbanian S. , Sutton L. , Andre R. , Arora P. , Moonga J. , Clarke A. R. , Schiavo G. , Jat P. et al. ( 2011;). Rapid cell-surface prion protein conversion revealed using a novel cell system. . Nat Commun 2: 281. [CrossRef] [PubMed]
    [Google Scholar]
  17. Gray B. C. , Siskova Z. , Perry V. H. , O'Connor V. . ( 2009;). Selective presynaptic degeneration in the synaptopathy associated with ME7-induced hippocampal pathology. . Neurobiol Dis 35: 63–74. [CrossRef] [PubMed]
    [Google Scholar]
  18. Hannaoui S. , Maatouk L. , Privat N. , Levavasseur E. , Faucheux B. A. , Haïk S. . ( 2013;). Prion propagation and toxicity occur in vitro with two-phase kinetics specific to strain and neuronal type. . J Virol 87: 2535–2548. [CrossRef] [PubMed]
    [Google Scholar]
  19. Horiuchi M. , Mochizuki M. , Ishiguro N. , Nagasawa H. , Shinagawa M. . ( 1997;). Epitope mapping of a monoclonal antibody specific to feline panleukopenia virus and mink enteritis virus. . J Vet Med Sci 59: 133–136.[PubMed] [CrossRef]
    [Google Scholar]
  20. Horiuchi M. , Karino A. , Furuoka H. , Ishiguro N. , Kimura K. , Shinagawa M. . ( 2009;). Generation of monoclonal antibody that distinguishes PrPSc from PrPC and neutralizes prion infectivity. . Virology 394: 200–207. [CrossRef] [PubMed]
    [Google Scholar]
  21. Jeffrey M. , Goodsir C. M. , Bruce M. E. , McBride P. A. , Scott J. R. , Halliday W. G. . ( 1992;). Infection specific prion protein (PrP) accumulates on neuronal plasmalemma in scrapie infected mice. . Neurosci Lett 147: 106–109. [CrossRef] [PubMed]
    [Google Scholar]
  22. Jeffrey M. , Goodsir C. M. , Bruce M. , McBride P. A. , Scott J. R. , Halliday W. G. . ( 1994;). Correlative light and electron microscopy studies of PrP localisation in 87V scrapie. . Brain Res 656: 329–343.[PubMed] [CrossRef]
    [Google Scholar]
  23. Jeffrey M. , Halliday W. G. , Bell J. , Johnston A. R. , MacLeod N. K. , Ingham C. , Sayers A. R. , Brown D. A. , Fraser J. R. . ( 2000;). Synapse loss associated with abnormal PrP precedes neuronal degeneration in the scrapie-infected murine hippocampus. . Neuropathol Appl Neurobiol 26: 41–54. [CrossRef] [PubMed]
    [Google Scholar]
  24. Kim C. L. , Umetani A. , Matsui T. , Ishiguro N. , Shinagawa M. , Horiuchi M. . ( 2004;). Antigenic characterization of an abnormal isoform of prion protein using a new diverse panel of monoclonal antibodies. . Virology 320: 40–51. [CrossRef] [PubMed]
    [Google Scholar]
  25. Korth C. , Stierli B. , Streit P. , Moser M. , Schaller O. , Fischer R. , Schulz-Schaeffer W. , Kretzschmar H. , Raeber A. et al. ( 1997;). Prion (PrPSc)-specific epitope defined by a monoclonal antibody. . Nature 390: 74–77. [CrossRef] [PubMed]
    [Google Scholar]
  26. Mallucci G. , Dickinson A. , Linehan J. , Klöhn P. C. , Brandner S. , Collinge J. . ( 2003;). Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. . Science 302: 871–874. [CrossRef] [PubMed]
    [Google Scholar]
  27. Marijanovic Z. , Caputo A. , Campana V. , Zurzolo C. . ( 2009;). Identification of an intracellular site of prion conversion. . PLoS Pathog 5: e1000426. [CrossRef] [PubMed]
    [Google Scholar]
  28. Masujin K. , Kaku-Ushiki Y. , Miwa R. , Okada H. , Shimizu Y. , Kasai K. , Matsuura Y. , Yokoyama T. . ( 2013;). The N-terminal sequence of prion protein consists an epitope specific to the abnormal isoform of prion protein (PrP(Sc)). . PLoS One 8: e58013. [CrossRef] [PubMed]
    [Google Scholar]
  29. Nakamitsu S. , Kurokawa A. , Yamasaki T. , Uryu M. , Hasebe R. , Horiuchi M. . ( 2010;). Cell density-dependent increase in the level of protease-resistant prion protein in prion-infected Neuro2a mouse neuroblastoma cells. . J Gen Virol 91: 563–569. [CrossRef] [PubMed]
    [Google Scholar]
  30. Naslavsky N. , Stein R. , Yanai A. , Friedlander G. , Taraboulos A. . ( 1997;). Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. . J Biol Chem 272: 6324–6331.[PubMed] [CrossRef]
    [Google Scholar]
  31. Paramithiotis E. , Pinard M. , Lawton T. , LaBoissiere S. , Leathers V. L. , Zou W. Q. , Estey L. A. , Lamontagne J. , Lehto M. T. et al. ( 2003;). A prion protein epitope selective for the pathologically misfolded conformation. . Nat Med 9: 893–899. [CrossRef] [PubMed]
    [Google Scholar]
  32. Pimpinelli F. , Lehmann S. , Maridonneau-Parini I. . ( 2005;). The scrapie prion protein is present in flotillin-1-positive vesicles in central- but not peripheral-derived neuronal cell lines. . Eur J Neurosci 21: 2063–2072. [CrossRef] [PubMed]
    [Google Scholar]
  33. Prusiner S. B. . ( 1998;). Prions. . Proc Natl Acad Sci U S A 95: 13363–13383.[PubMed] [CrossRef]
    [Google Scholar]
  34. Reiniger L. , Mirabile I. , Lukic A. , Wadsworth J. D. , Linehan J. M. , Groves M. , Lowe J. , Druyeh R. , Rudge P. et al. ( 2013;). Filamentous white matter prion protein deposition is a distinctive feature of multiple inherited prion diseases. . Acta Neuropathol Commun 1: 8. [CrossRef] [PubMed]
    [Google Scholar]
  35. Rouvinski A. , Karniely S. , Kounin M. , Moussa S. , Goldberg M. D. , Warburg G. , Lyakhovetsky R. , Papy-Garcia D. , Kutzsche J. et al. ( 2014;). Live imaging of prions reveals nascent PrPSc in cell-surface, raft-associated amyloid strings and webs. . J Cell Biol 204: 423–441. [CrossRef] [PubMed]
    [Google Scholar]
  36. Sakai K. , Hasebe R. , Takahashi Y. , Song C. H. , Suzuki A. , Yamasaki T. , Horiuchi M. . ( 2013;). Absence of CD14 delays progression of prion diseases accompanied by increased microglial activation. . J Virol 87: 13433–13445. [CrossRef] [PubMed]
    [Google Scholar]
  37. Schätzl H. M. , Laszlo L. , Holtzman D. M. , Tatzelt J. , DeArmond S. J. , Weiner R. I. , Mobley W. C. , Prusiner S. B. . ( 1997;). A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. . J Virol 71: 8821–8831.[PubMed]
    [Google Scholar]
  38. Shindoh R. , Kim C. L. , Song C. H. , Hasebe R. , Horiuchi M. . ( 2009;). The region approximately between amino acids 81 and 137 of proteinase K-resistant PrPSc is critical for the infectivity of the Chandler prion strain. . J Virol 83: 3852–3860. [CrossRef] [PubMed]
    [Google Scholar]
  39. Taraboulos A. , Serban D. , Prusiner S. B. . ( 1990;). Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells. . J Cell Biol 110: 2117–2132.[PubMed] [CrossRef]
    [Google Scholar]
  40. Taraboulos A. , Raeber A. J. , Borchelt D. R. , Serban D. , Prusiner S. B. . ( 1992;). Synthesis and trafficking of prion proteins in cultured cells. . Mol Biol Cell 3: 851–863.[PubMed] [CrossRef]
    [Google Scholar]
  41. Uryu M. , Karino A. , Kamihara Y. , Horiuchi M. . ( 2007;). Characterization of prion susceptibility in Neuro2a mouse neuroblastoma cell subclones. . Microbiol Immunol 51: 661–669.[PubMed] [CrossRef]
    [Google Scholar]
  42. Ushiki-Kaku Y. , Endo R. , Iwamaru Y. , Shimizu Y. , Imamura M. , Masujin K. , Yamamoto T. , Hattori S. , Itohara S. et al. ( 2010;). Tracing conformational transition of abnormal prion proteins during interspecies transmission by using novel antibodies. . J Biol Chem 285: 11931–11936. [CrossRef] [PubMed]
    [Google Scholar]
  43. Veith N. M. , Plattner H. , Stuermer C. A. , Schulz-Schaeffer W. J. , Bürkle A. . ( 2009;). Immunolocalisation of PrPSc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy. . Eur J Cell Biol 88: 45–63. [CrossRef] [PubMed]
    [Google Scholar]
  44. Vey M. , Pilkuhn S. , Wille H. , Nixon R. , DeArmond S. J. , Smart E. J. , Anderson R. G. , Taraboulos A. , Prusiner S. B. . ( 1996;). Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. . Proc Natl Acad Sci U S A 93: 14945–14949.[PubMed] [CrossRef]
    [Google Scholar]
  45. Wolf H. , Hossinger A. , Fehlinger A. , Büttner S. , Sim V. , McKenzie D. , Vorberg I. M. . ( 2015;). Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie. . Front Neurosci 9: 410. [CrossRef] [PubMed]
    [Google Scholar]
  46. Yamasaki T. , Suzuki A. , Shimizu T. , Watarai M. , Hasebe R. , Horiuchi M. . ( 2012;). Characterization of intracellular localization of PrPSc in prion-infected cells using a mAb that recognizes the region consisting of aa 119–127 of mouse PrP. . J Gen Virol 93: 668–680. [CrossRef] [PubMed]
    [Google Scholar]
  47. Yamasaki T. , Baron G. S. , Suzuki A. , Hasebe R. , Horiuchi M. . ( 2014a;). Characterization of intracellular dynamics of inoculated PrP-res and newly generated PrPSc during early stage prion infection in Neuro2a cells. . Virology 450–451: 324–335.[CrossRef]
    [Google Scholar]
  48. Yamasaki T. , Suzuki A. , Hasebe R. , Horiuchi M. . ( 2014b;). Comparison of the anti-prion mechanism of four different anti-prion compounds, anti-PrP monoclonal antibody 44B1, pentosan polysulfate, chlorpromazine, and U18666A, in prion-infected mouse neuroblastoma cells. . PLoS One 9: e106516. [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000514
Loading
/content/journal/jgv/10.1099/jgv.0.000514
Loading

Data & Media loading...

Supplements

Supplementary File 1



PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error