Originally published In Press as doi:10.1074/jbc.M412441200 on January 4, 2005
J. Biol. Chem., Vol. 280, Issue 12, 11320-11328, March 25, 2005
Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations*
Luana Fioriti
¶,
Sara Dossena,
Leanne R. Stewart||,
Richard S. Stewart||,
David A. Harris||,
Gianluigi Forloni, and
Roberto Chiesa**
From the
Dulbecco Telethon Institute (DTI) and Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, Milano 20157, Italy and the ||Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781–1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.
Received for publication, November 3, 2004
, and in revised form, December 27, 2004.
* This work was supported by Telethon-Italy (S00083, to R. C.) and the European Community (QLG-CT-2001-2353, to R. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a fellowship from the Fondazione Monzino.
** An Assistant Telethon Scientist (DTI, Fondazione Telethon). To whom correspondence should be addressed: Dulbecco Telethon Institute and Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy. Tel.: 39-02-39014428; Fax: 39-02-3546277; E-mail: chiesa{at}marionegri.it.
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Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
