Neurochemical Research

, 36:1863 | Cite as

Neurotoxic Effect of the Complex of the Ovine Prion Protein (OvPrPC) and RNA on the Cultured Rat Cortical Neurons

Original paper


Prion diseases are conformational diseases, many factors are involved in altering the conformation of prion, such as RNA, DNA, pH, and copper etc. However the neurotoxic mechanism of prion diseases is not clear yet. The aim of this study is to investigate the effect of the nucleoprotein complex of RNA and recombinant ovine prion protein (OvPrPC) on the cultured rat cortical neurons in vitro. Our previous study revealed that the nucleoprotein complex (OvPrPC-RNA) is characterized with high β sheet conformation and proteinase K resistance. Here we found that the OvPrPC-RNA induced marked neuronal cell death by the MTT (3-(4,5-dimethyl-thiazole -2-yl)-2,5-diphenyl –tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and the neurotoxic effects were confirmed by testing the content of Bcl-2 Associated X protein (Bax) in the immunoprecipitation assay and Western blot assay. Compared to the control group, there is no significant difference of active Bax or total Bax after RNA alone treatment or OvPrPC alone treatment, but the OvPrPC-RNA induced significant increases of active Bax level, while the contents of total Bax had no obvious changes after OvPrPC-RNA treatment. The results suggested that OvPrPC-RNA is neurotoxic in vitro, which added further evidence to the current understanding of mechanism of cellular injury by RNA molecules for transformation of the PrPC to PrPSc.


Prion OvPrPC-RNA Neuronal injury neurotoxicity Bax 



This work was supported by the NSFC (No. 30871854), NSFC Research Grant (10925208) and by Research Fund for the Doctoral Program of Higher Education of China (20091102110031) and also supported by the Fundamental Research Funds for the Central Universities (YWF-11-03-Q-056).


  1. 1.
    Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95:13363–13383PubMedCrossRefGoogle Scholar
  2. 2.
    Xiong LW, Raymond LD, Hayes SF, Raymond GJ, Caughey B (2001) Conformational change aggregation and fibril formation induced by detergent treatments of cellular prion protein. J Neurochem 79:669–678PubMedCrossRefGoogle Scholar
  3. 3.
    Adler V, Zeiler B, Kryukov V, Kascsak R, Rubenstein R, Grossman A (2003) Small, highly structured RNAs participate in the conversion of human recombinant PrP(Sen) to PrP(Res) in vitro. J Mol Biol 332:47–57PubMedCrossRefGoogle Scholar
  4. 4.
    Deleault NR, Lucassen RW, Supattapone S (2003) RNA molecules stimulate prion protein conversion. Nature 425:717–720PubMedCrossRefGoogle Scholar
  5. 5.
    Yang S, Thackray AM, Fitzmaurice TJ, Bujdoso R (2008) Copper-induced structural changes in the ovine prion protein are influenced by a polymorphism at codon 112. Biochim Biophys Acta 1784:683–692PubMedGoogle Scholar
  6. 6.
    Liang J, Pan Y, Zhang D, Guo C, Shi Y, Wang J, Chen Y, Wang X, Liu J, Guo X, Chen Z, Qiao T, Fan D (2007) Cellular prion protein promotes proliferation and G1/S transition of human gastric cancer cells SGC7901 and AGS. FASEB J 21:2247–2256PubMedCrossRefGoogle Scholar
  7. 7.
    Pokrovskii VI, Kiselev OI (1998) Molecular bases of prion diseases. Vestn Ross Akad Med Nauk 10:45–55PubMedGoogle Scholar
  8. 8.
    Liu ML, Li YX, Zhou XM, Zhao DM (2008) Copper(II) inhibits in vitro conformational conversion of ovine prion protein triggered by low pH. J Biochem 143:333–337PubMedCrossRefGoogle Scholar
  9. 9.
    Weissmann C, Enari M, Klohn PC, Rossi D, Flechsig E (2002) Transmission of prions. J Infect Dis 186(Suppl 2):S157–S165PubMedCrossRefGoogle Scholar
  10. 10.
    Aguzzi A, Weissmann C (1997) Prion research: the next frontiers. Nature 389:795–798PubMedCrossRefGoogle Scholar
  11. 11.
    Chiesa R, Harris DA (2001) Prion diseases: what is the neurotoxic molecule? Neurobiol Dis 8:743–763PubMedCrossRefGoogle Scholar
  12. 12.
    Saborio GP, Soto C, Kascsak RJ, Levy E, Kascsak R, Harris DA, Frangione B (1999) Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone. Biochem Biophys Res Commun 258:470–475PubMedCrossRefGoogle Scholar
  13. 13.
    Weiss S, Proske D, Neumann M, Groschup MH, Kretzschmar HA, Famulok M, Winnacker EL (1997) RNA aptamers specifically interact with the prion protein PrP. J Virol 71:8790–8797PubMedGoogle Scholar
  14. 14.
    Zeiler B, Adler V, Kryukov V, Grossman A (2003) Concentration and removal of prion proteins from biological solutions. Biotechnol Appl Biochem 37:173–182PubMedCrossRefGoogle Scholar
  15. 15.
    Gomes MP, Millen TA, Ferreira PS, e Silva NL, Vieira TC, Almeida MS, Silva JL, Cordeiro Y (2008) Prion protein complexed to N2a cellular RNAs through its N-terminal domain forms aggregates and is toxic to murine neuroblastoma cells. J Biol Chem 283:19616–19625PubMedCrossRefGoogle Scholar
  16. 16.
    Liu M, Yu S, Yang J, Yin X, Zhao D (2007) RNA and CuCl2 induced conformational changes of the recombinant ovine prion protein. Mol Cell Biochem 294:197–203PubMedCrossRefGoogle Scholar
  17. 17.
    Westphal D, Ledgerwood EC, Hibma MH, Fleming SB, Whelan EM, Mercer AA (2007) A novel Bcl-2-like inhibitor of apoptosis is encoded by the parapoxvirus ORF virus. J Virol 81:7178–7188PubMedCrossRefGoogle Scholar
  18. 18.
    Tomiyama A, Serizawa S, Tachibana K, Sakurada K, Samejima H, Kuchino Y, Kitanaka C (2006) Critical role for mitochondrial oxidative phosphorylation in the activation of tumor suppressors Bax and Bak. J Natl Cancer Inst 98:1462–1473PubMedCrossRefGoogle Scholar
  19. 19.
    Shibuta S, Kosaka J, Mashimo T, Fukuda Y, Yoshiya I (1998) Nitric oxide-induced cytotoxicity attenuation by thiopentone sodium but not pentobarbitone sodium in primary brain cultures. Br J Pharmacol 124:804–810PubMedCrossRefGoogle Scholar
  20. 20.
    Varathan S, Shibuta S, Varathan V, Takemura M, Yonehara N, Mashimo T (2003) Effects of deep hypothermia on nitric oxide-induced cytotoxicity in primary cultures of cortical neurons. J Neurosci Res 72:613–621PubMedCrossRefGoogle Scholar
  21. 21.
    Eghiaian F, Grosclaude J, Lesceu S, Debey P, Doublet B, Treguer E, Rezaei H, Knossow M (2004) Insight into the PrPC–>PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants. Proc Natl Acad Sci USA 101:10254–10259PubMedCrossRefGoogle Scholar
  22. 22.
    Wang C, Sadovova N, Ali HK, Duhart HM, Fu X, Zou X, Patterson TA, Binienda ZK, Virmani A, Paule MG, Slikker W Jr, Ali SF (2007) L-carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture. Neuroscience 144:46–55PubMedCrossRefGoogle Scholar
  23. 23.
    Gupta R, Steward O (2003) Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells. J Comp Neurol 461:174–186PubMedCrossRefGoogle Scholar
  24. 24.
    Sekiya T, Yagihashi A, Shimamura N, Asano K, Suzuki S, Matsubara A, Namba A, Shinkawa H (2003) Apoptosis of auditory neurons following central process injury. Exp Neurol 184:648–658PubMedCrossRefGoogle Scholar
  25. 25.
    Donne DG, Viles JH, Groth D, Mehlhorn I, James TL, Cohen FE, Prusiner SB, Wright PE, Dyson HJ (1997) Structure of the recombinant full-length hamster prion protein PrP(29–231): the N terminus is highly flexible. Proc Natl Acad Sci USA 94:13452–13457PubMedCrossRefGoogle Scholar
  26. 26.
    Zou WQ, Cashman NR (2002) Acidic pH and detergents enhance in vitro conversion of human brain PrPC to a PrPSc-like form. J Biol Chem 277:43942–43947PubMedCrossRefGoogle Scholar
  27. 27.
    Nandi PK, Sizaret PY (2001) Murine recombinant prion protein induces ordered aggregation of linear nucleic acids to condensed globular structures. Arch Virol 146:327–345PubMedCrossRefGoogle Scholar
  28. 28.
    Horonchik L, Tzaban S, Ben-Zaken O, Yedidia Y, Rouvinski A, Papy-Garcia D, Barritault D, Vlodavsky I, Taraboulos A (2005) Heparan sulfate is a cellular receptor for purified infectious prions. J Biol Chem 280:17062–17067PubMedCrossRefGoogle Scholar
  29. 29.
    Cordeiro Y, Machado F, Juliano L, Juliano MA, Brentani RR, Foguel D, Silva JL (2001) DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation. J Biol Chem 276:49400–49409PubMedCrossRefGoogle Scholar
  30. 30.
    Silva JL, Lima LM, Foguel D, Cordeiro Y (2008) Intriguing nucleic-acid-binding features of mammalian prion protein. Trends Biochem Sci 33:132–140PubMedCrossRefGoogle Scholar
  31. 31.
    Cordeiro Y, Silva JL (2005) The hypothesis of the catalytic action of nucleic acid on the conversion of prion protein. Protein Pept Lett 12:251–255PubMedCrossRefGoogle Scholar
  32. 32.
    Baskakov IV, Legname G, Prusiner SB, Cohen FE (2001) Folding of prion protein to its native alpha-helical conformation is under kinetic control. J Biol Chem 276:19687–19690PubMedCrossRefGoogle Scholar
  33. 33.
    Caughey B, Brown K, Raymond GJ, Katzenstein GE, Thresher W (1994) Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and congo red [corrected]. J Virol 68:2135–2141PubMedGoogle Scholar
  34. 34.
    S. Gilch, and H.M. Schatzl (2009) Aptamers against prion proteins and prions. Cell Mol Life SciGoogle Scholar
  35. 35.
    Wang S, Lv X, Zhang K, Lin T, Liu X, Yuan J, Dai Y, Li N (2010) Knockdown of the prion gene expression by RNA interference in bovine fibroblast cells. Mol Biol Rep 37:3193–3198PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Key Laboratory for Biomechanics and Mechanobiology of Ministry of EducationSchool of Biological Science and Medical Engineering, Beihang UniversityBeijingChina
  2. 2.National Animal Transmissible Spongiform Encephalopathies LaboratoryCollege of Veterinary Medicine, China Agricultural UniversityBeijingChina
  3. 3.Department of GastroenterologyGan Zhou People’s HospitalGanzhouChina

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