Mouse-adapted sporadic human Creutzfeldt–Jakob disease prions propagate in cell culture

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Abstract

Cell based models used for the study of prion diseases have traditionally employed mouse-adapted strains of sheep scrapie prions. To date, attempts to generate human prion propagation in cell culture have been unsuccessful. Rabbit kidney epithelial cells (RK13) are permissive to infection with prions from a variety of species upon expression of cognate PrP transgenes. We explored RK13 cells expressing human PrP for their utility as a cell line capable of sustaining infection with human prions. RK13 cells processed exogenously expressed human PrP similarly to exogenously expressed mouse PrP but were not permissive to infection when exposed to sporadic Creutzfeldt–Jakob disease prions. Transmission of the same sporadic Creutzfeldt Jakob disease prions to wild-type mice generated a strain of mouse-adapted human prions, which efficiently propagated in RK13 cells expressing mouse PrP, demonstrating these cells are permissive to infection by mouse-adapted human prions. Our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, additional unidentified cofactors or subcellular environment are critical for the generation of human prions.

Introduction

Prion diseases are fatal, transmissible neurodegenerative disorders affecting humans and animals and include Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep and bovine spongiform encephalopathy (BSE) in cattle. According to the protein only hypothesis, an abnormal isoform of the host encoded prion protein (PrPC), referred to as PrPSc, is the sole or major component of the infectious agent (the “prion”) (Prusiner, 1982). During prion disease pathogenesis, PrPSc is predominantly found in the central nervous system and lymphoreticular tissues despite PrPC expression in most tissues of the body. While an explanation for the tissue specific distribution of PrPSc is still forthcoming, a similar phenomenon is seen in cell culture models whereby prion propagation has been mainly reported for neuronal cell lines of murine origin (notably N2a, and GT1-7) (Solassol et al., 2003) and some non-neuronal lines (3T3, L29) (Vorberg et al., 2004)). However, it is unclear why these cells are susceptible to prion infection whilst many others are not (Gibson et al., 1972, Clarke and Haig, 1976, Race, 1991, Elleman, 1984, Chesebro et al., 1993).

A common feature of cell lines that are permissive to prion infection is that they support propagation of mouse-adapted strains of sheep scrapie prions (Bosque and Prusiner, 2000, Schätzl et al., 1997, Vorberg et al., 2004, Maas et al., 2007). An exception to this is the development of a heterologous system whereby rabbit kidney epithelial cells (RK13) are susceptible to direct infection with sheep scrapie isolates when they overexpress ovine PrPC (Vilette et al., 2001) or the recent propagation of mouse and bank vole isolates upon expression of their respective PrP genes (Vella et al., 2007, Courageot et al., 2008). Propagation of human prions in SYSY-5Y cells has been reported (Ladogana et al., 1995) although this finding has not been repeated by others. Mouse-adapted GSS and CJD prions of unknown strain-type origin have been shown to replicate in a GT1 cell model (Arjona et al., 2004, Milhavet et al., 2000).

Here we have investigated RK13 cells expressing human PrP for their ability to sustain infection with human prions directly derived from a patient with sporadic CJD. RK13 cells processed the exogenously expressed human PrP similarly to exogenously expressed mouse PrP, but were not permissive to infection when directly exposed to sporadic CJD prions. Primary transmission of the same CJD prions to wild-type mice generated mouse-adapted CJD prions, which then efficiently propagated in moRK13 cells providing a model to study prion biogenesis and the screening of potential anti-prion therapeutics. Additionally, our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, yet to be identified cofactors and/or subcellular environment appear critical for the generation of human prions.

Section snippets

Reagents and antibodies

Proteinase K (PK) was obtained from Invitrogen. The anti-PrP monoclonal antibodies used were ICSM18 (D-Gen) and 3F4 (Covance). Polyclonal antibodies were raised in rabbits to synthetic peptides (Mimotopes, Victoria, Australia) corresponding to amino acid residues 23–37, 89–103 and 218–232 of mouse PrP sequence using previously described methods (Caughey et al., 1991). For each sera tested 03R17 (23–37), 03R19 (89–103) and 03R22 (218–232) a PrP specific signal was detected between 28 and 38 kDa

Characterising expression of moPrP and huPrP in RK13 cells

Following transfection of the vectors containing either human PrP (encoding methionine at codon 129) or mouse PrP into the RK13 cell line, cells were treated with puromycin to select for PrP expressing cells. Brain homogenates from the same species were electrophoresed alongside moRK13 and huRK13 cell lysate on an SDS-PAGE gel and immunoblotted for PrPC using two antibodies with different species affinity. The PrP antibody 03R19 detects both mouse and human PrP, whilst the 3F4 antibody is

Discussion

Despite several cell culture models of prion propagation having been established for sheep derived scrapie strains, an in vitro system for studying human prion strains has not yet been developed. By introducing human PrP into the generally permissive RK13 cells we attempted to create a human prion susceptible cell line. RK13 cells can propagate prions from a variety of species when they express PrPC from the donor species (Vilette et al., 2001, Courageot et al., 2008, Vella et al., 2007).

Note added in proof

A recent publication has demonstrated that cerebellar granule neurons taken from transgenic mice overexpressing a human PrP transgene are permissive to infection with human CJD prions upon propagation of the human strain in transgenic mice (Cronier et al., 2007), providing a model for investigating human prion propagation in the presence of mouse co-factors.

Conflict of interest

AFH is a shareholder in and consultant to D-Gen Limited, an academic spin-out company working in the field of prion disease diagnosis, therapeutics, and decontamination. D-Gen markets the ICSM18 antibody used in this study.

Acknowledgements

We thank Professor Charles Weissmann for the gift of the Tga20 mice, Professor John Collinge for the gift of monoclonal antibody ICSM18 and Dr Bruce Chesebro for the gift of the monoclonal antibody 3F4. We thank the animal facility staff of the Department of Pathology, Ms. L. Leone and Ms. L. Forester for performing histology and immunohistochemistry, and Mr. B. Kreunen for preparation of figures. This work was supported by an NHMRC Program Grant #400202. LJV is the recipient of a University of

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    These authors contributed equally to this study.

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