Skip to main content
SpringerLink
Log in

Cell Biology Approaches to Studying Prion Diseases

  • Protocol
  • First Online:
Prions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1658))

Abstract

During the course of prion infection, the normally soluble and protease-sensitive mammalian prion protein (PrPC) is refolded into an insoluble, partially protease-resistant, and infectious form called PrPSc. The conformational conversion of PrPC to PrPSc is a critical event during prion infection and is essential for the production of prion infectivity. This chapter briefly summarizes the ways in which cell biological approaches have enhanced our understanding of how PrP contributes to different aspects of prion pathogenesis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Grassmann A, Wolf H, Hofmann J, Graham J, Vorberg I (2013) Cellular aspects of prion replication in vitro. Virus 5:374–405

    Article  CAS  Google Scholar 

  2. Cronier S, Laude H, Peyrin JM (2004) Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death. Proc Natl Acad Sci U S A 101:12271–12276

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Falsig J, Julius C, Margalith I, Schwarz P, Heppner FL, Aguzzi A (2008) A versatile prion replication assay in organotypic brain slices. Nat Neurosci 11:109–117

    Article  CAS  PubMed  Google Scholar 

  4. Giri RK, Young R, Pitstick R, Dearmond SJ, Prusiner SB, Carlson GA (2006) Prion infection of mouse neurospheres. Proc Natl Acad Sci U S A 103:3875–3880

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Butler DA, Scott MR, Bockman JM et al (1988) Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J Virol 62:1558–1564

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Race RE, Caughey B, Graham K, Ernst D, Chesebro B (1988) Analyses of frequency of infection, specific infectivity, and prion protein biosynthesis in scrapie-infected neuroblastoma cell clones. J Virol 62:2845–2849

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Haraguchi T, Fisher S, Olofsson S et al (1989) Asparagine-linked glycosylation of the scrapie and cellular prion proteins. Arch Biochem Biophys 274:1–13

    Article  CAS  PubMed  Google Scholar 

  8. Hope J, Morton LJ, Farquhar CF, Multhaup G, Beyreuther K, Kimberlin RH (1986) The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO J 5:2591–2597

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Stahl N, Borchelt DR, Hsiao K, Prusiner SB (1987) Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51:229–240

    Article  CAS  PubMed  Google Scholar 

  10. Borchelt DR, Rogers M, Stahl N, Telling G, Prusiner SB (1993) Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor. Glycobiology 3:319–329

    Article  CAS  PubMed  Google Scholar 

  11. Rane NS, Yonkovich JL, Hegde RS (2004) Protection from cytosolic prion protein toxicity by modulation of protein translocation. EMBO J 23:4550–4559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Vincent B, Paitel E, Saftig P et al (2001) The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein. J Biol Chem 276:37743–37746

    Article  CAS  PubMed  Google Scholar 

  13. Caughey BG, Raymond 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

    CAS  PubMed  Google Scholar 

  14. Sunyach C, Jen A, Deng J et al (2003) The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein. EMBO J 22:3591–3601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Shyng SL, Huber MT, Harris DA (1993) A prion protein cycles between the cell surface and an endocytic compartment in cultured neuroblastoma cells. J Biol Chem 268:15922–15928

    CAS  PubMed  Google Scholar 

  16. Magalhaes AC, Silva JA, Lee KS et al (2002) Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein. J Biol Chem. 277:33311–33318

    Article  CAS  PubMed  Google Scholar 

  17. Caughey B, Raymond GJ, Ernst D, Race RE (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

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Marijanovic Z, Caputo A, Campana V, Zurzolo C (2009) Identification of an intracellular site of prion conversion. PLoS Pathog 5:e1000426

    Article  PubMed  PubMed Central  Google Scholar 

  19. Yamasaki T, Baron GS, Suzuki A, Hasebe R, Horiuchi M (2014) Characterization of intracellular dynamics of inoculated PrP-res and newly generated PrP(Sc) during early stage prion infection in Neuro2a cells. Virology 450-451:324–335

    Article  CAS  PubMed  Google Scholar 

  20. Dron M, Moudjou M, Chapuis J et al (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Borchelt DR, Scott M, Taraboulos A, Stahl N, Prusiner SB (1990) Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol 110:743–752

    Article  CAS  PubMed  Google Scholar 

  22. Lehmann S, Harris DA (1996) Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform. Proc Natl Acad Sci U S A 93:5610–5614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Priola SA, Chesebro B (1998) Abnormal properties of prion protein with insertional mutations in different cell types. J Biol Chem 273:11980–11985

    Article  CAS  PubMed  Google Scholar 

  24. Lehmann S, Harris DA (1996) Mutant and infectious prion proteins display common biochemical properties in cultured cells. J Biol Chem 271:1633–1637

    Article  CAS  PubMed  Google Scholar 

  25. Petersen RB, Parchi P, Richardson SL, Urig CB, Gambetti P (1996) Effect of the D178N mutation and the codon 129 polymorphism on the metabolism of the prion protein. J Biol Chem 271:12661–12668

    Article  CAS  PubMed  Google Scholar 

  26. Hsiao KK, Scott M, Foster D, Groth DF, Dearmond SJ, Prusiner SB (1990) Spontaneous neurodegeneration in transgenic mice with mutant prion protein. Science 250:1587–1590

    Article  CAS  PubMed  Google Scholar 

  27. Chiesa R, Piccardo P, Quaglio E et al (2003) Molecular distinction between pathogenic and infectious properties of the prion protein. J Virol 77:7611–7622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Jackson WS, Borkowski AW, Faas H et al (2009) Spontaneous generation of prion infectivity in fatal familial insomnia knockin mice. Neuron 63:438–450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Magalhaes AC, Baron GS, Lee KS et al (2005) Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells. J Neurosci 25:5207–5216

    Article  CAS  PubMed  Google Scholar 

  30. Paquet S, Daude N, Courageot MP, Chapuis J, Laude H, Vilette D (2007) PrPc does not mediate internalization of PrPSc but is required at an early stage for de novo prion infection of Rov cells. J Virol 81:10786–10791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Greil CS, Vorberg IM, Ward AE, Meade-White KD, Harris DA, Priola SA (2008) Acute cellular uptake of abnormal prion protein is cell type and scrapie-strain independent. Virology 379:284–293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Wadia JS, Schaller M, Williamson RA, Dowdy SF (2008) Pathologic prion protein infects cells by lipid-raft dependent macropinocytosis. PLoS One 3:e3314

    Article  PubMed  PubMed Central  Google Scholar 

  33. Vorberg I, Raines A, Priola SA (2004) Acute formation of protease-resistant prion protein does not always lead to persistent scrapie infection in vitro. J Biol Chem 279:29218–29225

    Article  CAS  PubMed  Google Scholar 

  34. Goold R, Rabbanian S, Sutton L et al (2011) Rapid cell-surface prion protein conversion revealed using a novel cell system. Nat Commun 2:281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Goold R, McKinnon C, Rabbanian S, Collinge J, Schiavo G, Tabrizi SJ (2013) Alternative fates of newly formed PrPSc upon prion conversion on the plasma membrane. J Cell Sci 126:3552–3562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. McNally KL, Ward AE, Priola SA (2009) Cells expressing anchorless prion protein are resistant to scrapie infection. J Virol 83:4469–4475

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Marbiah MM, Harvey A, West BT et al (2014) Identification of a gene regulatory network associated with prion replication. EMBO J 33:1527–1547

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Scott MR, Kohler R, Foster D, Prusiner SB (1992) Chimeric prion protein expression in cultured cells and transgenic mice. Protein Sci 1:986–997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Priola SA, Chesebro B (1995) A single hamster PrP amino acid blocks conversion to protease-resistant PrP in scrapie-infected mouse neuroblastoma cells. J Virol 69:7754–7758

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Scott M, Foster D, Mirenda C et al (1989) Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59:847–857

    Article  CAS  PubMed  Google Scholar 

  41. Scott M, Groth D, Foster D et al (1993) Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell 73:979–988

    Article  CAS  PubMed  Google Scholar 

  42. Vilette D, Andreoletti O, Archer F et al (2001) Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein. Proc Natl Acad Sci U S A 98:4055–4059

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Courageot MP, Daude N, Nonno R et al (2008) A cell line infectible by prion strains from different species. J Gen Virol 89:341–347

    Article  CAS  PubMed  Google Scholar 

  44. Bian J, Napier D, Khaychuck V, Angers R, Graham C, Telling G (2010) Cell-based quantification of chronic wasting disease prions. J Virol 84:8322–8326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Priola SA, Caughey B, Race RE, Chesebro B (1994) Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapie-infected murine neuroblastoma cells. J Virol 68:4873–4878

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Zulianello L, Kaneko K, Scott M et al (2000) Dominant-negative inhibition of prion formation diminished by deletion mutagenesis of the prion protein. J Virol 74:4351–4360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Vorberg I, Chan K, Priola SA (2001) Deletion of beta-strand and alpha-helix secondary structure in normal prion protein inhibits formation of its protease-resistant isoform. J Virol 75:10024–10032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Taguchi Y, Mistica AM, Kitamoto T, Schatzl HM (2013) Critical significance of the region between helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. PLoS Pathog 9:e1003466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Williamson RA, Peretz D, Pinilla C et al (1998) Mapping the prion protein using recombinant antibodies. J Virol 72:9413–9418

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Abalos GC, Cruite JT, Bellon A et al (2008) Identifying key components of the PrPC-PrPSc replicative interface. J Biol Chem 283:34021–34028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Korth C, Kaneko K, Prusiner SB (2000) Expression of unglycosylated mutated prion protein facilitates PrP(Sc) formation in neuroblastoma cells infected with different prion strains. J Gen Virol 81:2555–2563

    Article  CAS  PubMed  Google Scholar 

  52. Neuendorf E, Weber A, Saalmueller A et al (2004) Glycosylation deficiency at either one of the two glycan attachment sites of cellular prion protein preserves susceptibility to bovine spongiform encephalopathy and scrapie infections. J Biol Chem 279:53306–53316

    Article  CAS  PubMed  Google Scholar 

  53. Salamat MK, Dron M, Chapuis J, Langevin C, Laude H (2011) Prion propagation in cells expressing PrP glycosylation mutants. J Virol 85:3077–3085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Birkett CR, Hennion RM, Bembridge DA et al (2001) Scrapie strains maintain biological phenotypes on propagation in a cell line in culture. EMBO J 20:3351–3358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Mahal SP, Baker CA, Demczyk CA, Smith EW, Julius C, Weissmann C (2007) Prion strain discrimination in cell culture: the cell panel assay. Proc Natl Acad Sci U S A 104:20908–20913

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Browning S, Baker CA, Smith E et al (2011) Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication. J Biol Chem 286:40962–40973

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Li J, Browning S, Mahal SP, Oelschlegel AM, Weissmann C (2010) Darwinian evolution of prions in cell culture. Science 327:869–872

    Article  CAS  PubMed  Google Scholar 

  58. Trevitt CR, Collinge J (2006) A systematic review of prion therapeutics in experimental models. Brain 129:2241–2265

    Article  PubMed  Google Scholar 

  59. Caughey WS, Raymond LD, Horiuchi M, Caughey B (1998) Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines. Proc Natl Acad Sci U S A 95:12117–12122

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Priola SA, Raines A, Caughey WS (2000) Porphyrin and phthalocyanine antiscrapie compounds. Science 287:1503–1506

    Article  CAS  PubMed  Google Scholar 

  61. Forloni G, Artuso V, Roiter I, Morbin M, Tagliavini F (2013) Therapy in prion diseases. Curr Top Med Chem 13:2465–2476

    Article  CAS  PubMed  Google Scholar 

  62. Klohn PC, Stoltze L, Flechsig E, Enari M, Weissmann C (2003) A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions. Proc Natl Acad Sci U S A 100:11666–11671

    Article  PubMed  PubMed Central  Google Scholar 

  63. Arellano-Anaya ZE, Savistchenko J, Mathey J et al (2011) A simple, versatile and sensitive cell-based assay for prions from various species. PLoS One 6:e20563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Tattum MH, Jones S, Pal S, Collinge J, Jackson GS (2010) Discrimination between prion-infected and normal blood samples by protein misfolding cyclic amplification. Transfusion 50:996–1002

    Article  CAS  PubMed  Google Scholar 

  65. Orru CD, Bongianni M, Tonoli G et al (2014) A test for Creutzfeldt-Jakob disease using nasal brushings. N Engl J Med 371:519–529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Halliez S, Jaumain E, Huor A et al (2014) White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays. PLoS One 9:e104287

    Article  PubMed  PubMed Central  Google Scholar 

  67. Sowemimo-Coker SO, Demczyk CA, Andrade F, Baker CA (2010) Evaluation of removal of prion infectivity from red blood cells with prion reduction filters using a new rapid and highly sensitive cell culture-based infectivity assay. Transfusion 50:980–988

    Article  PubMed  Google Scholar 

  68. You B, Aubin JT, Le-Hir G, Arzel A, Laude H, Flan B (2010) In vitro infectivity assay for prion titration for application to the evaluation of the prion removal capacity of biological products manufacturing processes. J Virol Methods 164:1–6

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Disease. The author would like to thank Anita Mora for the graphical assistance.

Author information

Authors and Affiliations

  1. Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th Street, Hamilton, MT, 59840, USA

    Suzette A. Priola

Authors
  1. Suzette A. Priola
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Suzette A. Priola .

Editor information

Editors and Affiliations

  1. Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia

    Victoria A. Lawson

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Priola, S.A. (2017). Cell Biology Approaches to Studying Prion Diseases. In: Lawson, V. (eds) Prions. Methods in Molecular Biology, vol 1658. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7244-9_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7244-9_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7242-5

  • Online ISBN: 978-1-4939-7244-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation