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Molecular Neurodegeneration
Published in: Molecular Neurodegeneration 1/2012

Open Access 01-12-2012 | Research article

Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPreslevel disparity

Authors: Victoria Lewis, Cathryn L Haigh, Colin L Masters, Andrew F Hill, Victoria A Lawson, Steven J Collins

Published in: Molecular Neurodegeneration | Issue 1/2012

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Abstract

Background

Prion disease transmission and pathogenesis are linked to misfolded, typically protease resistant (PrPres) conformers of the normal cellular prion protein (PrPC), with the former posited to be the principal constituent of the infectious 'prion'. Unexplained discrepancies observed between detectable PrPres and infectivity levels exemplify the complexity in deciphering the exact biophysical nature of prions and those host cell factors, if any, which contribute to transmission efficiency. In order to improve our understanding of these important issues, this study utilized a bioassay validated cell culture model of prion infection to investigate discordance between PrPres levels and infectivity titres at a subcellular resolution.

Findings

Subcellular fractions enriched in lipid rafts or endoplasmic reticulum/mitochondrial marker proteins were equally highly efficient at prion transmission, despite lipid raft fractions containing up to eight times the levels of detectable PrPres. Brain homogenate infectivity was not differentially enhanced by subcellular fraction-specific co-factors, and proteinase K pre-treatment of selected fractions modestly, but equally reduced infectivity. Only lipid raft associated infectivity was enhanced by sonication.

Conclusions

This study authenticates a subcellular disparity in PrPres and infectivity levels, and eliminates simultaneous divergence of prion strains as the explanation for this phenomenon. On balance, the results align best with the concept that transmission efficiency is influenced more by intrinsic characteristics of the infectious prion, rather than cellular microenvironment conditions or absolute PrPres levels.
Appendix
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Literature
1.
2.
Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, Groth D, Mehlhorn I, Huang Z, Fletterick RJ, Cohen FE, et al: Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA. 1993, 90: 10962-10966.PubMedCentralCrossRefPubMed
3.
Caughey BW, Dong A, Bhat KS, Ernst D, Hayes SF, Caughey WS: Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy. Biochemistry. 1991, 30: 7672-7680.CrossRefPubMed
4.
Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, Glenner GG: Scrapie prions aggregate to form amyloid-like birefringent rods. Cell. 1983, 35: 349-358.CrossRefPubMed
5.
Prusiner SB, Bolton DC, Groth DF, Bowman KA, Cochran SP, McKinley MP: Further purification and characterization of scrapie prions. Biochemistry. 1982, 21: 6942-6950.CrossRefPubMed
6.
McKinley MP, Bolton DC, Prusiner SB: A protease-resistant protein is a structural component of the scrapie prion. Cell. 1983, 35: 57-62.CrossRefPubMed
7.
Collins SJ, Lewis V, Brazier MW, Hill AF, Lawson VA, Klug GM, Masters CL: Extended period of asymptomatic prion disease after low dose inoculation: assessment of detection methods and implications for infection control. Neurobiol Dis. 2005, 20: 336-346.CrossRefPubMed
8.
Balkema-Buschmann A, Eiden M, Hoffmann C, Kaatz M, Ziegler U, Keller M, Groschup MH: BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle. J Gen Virol. 2010, doi:10.1099/vir.0.025387-0
9.
Tixador P, Herzog L, Reine F, Jaumain E, Chapuis J, Le Dur A, Laude H, Beringue V: The physical relationship between infectivity and prion protein aggregates is strain-dependent. PLoS Pathog. 2010, 6: e1000859-PubMedCentralCrossRefPubMed
10.
Hill AF, Joiner S, Linehan J, Desbruslais M, Lantos PL, Collinge J: Species-barrier-independent prion replication in apparently resistant species. Proc Natl Acad Sci USA. 2000, 97: 10248-10253.PubMedCentralCrossRefPubMed
11.
Manuelidis L, Fritch W, Xi YG: Evolution of a strain of CJD that induces BSE-like plaques. Science. 1997, 277: 94-98.CrossRefPubMed
12.
Lasmezas CI, Deslys JP, Robain O, Jaegly A, Beringue V, Peyrin JM, Fournier JG, Hauw JJ, Rossier J, Dormont D: Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science. 1997, 275: 402-405.CrossRefPubMed
13.
Czub M, Braig HR, Diringer H: Pathogenesis of scrapie: study of the temporal development of clinical symptoms, of infectivity titres and scrapie-associated fibrils in brains of hamsters infected intraperitoneally. J Gen Virol. 1986, 67 (Pt 9): 2005-2009.CrossRefPubMed
14.
Barron RM, Campbell SL, King D, Bellon A, Chapman KE, Williamson RA, Manson JC: High titers of transmissible spongiform encephalopathy infectivity associated with extremely low levels of PrPSc in vivo. J Biol Chem. 2007, 282: 35878-35886.CrossRefPubMed
15.
Castilla J, Saa P, Hetz C, Soto C: In vitro generation of infectious scrapie prions. Cell. 2005, 121: 195-206.CrossRefPubMed
16.
Lewis V, Hooper NM: The role of lipid rafts in prion protein biology. Front Biosci. 2011, 16: 151-168.CrossRef
17.
Kaneko K, Zulianello L, Scott M, Cooper CM, Wallace AC, James TL, Cohen FE, Prusiner SB: Evidence for protein × binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proc Natl Acad Sci USA. 1997, 94: 10069-10074.PubMedCentralCrossRefPubMed
18.
Telling GC, Scott M, Mastrianni J, Gabizon R, Torchia M, Cohen FE, DeArmond SJ, Prusiner SB: Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell. 1995, 83: 79-90.CrossRefPubMed
19.
Yehiely F, Bamborough P, Da Costa M, Perry BJ, Thinakaran G, Cohen FE, Carlson GA, Prusiner SB: Identification of candidate proteins binding to prion protein. Neurobiol Dis. 1997, 3: 339-355.CrossRefPubMed
20.
Gomes MP, Millen TA, Ferreira PS, Cunha ESNL, Vieira TC, Almeida MS, Silva JL, Cordeiro Y: Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells. J Biol Chem. 2008, 283: 19616-19625.PubMedCentralCrossRefPubMed
21.
Deleault NR, Lucassen RW, Supattapone S: RNA molecules stimulate prion protein conversion. Nature. 2003, 425: 717-720.CrossRefPubMed
22.
Adler V, Zeiler B, Kryukov V, Kascsak R, Rubenstein R, Grossman A: Small, highly structured RNAs participate in the conversion of human recombinant PrP(Sen) to PrP(Res) in vitro. J Mol Biol. 2003, 332: 47-57.CrossRefPubMed
23.
Geoghegan JC, Valdes PA, Orem NR, Deleault NR, Williamson RA, Harris BT, Supattapone S: Selective incorporation of polyanionic molecules into hamster prions. J Biol Chem. 2007, 282: 36341-36353.PubMedCentralCrossRefPubMed
24.
Liu M, Yu S, Yang J, Yin X, Zhao D: RNA and CuCl2 induced conformational changes of the recombinant ovine prion protein. Mol Cell Biochem. 2007, 294: 197-203.CrossRefPubMed
25.
Schonberger O, Horonchik L, Gabizon R, Papy-Garcia D, Barritault D, Taraboulos A: Novel heparan mimetics potently inhibit the scrapie prion protein and its endocytosis. Biochem Biophys Res Commun. 2003, 312: 473-479.CrossRefPubMed
26.
Ben-Zaken O, Tzaban S, Tal Y, Horonchik L, Esko JD, Vlodavsky I, Taraboulos A: Cellular heparan sulfate participates in the metabolism of prions. J Biol Chem. 2003, 278: 40041-40049.CrossRefPubMed
27.
Mayer-Sonnenfeld T, Zeigler M, Halimi M, Dayan Y, Herzog C, Lasmezas CI, Gabizon R: The metabolism of glycosaminoglycans is impaired in prion diseases. Neurobiol Dis. 2005, 20: 738-743.CrossRefPubMed
28.
Taylor DR, Whitehouse IJ, Hooper NM: Glypican-1 mediates both prion protein lipid raft association and disease isoform formation. PLoS Pathog. 2009, 5: e1000666-PubMedCentralCrossRefPubMed
29.
Lawson VA, Lumicisi B, Welton J, Machalek D, Gouramanis K, Klemm HM, Stewart JD, Masters CL, Hoke DE, Collins SJ, Hill AF: Glycosaminoglycan sulphation affects the seeded misfolding of a mutant prion protein. PLoS One. 2010, 5: e12351-PubMedCentralCrossRefPubMed
30.
31.
Weber P, Reznicek L, Mitteregger G, Kretzschmar H, Giese A: Differential effects of prion particle size on infectivity in vivo and in vitro. Biochem Biophys Res Commun. 2008, 369: 924-928.CrossRefPubMed
32.
Borchelt DR, Scott M, Taraboulos A, Stahl N, Prusiner SB: Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol. 1990, 110: 743-752.CrossRefPubMed
33.
Shyng SL, Huber MT, Harris DA: A prion protein cycles between the cell surface and an endocytic compartment in cultured neuroblastoma cells. J Biol Chem. 1993, 268: 15922-15928.PubMed
34.
Gilch S, Winklhofer KF, Groschup MH, Nunziante M, Lucassen R, Spielhaupter C, Muranyi W, Riesner D, Tatzelt J, Schätzl HM: Intracellular re-routing of prion protein prevents propagation of PrPSc and delays onset of prion disease. EMBO J. 2001, 20: 3957-3966.PubMedCentralCrossRefPubMed
35.
Macdonald JL, Pike LJ: A simplified method for the preparation of detergent-free lipid rafts. J Lipid Res. 2005, 46: 1061-1067.CrossRefPubMed
36.
Naslavsky N, Shmeeda H, Friedlander G, Yanai A, Futerman AH, Barenholz Y, Taraboulos A: Sphingolipid depletion increases formation of the scrapie prion protein in neuroblastoma cells infected with prions. J Biol Chem. 1999, 274: 20763-20771.CrossRefPubMed
37.
Liberski PP, Ironside JW: An outline of the neuropathology of transmissible spongiform encephalopathies (prion diseases). Folia Neuropathol. 2004, 42:
38.
Bruce ME, Boyle A, Cousens S, McConnell I, Foster J, Goldmann W, Fraser H: Strain characterization of natural sheep scrapie and comparison with BSE. J Gen Virol. 2002, 83: 695-704.CrossRefPubMed
39.
Bruce ME, McConnell I, Fraser H, Dickinson AG: The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. J Gen Virol. 1991, 72 (Pt 3): 595-603.CrossRefPubMed
40.
Brazier MW, Lewis V, Ciccotosto GD, Klug GM, Lawson VA, Cappai R, Ironside JW, Masters CL, Hill AF, White AR, Collins S: Correlative studies support lipid peroxidation is linked to PrP(res) propagation as an early primary pathogenic event in prion disease. Brain Res Bull. 2006, 68: 346-354.CrossRefPubMed
41.
Lawson VA, Vella LJ, Stewart JD, Sharples RA, Klemm H, Machalek DM, Masters CL, Cappai R, Collins SJ, Hill AF: Mouse-adapted sporadic human Creutzfeldt-Jakob disease prions propagate in cell culture. Int J Biochem Cell Biol. 2008, 40: 2793-2801.CrossRefPubMed
42.
Bessen RA, Marsh RF: Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol. 1994, 68: 7859-7868.PubMedCentralPubMed
43.
Somerville RA, Chong A, Mulqueen OU, Birkett CR, Wood SC, Hope J: Biochemical typing of scrapie strains. Nature. 1997, 386: 564-CrossRefPubMed
44.
Caughey B, Raymond GJ, Bessen RA: Strain-dependent differences in beta-sheet conformations of abnormal prion protein. J Biol Chem. 1998, 273: 32230-32235.CrossRefPubMed
45.
Parchi P, Castellani R, Capellari S, Ghetti B, Young K, Chen SG, Farlow M, Dickson DW, Sima AA, Trojanowski JQ, et al: Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease. Ann Neurol. 1996, 39: 767-778.CrossRefPubMed
46.
Collinge J, Sidle KC, Meads J, Ironside J, Hill AF: Molecular analysis of prion strain variation and the aetiology of 'new variant' CJD. Nature. 1996, 383: 685-690.CrossRefPubMed
47.
Tzaban S, Friedlander G, Schonberger O, Horonchik L, Yedidia Y, Shaked G, Gabizon R, Taraboulos A: Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes. Biochemistry. 2002, 41: 12868-12875.CrossRefPubMed
48.
Thackray AM, Hopkins L, Bujdoso R: Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay. Biochem J. 2007, 401: 475-483.PubMedCentralCrossRefPubMed
49.
Pastrana MA, Sajnani G, Onisko B, Castilla J, Morales R, Soto C, Requena JR: Isolation and characterization of a proteinase K-sensitive PrPSc fraction. Biochemistry. 2006, 45: 15710-15717.CrossRefPubMed
50.
Gambetti P, Dong Z, Yuan J, Xiao X, Zheng M, Alshekhlee A, Castellani R, Cohen M, Barria MA, Gonzalez-Romero D, et al: A novel human disease with abnormal prion protein sensitive to protease. Ann Neurol. 2008, 63: 697-708.PubMedCentralCrossRefPubMed
51.
Colby DW, Wain R, Baskakov IV, Legname G, Palmer CG, Nguyen HO, Lemus A, Cohen FE, DeArmond SJ, Prusiner SB: Protease-sensitive synthetic prions. PLoS Pathog. 2010, 6: e1000736-PubMedCentralCrossRefPubMed
52.
Stathopulos PB, Scholz GA, Hwang YM, Rumfeldt JA, Lepock JR, Meiering EM: Sonication of proteins causes formation of aggregates that resemble amyloid. Protein Sci. 2004, 13: 3017-3027.PubMedCentralCrossRefPubMed
53.
Piening N, Weber P, Giese A, Kretzschmar H: Breakage of PrP aggregates is essential for efficient autocatalytic propagation of misfolded prion protein. Biochem Biophys Res Commun. 2005, 326: 339-343.CrossRefPubMed
54.
Deleault NR, Harris BT, Rees JR, Supattapone S: Formation of native prions from minimal components in vitro. Proc Natl Acad Sci USA. 2007, 104: 9741-9746.PubMedCentralCrossRefPubMed
55.
Lewis V, Hill AF, Klug GM, Boyd A, Masters CL, Collins SJ: Australian sporadic CJD analysis supports endogenous determinants of molecular-clinical profiles. Neurology. 2005, 65: 113-118.CrossRefPubMed
56.
Hill AF, Joiner S, Wadsworth JD, Sidle KC, Bell JE, Budka H, Ironside JW, Collinge J: Molecular classification of sporadic Creutzfeldt-Jakob disease. Brain. 2003, 126: 1333-1346.CrossRefPubMed
57.
58.
Benestad SL, Sarradin P, Thu B, Schonheit J, Tranulis MA, Bratberg B: Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec. 2003, 153: 202-208.CrossRefPubMed
59.
Arsac JN, Biacabe AG, Nicollo J, Bencsik A, Baron T: Biochemical identification of bovine spongiform encephalopathies in cattle. Acta neuropathologica. 2007, 114: 509-516.CrossRefPubMed
60.
Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, Monaco S, Caramelli M: Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc Natl Acad Sci USA. 2004, 101: 3065-3070.PubMedCentralCrossRefPubMed
61.
DeMarco ML, Daggett V: Local environmental effects on the structure of the prion protein. C R Biol. 2005, 328: 847-862.CrossRefPubMed
62.
Kimberlin RH, Walker CA: Evidence that the transmission of one source of scrapie agent to hamsters involves separation of agent strains from a mixture. J Gen Virol. 1978, 39: 487-496.CrossRefPubMed
63.
Puoti G, Giaccone G, Rossi G, Canciani B, Bugiani O, Tagliavini F: Sporadic Creutzfeldt-Jakob disease: co-occurrence of different types of PrP(Sc) in the same brain. Neurology. 1999, 53: 2173-2176.CrossRefPubMed
64.
Hirogari Y, Kubo M, Kimura KM, Haritani M, Yokoyama T: Two different scrapie prions isolated in Japanese sheep flocks. Microbiol Immunol. 2003, 47: 871-876.CrossRefPubMed
65.
Hecker R, Taraboulos A, Scott M, Pan KM, Yang SL, Torchia M, Jendroska K, DeArmond SJ, Prusiner SB: Replication of distinct scrapie prion isolates is region specific in brains of transgenic mice and hamsters. Genes Dev. 1992, 6: 1213-1228.CrossRefPubMed
66.
Bartz JC, Aiken JM, Bessen RA: Delay in onset of prion disease for the HY strain of transmissible mink encephalopathy as a result of prior peripheral inoculation with the replication-deficient DY strain. J Gen Virol. 2004, 85: 265-273.CrossRefPubMed
67.
Courageot MP, Daude N, Nonno R, Paquet S, Di Bari MA, Le Dur A, Chapuis J, Hill AF, Agrimi U, Laude H, Vilette D: A cell line infectible by prion strains from different species. J Gen Virol. 2008, 89: 341-347.CrossRefPubMed
68.
Gonzalez-Montalban N, Makarava N, Ostapchenko VG, Savtchenk R, Alexeeva I, Rohwer RG, Baskakov IV: Highly efficient protein misfolding cyclic amplification. PLoS Pathog. 2011, 7: e1001277-PubMedCentralCrossRefPubMed
69.
Piro JR, Wang F, Walsh DJ, Rees JR, Ma J, Supattapone S: Seeding specificity and ultrastructural characteristics of infectious recombinant prions. Biochemistry. 2011, 50: 7111-7116.PubMedCentralCrossRefPubMed
70.
Gonzalez-Montalban N, Makarava N, Savtchenko R, Baskakov IV: Relationship between Conformational Stability and Amplification Efficiency of Prions. Biochemistry. 2011, 50: 7933-7940.PubMedCentralCrossRefPubMed
71.
Safar JG, Scott M, Monaghan J, Deering C, Didorenko S, Vergara J, Ball H, Legname G, Leclerc E, Solforosi L, et al: Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice. Nat Biotechnol. 2002, 20: 1147-1150.CrossRefPubMed
72.
Aiken JM, Williamson JL, Marsh RF: Evidence of mitochondrial involvement in scrapie infection. J Virol. 1989, 63: 1686-1694.PubMedCentralPubMed
73.
Akao Y, Otsuki Y, Kataoka S, Ito Y, Tsujimoto Y: Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res. 1994, 54: 2468-2471.PubMed
74.
75.
76.
Millson GC, Hunter GD, Kimberlin RH: An experimental examination of the scrapie agent in cell membrane mixtures. II. The association of scrapie activity with membrane fractions. Comp Pathol. 1971, 81: 255-265.CrossRef
77.
Alais S, Simoes S, Baas D, Lehmann S, Raposo G, Darlix JL, Leblanc P: Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles. Biol Cell. 2008, 100: 603-615.CrossRefPubMed
78.
Vella LJ, Sharples RA, Lawson VA, Masters CL, Cappai R, Hill AF: Packaging of prions into exosomes is associated with a novel pathway of PrP processing. J Pathol. 2007, 211: 582-590.CrossRefPubMed
79.
Lewis V, Hill AF, Haigh CL, Klug GM, Masters CL, Lawson VA, Collins SJ: Increased Proportions of C1 Truncated Prion Protein Protect Against Cellular M1000 Prion Infection. J Neuropathol Exp Neurol. 2009, 68: 1125-1135.CrossRefPubMed
80.
Naslavsky N, Stein R, Yanai A, Friedlander G, Taraboulos A: Characterization of detergent-insoluble complexes containing the cellular prion protein and its scrapie isoform. J Biol Chem. 1997, 272: 6324-6331.CrossRefPubMed
81.
Lawson VA, Stewart JD, Masters CL: Enzymatic detergent treatment protocol that reduces protease-resistant prion protein load and infectivity from surgical-steel monofilaments contaminated with a human-derived prion strain. J Gen Virol. 2007, 88: 2905-2914.CrossRefPubMed
82.
Fischer M, Rulicke T, Raeber A, Sailer A, Moser M, Oesch B, Brandner S, Aguzzi A, Weissmann C: Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. EMBO J. 1996, 15: 1255-1264.PubMedCentralPubMed
Metadata
Title
Prion subcellular fractionation reveals infectivity spectrum, with a high titre-low PrPreslevel disparity
Authors
Victoria Lewis
Cathryn L Haigh
Colin L Masters
Andrew F Hill
Victoria A Lawson
Steven J Collins
Publication date
01-12-2012
Publisher
BioMed Central
Published in
Molecular Neurodegeneration / Issue 1/2012
Electronic ISSN: 1750-1326
DOI
https://doi.org/10.1186/1750-1326-7-18

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