Skip to main content

Advertisement

Log in

Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Barret A, Forestier L, Deslys JP, Julien R, Gallet PF (2005) Glycosylation-related gene expression in prion diseases: PrPSc accumulation in scrapie infected GT1 cells depends on beta-1, 4-linked GalNAc-4-SO4 hyposulfation. J Biol Chem 280:10516–10523

    Article  PubMed  CAS  Google Scholar 

  2. Borchelt DR, Taraboulos A, Prusiner SB (1992) Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J Biol Chem 267:16188–16199

    PubMed  CAS  Google Scholar 

  3. Bosque PJ, Prusiner SB (2000) Cultured cell sublines highly susceptible to prion infection. J Virol 74:4377–4386

    Article  PubMed  CAS  Google Scholar 

  4. Brown DR, Schmidt B, Kretzschmar HA (1997) Effects of oxidative stress on prion protein expression in PC12 cells. Int J Dev Neurosci 15:961–972

    Article  PubMed  CAS  Google Scholar 

  5. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, Aguet M, Weissmann C (1993) Mice devoid of PrP are resistant to scrapie. Cell 73:1339–1347

    Article  PubMed  CAS  Google Scholar 

  6. Chich JF, Schaeffer B, Bouin AP, Mouthon F, Labas V, Larramendy C, Deslys JP, Grosclaude J (2007) Prion infection-impaired functional blocks identified by proteomics enlighten the targets and the curing pathways of an anti-prion drug. Biochim Biophys Acta 1774:154–167

    PubMed  CAS  Google Scholar 

  7. Chiesa R, Piccardo P, Dossena S, Nowoslawski L, Roth KA, Ghetti B, Harris DA (2005) Bax deletion prevents neuronal loss but not neurological symptoms in a transgenic model of inherited prion disease. Proc Natl Acad Sci USA 102:238–243

    Article  PubMed  CAS  Google Scholar 

  8. Coulpier M, Messiaen S, Hamel R, Fernandez de Marco M, Lilin T, Eloit M (2006) Bax deletion does not protect neurons from BSE-induced death. Neurobiol Dis 23:603–611

    Google Scholar 

  9. Doh-ura K, Perryman S, Race R, Chesebro B (1995) Identification of differentially expressed genes in scrapie-infected mouse neuroblastoma cells. Microb Pathog 18:1–9

    PubMed  CAS  Google Scholar 

  10. Ehehalt R, Keller P, Haass C, Thiele C, Simons K (2003) Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J Cell Biol 160:113–123

    Article  PubMed  CAS  Google Scholar 

  11. Fischer M, Rulicke T, Raeber A, Sailer A, Moser M, Oesch B, Brandner S, Aguzzi A, Weissmann C (1996) Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. EMBO J 15:1255–1264

    PubMed  CAS  Google Scholar 

  12. Ghaemmaghami S, Phuan PW, Perkins B, Ullman J, May BC, Cohen FE, Prusiner SB (2007) Cell division modulates prion accumulation in cultured cells. Proc Natl Acad Sci USA 104:17971–17976

    Article  PubMed  CAS  Google Scholar 

  13. Greenwood AD, Horsch M, Stengel A, Vorberg I, Lutzny G, Maas E, Schadler S, Erfle V, Beckers J, Schatzl H, Leib-Mosch C (2005) Cell line dependent RNA expression profiles of prion-infected mouse neuronal cells. J Mol Biol 349:487–500

    Article  PubMed  CAS  Google Scholar 

  14. Hsich G, Kenney K, Gibbs CJ, Lee KH, Harrington MG (1996) The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies. N Engl J Med 335:924–930

    Article  PubMed  CAS  Google Scholar 

  15. Kawashima T, Doh-ura K, Torisu M, Uchida Y, Furuta A, Iwaki T (2000) Differential expression of metallothioneins in human prion diseases. Dement Geriatr Cogn Disord 11:251–262

    Article  PubMed  CAS  Google Scholar 

  16. Klebe RJ, Ruddle FH (1969) Neuroblastoma: cell culture analysis of a differentiating stem cell system. J Cell Biol 43:69

    Google Scholar 

  17. Kocisko DA, Caughey B (2006) Searching for anti-prion compounds: cell-based high-throughput in vitro assays and animal testing strategies. Methods Enzymol 412:223–234

    Article  PubMed  CAS  Google Scholar 

  18. Leucht C, Simoneau S, Rey C, Vana K, Rieger R, Lasmezas CI, Weiss S (2003) The 37 kDa/67 kDa laminin receptor is required for PrP(Sc) propagation in scrapie-infected neuronal cells. EMBO Rep 4:290–295

    Google Scholar 

  19. Lloyd SE, Uphill JB, Targonski PV, Fisher EM, Collinge J (2002) Identification of genetic loci affecting mouse-adapted bovine spongiform encephalopathy incubation time in mice. Neurogenetics 4:77–81

    Article  PubMed  CAS  Google Scholar 

  20. 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 USA 104:20908–20913

    Article  PubMed  CAS  Google Scholar 

  21. Mange A, Crozet C, Lehmann S, Beranger F (2004) Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin. J Cell Sci 117:2411–2416

    Article  PubMed  CAS  Google Scholar 

  22. Marella M, Gaggioli C, Batoz M, Deckert M, Tartare-Deckert S, Chabry J (2005) Pathological prion protein exposure switches on neuronal mitogen-activated protein kinase pathway resulting in microglia recruitment. J Biol Chem 280:1529–1534

    Article  PubMed  CAS  Google Scholar 

  23. Milhavet O, McMahon HE, Rachidi W, Nishida N, Katamine S, Mange A, Arlotto M, Casanova D, Riondel J, Favier A, Lehmann S (2000) Prion infection impairs the cellular response to oxidative stress. Proc Natl Acad Sci USA 97:13937–13942

    Article  PubMed  CAS  Google Scholar 

  24. Moreno CR, Lantier F, Lantier I, Sarradin P, Elsen JM (2003) Detection of new quantitative trait Loci for susceptibility to transmissible spongiform encephalopathies in mice. Genetics 165:2085–2091

    PubMed  CAS  Google Scholar 

  25. Mouillet-Richard S, Ermonval M, Chebassier C, Laplanche JL, Lehmann S, Launay JM, Kellermann O (2000) Signal transduction through prion protein. Science 289:1925–1928

    Article  PubMed  CAS  Google Scholar 

  26. Neuvial P, Hupe P, Brito I, Liva S, Manie E, Brennetot C, Radvanyi F, Aurias A, Barillot E (2006) Spatial normalization of array-CGH data. BMC Bioinformatics 7:264

    Article  PubMed  CAS  Google Scholar 

  27. Nishida N, Harris DA, Vilette D, Laude H, Frobert Y, Grassi J, Casanova D, Milhavet O, Lehmann S (2000) Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein. J Virol 74:320–325

    Article  PubMed  CAS  Google Scholar 

  28. Paitel E, Alves da Costa C, Vilette D, Grassi J, Checler F (2002) Overexpression of PrPc triggers caspase 3 activation: potentiation by proteasome inhibitors and blockade by anti-PrP antibodies. J Neurochem 83:1208–1214

    Google Scholar 

  29. Paitel E, Fahraeus R, Checler F (2003) Cellular prion protein sensitizes neurons to apoptotic stimuli through Mdm2-regulated and p53-dependent caspase 3-like activation. J Biol Chem 278:10061–10066

    Article  PubMed  CAS  Google Scholar 

  30. Park SK, Choi SI, Jin JK, Choi EK, Kim JI, Carp RI, Kim YS (2000) Differential expression of Bax and Bcl-2 in the brains of hamsters infected with 263K scrapie agent. Neuroreport 11:1677–1682

    Article  PubMed  CAS  Google Scholar 

  31. Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216:136–144

    Article  PubMed  CAS  Google Scholar 

  32. Prusiner SB, Scott MR, Dearmond SJ, Cohen FE (1998) Prion protein biology. Cell 93:337–348

    Article  PubMed  CAS  Google Scholar 

  33. Rangon CM, Haik S, Faucheux BA, Metz-Boutigue MH, Fierville F, Fuchs JP, Hauw JJ, Aunis D (2003) Different chromogranin immunoreactivity between prion and a-beta amyloid plaque. Neuroreport 14:755–758

    Article  PubMed  CAS  Google Scholar 

  34. Rieger R, Edenhofer F, Lasmezas CI, Weiss S (1997) The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells. Nat Med 3:1383–1388

    Article  PubMed  CAS  Google Scholar 

  35. Roucou X, Giannopoulos PN, Zhang Y, Jodoin J, Goodyer CG, LeBlanc A (2005) Cellular prion protein inhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells. Cell Death Differ 12:783–795

    Article  PubMed  CAS  Google Scholar 

  36. Sandberg MK, Low P (2005) Altered interaction and expression of proteins involved in neurosecretion in scrapie-infected GT1-1 cells. J Biol Chem 280:1264–1271

    Article  PubMed  CAS  Google Scholar 

  37. Santuccione A, Sytnyk V, Leshchyns’ka I, Schachner M (2005) Prion protein recruits its neuronal receptor NCAM to lipid rafts to activate p59fyn and to enhance neurite outgrowth. J Cell Biol 169:341–354

    Article  PubMed  CAS  Google Scholar 

  38. Satoh J, Onoue H, Arima K, Yamamura T (2005) The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein. J Neuropathol Exp Neurol 64:858–868

    Article  PubMed  CAS  Google Scholar 

  39. Schmitt-Ulms G, Legname G, Baldwin MA, Ball HL, Bradon N, Bosque PJ, Crossin KL, Edelman GM, DeArmond SJ, Cohen FE, Prusiner SB (2001) Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein. J Mol Biol 314:1209–1225

    Article  PubMed  CAS  Google Scholar 

  40. Shyng SL, Heuser JE, Harris DA (1994) A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J Cell Biol 125:1239–1250

    Article  PubMed  CAS  Google Scholar 

  41. Simoneau S, Haik S, Leucht C, Dormont D, Deslys JP, Weiss S, Lasmezas C (2003) Different isoforms of the non-integrin laminin receptor are present in mouse brain and bind PrP. Biol Chem 384:243–246

    Article  PubMed  CAS  Google Scholar 

  42. Siso S, Puig B, Varea R, Vidal E, Acin C, Prinz M, Montrasio F, Badiola J, Aguzzi A, Pumarola M, Ferrer I (2002) Abnormal synaptic protein expression and cell death in murine scrapie. Acta Neuropathol 103:615–626

    Article  PubMed  CAS  Google Scholar 

  43. 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  CAS  Google Scholar 

  44. Vilette D (2007) Cell models of prion infection. Vet Res 39:10

    PubMed  Google Scholar 

  45. Vincent B, Paitel E, Saftig P, Frobert Y, Hartmann D, De Strooper B, Grassi J, Lopez-Perez E, Checler F (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

    Google Scholar 

  46. Warter S, Hermetet JC, Cieselski-Treska J (1974) Cytogenetic characterization of C1300 neuroblastoma cells. Experientia 30:291–292

    Article  PubMed  CAS  Google Scholar 

  47. Winer J, Jung CK, Shackel I, Williams PM (1999) Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270:41–49

    Article  PubMed  CAS  Google Scholar 

  48. Wong BS, Pan T, Liu T, Li R, Gambetti P, Sy MS (2000) Differential contribution of superoxide dismutase activity by prion protein in vivo. Biochem Biophys Res Commun 273:136–139

    Article  PubMed  CAS  Google Scholar 

  49. Zanata SM, Lopes MH, Mercadante AF, Hajj GN, Chiarini LB, Nomizo R, Freitas AR, Cabral AL, Lee KS, Juliano MA, de Oliveira E, Jachieri SG, Burlingame A, Huang L, Linden R, Brentani RR, Martins VR (2002) Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection. EMBO J 21:3307–3316

    Article  PubMed  CAS  Google Scholar 

  50. Zhang Y, Poirier GG, Burkle A (2002) In-situ analysis of cellular poly(ADP-ribose) production in scrapie-infected mouse neuroblastoma cells. Histochem J 34:357–363

    Article  PubMed  CAS  Google Scholar 

  51. Zhang Y, Spiess E, Groschup MH, Burkle A (2003) Up-regulation of cathepsin B and cathepsin L activities in scrapie-infected mouse Neuro2a cells. J Gen Virol 84:2279–2283

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from “GIS Infections à prions”. Isolation of susceptible and resistant cell clones was supported by a grants from the EU Commission (EU-PRIONS, QLRT-2000-01924; NEUROPRION, FOOD-CT-2004-506579). The construction of the mouse BAC-array was supported by grants from the “Carte d’Identité des Tumeurs (CIT)” program of the “Ligue Nationale Contre le Cancer”. Anti-prion antibodies were generously provided by J. Grassi and C. Creminon (CEA Saclay, SPI/CEA).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sylvain Lehmann.

Additional information

S. Chasseigneaux and M. Pastore contributed equally to the work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chasseigneaux, S., Pastore, M., Britton-Davidian, J. et al. Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines. Arch Virol 153, 1693–1702 (2008). https://doi.org/10.1007/s00705-008-0177-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-008-0177-8

Keywords

Navigation