Original Contribution
Acute exposure to prion infection induces transient oxidative stress progressing to be cumulatively deleterious with chronic propagation in vitro

https://doi.org/10.1016/j.freeradbiomed.2011.03.035Get rights and content

Abstract

Neuronal loss is a pathological feature of prion diseases for which increased reactive oxygen species (ROS) and consequent oxidative stress is one proposed mechanism. The processes underlying ROS production in prion disease and the precise relationship to misfolding of the prion protein remain obscure. Using cell culture models of prion infection we found that cells demonstrate a rapid, prion protein (PrP) dependent, increase in intracellular ROS following exposure to infectious inoculum. ROS production correlated with internalisation and increased intracellular protease resistant PrP (PrPRes). The ROS increase was predominantly lysosomal in origin but not sustained, with cells adapting within 48 hours. Overall ROS levels remained normal in the chronically prion infected cell population; however a subpopulation characterised by loss of membrane phosphatidylserine asymmetry exhibited highly peroxidised intracellular aggregates that localised with PrP and intense caspase activation. These apoptotic cells showed increased ROS closely correlating with increased PrPRes. Our findings demonstrate that a PrP-dependent, transient, increase in intracellular ROS is characteristic of acute cellular prion infection, while chronic phases of prion infection in vitro are associated with a significant subpopulation manifesting apoptosis accompanying heightened oxidative stress and increased PrPRes burden. Such observations strengthen the direct links between heightened ROS and ongoing prion propagation with eventual cellular demise.

Introduction

Transmissibility is a fundamental feature of prion diseases separating them from other neurodegenerative disorders such as Alzheimer's disease. Transmission and neuronal degeneration only occur when the native prion protein (PrPC) is expressed, allowing ongoing formation of abnormal conformers with increased beta-sheet content [1], [2]. Generally in animal models, the abnormal conformers can be detected in the brains, by means of their increased protease resistance, well before overt disease is evident. At the earliest time point when these abnormal, protease resistant conformers (referred to as PrPRes) can be weakly detected there is also a significant elevation in lipid peroxidation, a marker of increased ROS [3]. This finding raises the possibility that conversion of PrPC to the abnormal isoform occurs concurrently with elevated ROS production, especially before any cellular compensatory responses can be established. Recent studies have highlighted the role of methionine oxidation, caused by ROS, in destabilising PrPC conformation and so indicate that oxidation of PrPC itself might be part of the conversion mechanism to PrPRes[4].

PrPC has been linked with cellular anti-oxidant capacity. This protection may arise through an intrinsic superoxide dismutase-like activity of PrPC[5], [6], or through the activation of signal transduction pathways that stimulate anti-oxidant defence by other means [7]. Transgenic mice with the PrP gene ablated (Prnp PrPo/o) appear to have no significant impairment of central nervous system function [1], [8]; however such mice do show heightened levels of oxidative stress markers in the brain and peripheral organs [9]. Further, when challenged with large insults, such as transient cerebral ischemia, PrPo/o mice display much greater oxidative damage than their wild-type counterparts [9], [10], [11]. Prion diseased mice with ongoing PrPRes propagation show compromised antioxidant function and, specifically, attenuated cellular superoxide dismutase activity [12], [13]. The implication being that, during prion disease, a PrPC-related ROS protective function is lost either as a direct or indirect result of conversion of PrPC to PrPRes. An alternative to such ‘loss of function’ theories is the possibility that PrPC to PrPRes conformational change brings about an innate toxic gain of function directly contributing to increased cellular oxidative stress. Increased ROS have been found due to activation of NADPH oxidase when cells are exposed to PrPRes or the amyloidogenic and toxic 106–126 peptide derived from the core of PrP [14]. The authors reported that exposure to the ‘toxic’ agent initiates deleterious signalling through PrP, fyn and NADPH oxidase thereby promoting pathogenesis.

Considerable evidence exists supporting the occurrence and likely pathogenic role of heightened oxidative stress in prion disease evolution although whether this is a primary or initiating event or a later contributor after the development of neuronal dysfunction remains to be confidently resolved. Further, the processes underlying ROS production in prion disease and the precise relationship to misfolding of the prion protein remain obscure. At the earliest time point when protease resistant conformers were detected in the brains of prion infected mice, increased lipid peroxidation was detected concurrently [3]. The purpose of the present study was therefore to better understand the associations between PrPRes propagation, ROS production and toxicity at the cellular level. Accordingly this study investigated the ROS changes associated with acute and chronic phases of prion infection in cell cultures. We found that cellular adaptation quickly restores increased ROS levels following acute prion infection but appears to be progressively overwhelmed in a significant subpopulation with resultant toxicity and apoptosis during chronic propagation.

Section snippets

Cell culture

Rabbit kidney epithelial (RK13) cells, transfected (using FuGene 6; Roche, AUS) with empty vector, mouse PrP or human PrP, mouse olfactory bulb (OBL-21) cells [15], [16] and the GT1-7 mouse hypothalamic neuronal cell line were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen AUS) supplemented with 10% (v/v) foetal bovine serum (Lonza Australia Pty) and 50 U/ml pencillin/50 μg/ml streptomycin FBS; solution (Sigma, AUS) and maintained at 37 °C with 5% CO2 in a humidified incubator.

Stem cell harvest and culture

Exposure to M1000 infectious inocula induces a rapid, PrP-dependent, increase in intracellular ROS in moRK13 cells

The RK13 cell system used in this study has previously been characterised in detail [17], [18]. RK13 cells transfected with mouse PrPC readily propagate the M1000 mouse-adapted Gerstmann-Sträussler-Scheinker syndrome (GSS) prion strain [17], [21], [22]. Mixed population stable cell lines expressing mouse PrP (moRK13), human PrP (huRK13) and the empty vector control (vecRK13) were created by transfection. Infection of moRK13 cells with M1000 prions derived from the brains of mice in the

Discussion

This study has shown that intracellular ROS production changes in acute, adaptive and chronic phases of prion infection in vitro and further links ROS with prion propagation and cellular demise. Upon initiation of prion infection cellular adaptation to increased intracellular ROS occurs quickly within the overall cell population, a response probably essential for maintaining viability. The cells progress to a chronic phase of infection where the ROS response is kept below levels that would

Acknowledgments

The authors would like to acknowledge A/Prof AF Hill for the kind gift of the pIRES vector containing the PrP open reading frames and Dr Simon Drew for the SR-VAD-FMK reagent. The OBL-21 cell line used in this study were a kind gift of Dr Michael Oldstone, The Scripps Research Institute, La Jolla, California, USA. This study was funded by a Brain Foundation research grant. SJC is supported by an NH&MRC Practitioner Fellowship #400183 and an NH&MRC programme grant #400202. VL is supported by an

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