A Novel, Drug-based, Cellular Assay for the Activity of Neurotoxic Mutants of the Prion Protein

Tania Massignan,Richard S Stewart,Emiliano Biasini,Isaac H Solomon,Valentina Bonetto,Roberto Chiesa,David A Harris

doi:10.1074/jbc.m109.064949

Abstract

In prion diseases, the infectious isoform of the prion protein (PrP(Sc)) may subvert a normal, physiological activity of the cellular isoform (PrP(C)). A deletion mutant of the prion protein (Delta105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP(C) and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Delta105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Delta105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Delta105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.

Highlights

A great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the pathogenic mechanisms responsible for prion-induced neurodegeneration remain poorly understood [3]
Ectopic central nervous system expression of Doppel (Dpl), a PrP paralog that is structurally equivalent to ⌬32–134 PrP, produced a neurodegenerative phenotype in transgenic mice that was suppressed by co-expression of wild type (WT) PrP [11, 12]
WT PrP ameliorated the drug hypersensitivity induced by We investigated whether these neurotoxic molecules, like expression of ⌬CR PrP in HEK cells

Summary

Introduction

A great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the pathogenic mechanisms responsible for prion-induced neurodegeneration remain poorly understood [3]. Shmerling et al [9] originally reported that transgenic mice expressing PrP harboring either of two large, N-terminal deletions (⌬32–121 and ⌬32–134) developed a spontaneous neurodegenerative illness characterized by ataxia and massive degeneration of cerebellar granule neurons. This phenotype was only observed on the Prn-p0/0 (PrP-null) genetic background: co-expression of endogenous, wild type (WT) PrP from a single Prn-p allele completely abrogated clinical symptoms and neuropathology. The biochemical and cell biological properties of ⌬CR PrP are similar to those of WT PrP [16], suggesting that the neurotoxicity of the ⌬CR molecule results from an alteration of a normal activity of PrPC rather than from accumulation of misfolded protein aggregates or cellular mislocalization

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