British Medical Bulletin

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British Medical Bulletin 66:87-97 (2003)
© 2003 The British Council

Prion propagation in cultured cells

Jérôme Solassol, Carole Crozet and Sylvain Lehmann

Institut de Génétique Humaine, CNRS UPR 1142, Montpellier, France

	Abstract

Top Abstract Introduction Cellular cultures supporting TSE... Use of prion-infected cell... Conclusions Acknowledgements References

 
Cell cultures represent relevant and useful experimental models of transmissible spongiform encephalopathies (TSEs). They are able to promote, upon subpassaging, stable and persistent replication of PrP^Sc as well as infectivity. To date, only a few cell culture models permissive to prion replication are available. Among them, mouse neuroblastoma cell lines (N2a) are most commonly used. While they correspond to homologous models supporting propagation of mouse-adapted scrapie strains, recent studies have reported heterologous models sensitive to natural occurring disease. Infected cell culture models have provided some valuable insights into the biogenesis of PrP^Sc in terms of conversion, subcellular localisations, physiopathological consequences and species-barrier determinants. They have also contributed to the screening and the study of possible therapeutic compounds and to the development of new strategies for the investigation of TSE-specific diagnostic markers.

	Introduction

Top Abstract Introduction Cellular cultures supporting TSE... Use of prion-infected cell... Conclusions Acknowledgements References

 
Transmissible spongiform encephalopathies (TSEs) include mainly Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straüssler syndrome in humans, and scrapie and bovine spongiform encephalopathy (BSE) in animals¹. Also called prion diseases, these fatal neurodegenerative disorders are remarkable since they occur in humans in infectious, sporadic, and genetic forms. One of the main characteristics of these diseases is the accumulation in the brain of a pathological protein, PrP^Sc, that derives² from a normal protein of the host called PrP^C. According to the ‘protein only’ hypothesis, the key event in the disease pathology consists of the conversion of normal PrP^C into the pathogenic form PrP^Sc.

The normal biological function of PrP^C and the mechanisms involved in neurodegeneration and TSE transmission are not completely defined. During the past two decades, considerable efforts have been made to establish culture models of genetic and infectious forms of TSEs and especially cellular cultures supporting TSE agent replication. These models present, from both a fundamental and applied point of view, obvious advantages including: (i) the ability to analyse, at the molecular and cellular levels, the biological properties of PrP^C and PrP^Sc; (ii) the possibility of determining the nature of the infectious agent and the factors governing its propagation; (iii) the screening of drugs with potential therapeutic values; and (iv) the determination of biological markers of the infection with potential diagnostic and physiological interest.

	Cellular cultures supporting TSE agent replication

Top Abstract Introduction Cellular cultures supporting TSE... Use of prion-infected cell... Conclusions Acknowledgements References

 
The most straightforward approach to propagate prions ex vivo is to put infectious fractions in contact with cell culture and to check for the ‘replication’ of the agent. Different infectious fractions have been used, the simplest being represented by brain homogenates. In some instances, partially purified preparations (scrapie-associated fibrils, SAFs) have also been used³, but it is not clear if this results in a higher effectiveness of transmission. As for animal assays, pretreatment of the infectious fraction for 30 min at 80°C followed by sonication has been used to reduce the risk of transmission of conventional viral and bacterial agents. This method also reduces the toxicity of the fractions on cell cultures (personal observation). Following the ‘inoculation’ of the culture, the cells are generally washed extensively and passaged several times. Importantly, before the discovery of PrP^Sc, the success of prion transmission to cell culture was only assessed by the presence of infectivity. Animals were inoculated with cell extracts after sufficient passages in vitro to rule out the detection of the remaining infectivity from the inoculum.

With the demonstration of the close association between PrP^Sc production and prion propagation, the detection of this isoform became a useful marker in cell culture. It was then demonstrated⁴, using metabolic labelling, that infected cultures could generate new molecules of PrP^Sc. Several techniques of different sensitivities currently exist to detect the presence of PrP^Sc in prion-infected cells (Fig. 1). The most common and oldest technique is the SDS-PAGE analysis of cellular lysates treated with proteinase K, followed by Western blot analysis using carboxy-terminal anti-PrP antibodies. Recently, it has been shown⁵ for BSE diagnosis that the ELISA technique could increase the sensitivity of the detection of PrP^Sc. Another method, which seems quicker and more sensitive, is the cell blotting technique. In fact, Bosque et al. have shown that Western blotting could detect PrP^Sc when 10% of the cells of a small well were infected with prions, whereas cell blotting was positive with only 1% of the cells infected⁶. Another paradigm developed by Winklhofer et al. is represented by a filter retention assay for PrP^Sc that measures two diagnostic biochemical properties of PrP^Sc in combination – proteinase K resistance and the presence of a detergent-insoluble aggregated state⁷. Finally, Vilette et al. have used a post-embedded method able to detect single infected cells⁸. This method has the advantage of evaluating the percentage of infected cells present in a particular culture. It is significant that the first data obtained by dilution and subcloning of infected N2a cells revealed that only 1% of the cells were actually infected⁹. More efficient cell-culture models^6,10 (see below) seem to have up to 30% of cells actually accumulating PrP^Sc. The amount of infectivity present in the culture is also an important issue. Recent data on permissible cell lines revealed that cultures have the potential to accumulate as many infectious units per mg of protein as brain from affected animals⁸. In addition, the presence of infectivity has been detected in the culture medium of infected cells^10,11, confirming that these ex vivo infections are very efficient in propagating the infectious agent and represent a useful tool to study prion conversion and to produce prions for a variety of further studies.

View larger version (23K): [in this window] [in a new window]   Fig. 1 Different ways to detect PrPSc in prion-infected cells. (A) Western blot. Lysates of N2a or prion-infected N2a cells (ScN2a) were treated with proteinase K and analysed by SDS-PAGE and Western blotting with anti-PrP antibodies. (B) Slot blot. Cell lysates of N2a and ScN2a cells were filtered through nitrocellulose membranes with a slot blot device and PrPSc detected as described in (C). (C) Cell blot. N2a or ScN2a grown on cover-slips were directly transferred onto a nitrocellulose membrane and PrPSc was revealed, after proteinase K digestion, with anti PrP antibodies by Western blotting.

 
As detailed below, experimental models of prion propagation in cell culture can be arranged into different categories depending on the origin of the inoculum and of the cell culture (Table 1).

View this table: [in this window] [in a new window]   Table 1 Prion-susceptible cell lines

 

Homologous cell culture models for prion propagation
To avoid species-barrier phenomenon that decrease the effectiveness of prion propagation, mostly because of differences in primary amino sequences of PrPs, it made sense to construct ex vivo homologous cell culture models. In these models, PrP^Sc present in the inoculum has the same species origin as the one expressed by cultured cells. Moreover, it was also logical to focus initially on neuronal cells that seemed to be the main target for prions in vivo. The first attempt to obtain infected TSE culture cells was performed as early as 1970¹² and pursued successfully by the groups of Chesebro and Prusiner mainly using mouse neuroblastoma N2a cell lines (see Table 1)^3,13. These cells, which support the replication of the RML (Rocky Mountain Laboratory) strain, are currently the most intensively described and used model. Surprisingly, once infected, N2a cells display no obvious cytolytic changes¹³. In 1983, Markovits et al. described increased catecholamine levels and decreased free serotonin and noradrenalin levels in scrapie-positive N2a cultures compared to uninfected cells¹⁴. Several investigators have described various morphological differences and an increased rate of cell proliferation in scrapie-infected cells¹⁴, while others have described the opposite effect¹⁵. Unfortunately, it is not clear that the changes described were necessarily due only to the scrapie agent rather than to clonal differences or to other factors present in the inoculum used to infect the cells. In addition, scrapie-infected N2a cells showed alterations in bradykinin-mediated responses and in membrane fluidity, although these abnormalities did not seem to affect the growth or the viability of the cells^16,17.

As demonstrated in transgenic animals, transmission of the TSE agent in cultures might also be improved by increasing the level of PrP. In our laboratory, we have developed murine PrP overexpressed mouse neuroblastoma N2a#58 cell lines readily infected by three mouse PrP^Sc scrapie strains – Chandler, 139A and 22L¹⁰.

It is noteworthy that other neuroblastoma cell lines replicate prion agent with the same efficacy as N2a cells (Table 1)^3,9,10,14,18. Also widely used, the hypothalamic cell line called GT1 represents a reliable support for prion replication ex vivo¹¹. GT1 cells are well-differentiated, neuronal cell lines originating from the central nervous system and were established from gonadotropin-releasing hormone neurons immortalised by genetically targeted tumourigenesis in transgenic mice. Subclones of GT1 cells, that were stably transfected with the trkA gene encoding the high-affinity nerve growth factor (NGF) receptor, increased the viability of scrapie-infected cells and reduced the morphological and biochemical signs of vacuolisation and apoptosis present in untransfected GT1 cells¹¹. Recently, Nishida et al. have also developed GT1 cells that are able to support prion replication from 22L, Chandler, and 139A strains¹⁰. Since PrP^Sc accumulates mainly in central nervous system cells, the above cell lines have been chosen for their neuronal phenotype. However, peripheral nervous system cells such as Schwann cells are also able to support propagation of prions in culture^19,20. These models might be useful in the study of the peripheral steps of prion invasion^8,19,20.

From the different studies published so far, the following conclusions can be drawn regarding the transmission of prions in homologous models: (i) only some strains can replicate in one particular cell line; (ii) only some cells from a culture become infected and subcloning could improve the susceptibility to prions; (iii) the PrP expression level is an important factor for the detection of a successful infection; and (iv) the propagation of prions induces only subtle changes in the phenotype of the cultures.

Heterologous cell culture models for prion propagation
This section summarises some examples of successful ex vivo propagation of prions in which there was a mismatch between the species origin of the inoculum, the PrP expressed, or the cell line. In 1984, while testing the transmission of prions in various models, Rubenstein et al. showed for the first time that PC12 rat pheochromocytoma cells could be readily infected with mouse prions²¹. Prior to infection, cells were differentiated into neuron-like cells with nerve growth factor. Interestingly, scrapie agent replication in these cultures was accompanied by decreases in the activities of enzymes involved in the cholinergic, but not the adrenergic, neurotransmitter pathways²². This was the first demonstration that a species barrier could be overcome in cell culture.

A common feature of susceptible cell lines is that they only support the propagation of TSE strains that have been experimentally adapted to rodents. Recently, Vilette et al. developed a new heterologous model for naturally occurring sheep scrapie. This model was obtained by stable expression of the ovine PrP gene in a rabbit epithelial cell line (RK13)⁸. The authors showed that the expression of heterologous PrP in an otherwise refractory system, such as the rabbit system, is sufficient to cross the species barrier ex vivo.

These experimental systems are important since relevant cell culture models supporting naturally occurring TSE agents, including sheep scrapie or BSE, are needed when one considers the impact of these diseases on public health. It is noteworthy that the successful propagation of human CJD in cell cultures has only been reported once²³.

Animal-derived models for ex vivo prion propagation
An original approach to prion propagation in cell culture is the use of animal-derived cells. Several authors have tried to derive infected cultures directly from infected animals. This approach has been successful for the SMB cell line established from cultures derived from the brain of a mouse clinically affected by the Chandler scrapie strain^12,24. It has also been successful when combined with the immortalisation of cells derived directly from ovine PrP transgenic mice infected with natural sheep scrapie²⁰. One can also use cell lines derived directly from PrP knock-out mice eventually expressing heterologous PrP (ovine, bovine and human). Indeed, it has been shown that transgenic mice expressing human PrP became more susceptible to human CJD prions upon ablation of the mouse PrP gene, thus avoiding competitive interactions between PrP from different species²⁵. Developing this idea, work in our laboratory is being directed at attempts to infect hypothalamic PrP^o/o cells²⁶ transfected with human or bovine PrP.

	Use of prion-infected cell-culture models

Top Abstract Introduction Cellular cultures supporting TSE... Use of prion-infected cell... Conclusions Acknowledgements References

 
Considering the extensive studies that can be done in cell culture, prion-infected cultures that produce PrP^Sc represent a useful model to understand the cellular and molecular events leading to the formation of PrP^Sc.

Cell biology of PrP^Sc
From various cellular and biological studies of infected cells, it was demonstrated that PrP^C was the precursor of PrP^Sc and that the PrP^C endocytic pathway was essential for the generation of the abnormal isoform^4,27,28. PrP^Sc seems also to have a longer half-life than PrP^C and to accumulate inside of the cell in the lysosomal compartment²⁹. The exact subcellular localisation of the conversion of PrP^C into PrP^Sc is still not completely defined, but recent data suggest that the endoplasmic reticulum could play a role in this event³⁰.

Physiopathology of prion diseases
One of the most important issues in the study of cell cultures replicating the agent of prion disease was to look for a possible pathological consequence of the infection. Actually, in most cases, prion infection has little or no apparent effect on the cell line viability or phenotype. However, in differentiated PC12 cells, prion replication alters the catecholamine metabolism of the cells²². In GT1 cells also, a pathological phenotype was observed with an increase in the number of apoptotic cells in the infected cultures¹¹. A recent study by Milhavet et al.³¹ demonstrated that prion infection impaired the cellular response to oxidative stress in GT1 cells. This suggests that the normal function of PrP^C, that is also linked to this response³¹, may be altered by the infection. Since PrP^C seems to play a role in signal transduction, metabolism of metal ions or activities of anti-oxidant enzymes, further studies in infected cellular models will be necessary to evaluate, at a molecular level, the impact of infection on these functions.

Therapeutic research of prion diseases
Infected cell cultures have been widely used to discover inhibitors of scrapie formation, laying the groundwork for the development of anti-prion therapeutic agents. Most of these studies have been made in ScN2a cells. Many compounds decrease the amount of PrP^Sc produced in cell culture (for a review, see Brown³²) by a variety of different mechanisms^32,33. Some of these anti-prion agents have been tested in vivo and act on peripheral and/or central nervous system phase of TSE infection³².

Cell cultures can thus be considered as a relevant pre-screening step for the discovery of new anti-prion drugs. However, even if a drug does show promise in tissue culture experiments, it may still not be therapeutically useful as cellular systems are very simplified models compared to the in vivo situation.

Scrapie strains in cell culture
A better understanding of the molecular strain determinants could be obtained from studying infected cells. For example, it has been shown that SMB cells could be infected with different strains of scrapie and that the biological properties and prion-protein profiles characteristic of each of the original strains were propagated in this model³⁴. In addition, we have shown that N2a#58 cells carrying the murine Prnp-a allele could be infected with mouse PrP^Sc–A scrapie strains (Chandler, 139A and 22L) but not with PrP^Sc–B strains (87V and 22A). To determine whether this phenomenon is due to the Prnp allele chosen in our study, an interesting approach would be to test cell lines transfected with the Prnp-b allele¹⁰.

Diagnosis of prion diseases and public health research
An interesting potential for infected cell cultures may be the discovery of biological markers of prion infection, mainly by comparing control versus infected cultures. This type of experiment was first performed at a genetic level. With the development of sophisticated proteomic approaches, several groups are looking for differentially expressed proteins that could be used as diagnostic markers or at least could give some clue as to the physiopathological event leading to prion propagation. Having developed cell lines highly susceptible to prion infection^6,10, another potential of cell culture consists of the detection of infectivity in various biological samples. The basic idea is to test the presence of infectivity in susceptible cell lines. We were able for example to demonstrate that susceptible cell lines could detect infectivity over a 4-log range starting at 50 ID₅₀³⁵. In addition, Zobeley et al. have recently shown that cell cultures could also be used to assess the efficacy of sterilisation conditions on surface-bound prions using thin stainless-steel wire segments³⁶. In the future, cell culture based assays to titre prion infectivity will certainly be developed.

	Conclusions

Top Abstract Introduction Cellular cultures supporting TSE... Use of prion-infected cell... Conclusions Acknowledgements References

 
Studies made in scrapie-infected cell cultures have given relevant information concerning PrP^Sc propagation. It is clear that one cell line can be infected by several prion strains. Conversely, it appears that one strain can infect several different cell lines. In addition to the level and the type of PrP molecules expressed in cells, several other factors may also account for this phenomenon. First, it is possible that cell lines differ in the expression of co-factors necessary for the replication of the agent (chaperon, protein X)²⁵. Second, the general trafficking of PrP in the different lines can play a role in susceptibility. The endogenous cleavage and degradation pathways of PrP may also be involved. Finally, post-translational modifications of PrP (e.g. glycosylation) could modulate the conversion. From the applied research and public health point of view, cell cultures are very useful models for the screening and study of therapeutic compounds and for research into new diagnostic markers which are urgently needed.

	Acknowledgements

Top Abstract Introduction Cellular cultures supporting TSE... Use of prion-infected cell... Conclusions Acknowledgements References

 
We thank Marie Arlotto for the slot blotting experiment. JS is the recipient of a fellowship from the ALMP (Association de Lutte contre les Maladies à Prion, Paris, France) and the Carrefour International Foundation (Paris, France).

	Footnotes

 
Correspondence to: Dr Sylvain Lehmann, Institut de Génétique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, F-34396 Montpellier Cedex 5, France

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Top Abstract Introduction Cellular cultures supporting TSE... Use of prion-infected cell... Conclusions Acknowledgements References

 

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