Cytosolic Prion Protein (PrP) Is Not Toxic in N2a Cells and Primary Neurons Expressing Pathogenic PrP Mutations

Abstract

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.

Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells

“…It has been further proposed that enhanced accumulation of cytosolic PrP might be detrimental to the neurons, and that perturbation of PrP metabolism through the proteasomal pathway could play a pathogenic role in prion diseases (Ma & Lindquist, 1999;Yedidia et al, 2001;Grenier et al, 2006;Wang et al, 2006). For other authors, however, these observations have arisen essentially from experimental settings (Drisaldi et al, 2003;Fioriti et al, 2005). In addition, PrP was found in the cytosol in subpopulations of neurons in several areas of normal brain (Mironov et al, 2003), and a neurotoxic activity of cytosolic PrP C could not be demonstrated in various neuronal cell systems (Roucou et al, 2003;Fioriti et al, 2005).…”

“…Importantly, we found no evidence for the presence of an uncleaved signal peptide on the PrP 26K molecules produced in ALLN-treated CAD and N2A cells. The absence of a detectable signal peptide on insoluble PrP C argues that it was essentially mature protein, rather than a neo-translated protein, that failed to achieve ER translocation as proposed for cells overexpressing tagged PrP driven by a heterologous promoter (Drisaldi et al, 2003;Fioriti et al, 2005).…”

“…For other authors, however, these observations have arisen essentially from experimental settings (Drisaldi et al, 2003;Fioriti et al, 2005). In addition, PrP was found in the cytosol in subpopulations of neurons in several areas of normal brain (Mironov et al, 2003), and a neurotoxic activity of cytosolic PrP C could not be demonstrated in various neuronal cell systems (Roucou et al, 2003;Fioriti et al, 2005). Of note, the impact of such controversies seems to be limited as long as there is no clear evidence that cytosolic PrP increases during the course of a natural prion disease .…”

Disease-associated Mutations in the Prion Protein Impair Laminin-induced Process Outgrowth and Survival

“…However, data regarding the cellular and biochemical properties of prion proteins are controversial; disparities between groups may reflect differences in the cell types in which these proteins have been expressed (21,27,(62)(63)(64). In the current application, we have addressed this question in non-tumor-derived neural cells; a model may more faithfully replicate the effects of prion proteins in neurons.…”

Viability Assays for Cells in Culture

“…We performed a nonlinear regression analysis to extract IC 50 approximately 50% loss of viability at 10 μM MG132, as measured by the MTT assay 31 . Fioriti reported 60% loss of viability 4 hr after treatment with 50 μM MG132, again with the MTT assay 32 . Finally, Zhang and colleagues reported 60% loss of viability at 10 μM MG132, using counts of Hoechst-stained nuclei 33 .…”