Archives of Virology

, Volume 158, Issue 3, pp 651–658

Alteration of cell responses to PrPSc in prolonged cell culture and its effect on transmission of PrPSc to neural cells

Authors

    • Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Faculty of Applied Biological SciencesGifu University
    • Department of Applied Veterinary Sciences, United Graduate School of Veterinary SciencesGifu University
    • Department of Food Hygiene and Control, Faculty of Veterinary MedicineSuez Canal University
  • Yasuo Inoshima
    • Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Faculty of Applied Biological SciencesGifu University
  • Naotaka Ishiguro
    • Laboratory of Food and Environmental Hygiene, Department of Veterinary Medicine, Faculty of Applied Biological SciencesGifu University
Original Article

DOI: 10.1007/s00705-012-1540-3

Cite this article as:
Elhelaly, A.E., Inoshima, Y. & Ishiguro, N. Arch Virol (2013) 158: 651. doi:10.1007/s00705-012-1540-3

Abstract

The mechanisms and processes of the uptake, intracellular trafficking and intercellular spread of PrPSc and its transfer to neural cells are not clearly defined. The involvement of immune, intestinal, mast or peripheral neural cells in this process also remains unclear. The role of these cell types in the accumulation and transfer of PrPSc to neural cells was investigated following short and prolonged exposure to the Chandler and Obihiro strains of scrapie PrPSc for up to 28 days. Eight cell lines of murine immune, neural, intestinal and fibroblast cell types were tested. After transient degradation phases, certain immune, intestinal and neural cells accumulated PrPSc for up to 28 days postinfection. When co-cultured with N2a-3/EGFP neuroblastoma cells for 4 days followed by several passages, the immune, intestinal and the neural cell lines were able to transfer infection to neural cells. Our results suggest that some of these cell types may have a role in PrPSc accumulation and intercellular spread of PrPSc infection to neural cells in vivo.

Introduction

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are severe infectious and invariably fatal neurodegenerative diseases affecting animals and human. An abnormal form of the cellular prion (PrPC) called PrPSc is believed to be the infectious agent of TSE [1]. The biological functions of PrPC and the mechanisms of PrPSc involvement in neurodegeneration and TSE transmission are not completely defined. However, PrPC is believed to be the precursor of PrPSc and to be essential for the generation of the abnormal isoform [2, 3].

Ingested PrPSc particles may enter the host organism through the gut before invasion of gut-associated lymphoid tissues, where the first PrPSc amplification takes place [46]. PrPSc infection of the enteric nervous system has been reported to occur within fine nerve fibers directly below the villous or crypt cells of the epithelium [7]. PrPSc migrates asymptomatically along the peripheral nervous system by axonal transport mechanisms via the vagus and splanchnic nerves [6]. Upon finally reaching the central nervous system (CNS), PrPSc aggregates and accumulates as the neurodegeneration process advances [8, 9]. The sequential detection of PrPSc in lymphoreticular tissues and subsequently along neural projections to the CNS has led to the conclusion that these tissues are involved in the spread of PrPSc to the CNS [10].

Several studies have attempted to elucidate the mechanisms of uptake, accumulation and transfer of PrPSc to neural cells using different approaches [11, 12]. The most common and fastest ex vivo method is to incubate PrPSc with cell lines and check for its accumulation. From these studies, some cell culture models supporting PrPSc replication have been established [1315] and proved their importance as in vitro models for understanding the cell biology of PrPSc isoforms. Much effort has been directed towards determining whether follicular dendritic cells (FDC) and dendritic cells (DC) are main players in the transfer of PrPSc to neural cells [4, 16]. Accumulation of PrPSc was confirmed in DCs following infection [17] and co-culture of DCs with primary neural cells, resulting in transfer of infection [12, 18]. Mice lacking or having temporarily inactivated FDCs showed reduced or impaired PrPSc accumulation in lymphoid organs, and there was a delay in neuroinvasion [19]. However, the role of other cells such as macrophages, mast cells, intestinal cell or peripheral neural cells in the accumulation, establishment or transfer of prions remains unclear. In addition, the responses of these cells to PrPSc over time require further investigation.

This study was carried out to investigate the involvement of various cell types in the maintenance or accumulation of PrPSc and the potential role of these cells in the transfer of PrPSc from the intestine to enteric or central neurons. Therefore, the short-term and prolonged cellular responses to PrPSc up to 28 days were investigated using eight murine cell lines, including immune, neural and intestinal cell lines. Our in vitro results suggest that some immune, neural and intestinal cells may play a role in the accumulation and intercellular spread of PrPSc infection to neural cells in vivo.

Materials and methods

Cell lines

Eight cell lines of different types, including immune (P388-D1(IL-1) [20], J588L [21] and P1.HTR [22]), neural (N2a-3 [23, 24], GT1-7 [25] and TR6Bc1 [26]), intestinal (IEC-18 [27]) and fibroblast cells (NIH-3T3 [28]) were used in the study. These cell lines were maintained at 37 °C in 5 % CO2 in Dulbecco’s modified Eagle’s medium (DMEM; WAKO, Osaka, Japan) supplemented with 10 % fetal bovine serum (FBS; PAA Laboratories, GmbH, Haldenweg, Austria), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. A neuroblastoma cell line, N2a-3/EGFP, was used as recipient cells for the co-culture experiment. It is a highly PrPSc-susceptible subclone of mouse neuroblastoma N2a cells [23] and carries an enhanced green fluorescent protein (EGFP) gene. It was established by transfection of N2a-3 cells with pEGFP-C1 (Clontech, CA, USA), which contains a neomycin resistance gene. N2a-3/EGFP cells were then selected by addition of G148 at final concentration of 1 mg ml−1, and cells that were resistant to G418 for 50 passages were selected.

Prion strains

Brain homogenates were prepared from the brains of mice that were terminally infected with a mouse-adapted scrapie strain, Chandler [29] or Obihiro [30]. The infected brains were mechanically homogenized in phosphate-buffered saline (PBS, pH 7.4) and diluted to a final concentration of 10 % (w/v) in PBS, sonicated, and stored at −20 °C until use.

Infection of cells with prion

Cell lines were cultured in 60-mm culture dishes at the optimum cell numbers that provide 60-70 % confluence after overnight incubation. Chandler- or Obihiro-infected brain homogenates equivalent to 1 mg of infected brain tissue were added to the cell dishes and incubated for 24 h before rinsing to remove any unbound PrPSc particles. The cells were then incubated until they were harvested. The cells and supernatants were collected and centrifuged at 1,000 × g for 5 min. Pellets were then stored at −20 °C until proteinase K (PK) treatment and analysis by western blotting.

Co-cultures

At the terminal stage of incubation, the PrPSc-exposed cells were washed, trypsinized and harvested in a 15-ml tube. The cells were directly seeded into 10-cm dishes at a high density with N2a-3/EGFP cells at a ratio of one infected cell per one target cell. After 4 days of incubation, cells were split at a ratio of 1:5 every 4 days in the presence of G148 at a concentration of 1 mg ml−1 to remove the donor cells. At each split, 4/5 of the cells in each dish were collected, treated with PK, and analyzed by western blotting for PrPSc accumulation.

PrPC expression in cells

Duplicate samples of 5 × 105 cells of each cell line were collected into 2-ml tubes. Then, 200 μl of lysis buffer (5 mM EDTA, 0.5 % Triton X-100, 0.5 % sodium deoxycholate, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.5) was added to each tube and kept for 30 min at 4 °C. Cell debris was removed by centrifugation for 5 min at 1,000 × g. The PrPC was precipitated by adding 800 μl of methanol at −20 °C for 15 min before centrifugation at 20,000 × g for 30 min. The cell pellets were boiled in sample buffer (62.5 mM Tris-HCl, pH 6.8), 5 % glycerol, 3 mM EDTA, 5 % SDS, 4 M urea, 4 % β-mercaptoethanol, 0.04 % bromophenol blue) and separated by western blotting.

PK treatment

Cells were lysed with 300 μl of lysis buffer and kept at 4 °C for 30 min. Cell debris was removed by centrifugation for 5 min at 1000 × g. Then, 20 μg ml−1 PK (Roche Diagnostics, USA) was added to each sample, which was incubated for 20 min at 37 °C. Proteolysis was terminated by the addition of 1 mM Pefabloc (Roche Diagnostics, USA). The samples were then incubated with 0.3 % sodium phosphotungstic acid at 37 °C for 30 minutes instead of ultracentrifugation as described previously [31]. PK-treated samples were centrifuged at 20,000 × g for 45 min, and the resulting pellets were dissolved in sample buffer, boiled and stored at −20 °C until loading.

Western blot analysis

After denaturation with sample buffer, proteins were separated by 12 % SDS-polyacrylamide gel electrophoresis (Bio-Rad, Tokyo, Japan). The separated protein bands in the gel were transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA), blocked with 5 % non-fat milk in 0.1 % Tween-20 Tris-buffered saline, pH 7.5, probed with anti-prion monoclonal antibody (mAb) 31C6 [29] at a dilution of 1:4,000, and incubated with a peroxidase-conjugated anti-mouse antibody (GE Healthcare, Buckinghamshire, UK). Immunodetection was carried out using enhanced chemiluminescence (ECL Kit; GE Healthcare Bucks, UK) and exposure to x-ray film. Analysis of ECL images was performed using the public-domain ImageJ software (developed at the National Institutes of Health, Bethesda, Maryland, USA) according to the manufacturer’s instructions (URL: http://rsbweb.nih.gov/ij). A PK-treated brain homogenate from a mouse in the terminal stage of infection with the Chandler strain was used as control in western blot analysis.

Results

Characterization of short-term cellular responses to prion exposure

The short-term cellular responses to PrPSc in eight cell lines exposed to 1 mg of Chandler-infected brain homogenate were investigated. Treated cells were harvested at 1, 2, 3 and 5 days of exposure, treated with PK and assayed by Western blotting (Fig. 1). The cell lines varied in their response to infection with PrPSc. Neuroblastoma N2a-3 cells and the hippocampus GT1-7 cells showed incremental accumulation of PrPSc over the 5 days of incubation (Fig. 1). On the other hand, the TR6Bc1 cell line showed gradual clearance of PrPSc (Fig. 1a). Western blotting results for immune cells showed a general trend towards the degradation of PrPSc (J588L and P1.HTR) over the 5 days, except for P388-D1(IL-1) macrophages, which showed a gradual accumulation of PrPSc until day 5. A similar result was also observed in the IEC-18 intestinal epithelial cell line. These results prompted us to investigate whether these types of cell responses relate to cellular PrPC expression, and also to investigate whether these response patterns continue after the 5 days of incubation.
https://static-content.springer.com/image/art%3A10.1007%2Fs00705-012-1540-3/MediaObjects/705_2012_1540_Fig1_HTML.gif
Fig. 1

Kinetics of short-term PrPSc accumulation in cell lines in relation to PrPC expression. (a) Western blot and graphical representation showing PrPSc levels in infected cells over 5 days of incubation. Chandler-infected brain homogenate was used, and cells were harvested at 1, 2, 3, and 5 days and treated with PK. The monoclonal antibody 31C6 was used for detection of PrPSc. The amount of PrPSc is expressed in pixels and quantified by analysis of ECL-developed western blot images using the Image-J software. (b) Relative PrPC expression in each of the examined cell lines. Equal cell numbers of each cell line were prepared without PK treatment and analyzed by western blot using the mouse mAb 31C6. The relative amount of PrPSc is expressed in pixels and was quantified using the Image-J software

PrPC expression in different cell lines

To evaluate the relative expression of PrPC in the cell lines and its relationship to PrPSc-cell interaction, we analyzed 5 × 105 cells of each cell line by western blotting. All three neural of the cell lines expressed high levels of PrPC, while the immune cells showed low to undetectable levels of PrPC (Fig. 1b and Table 1). On the other hand, intestinal epithelial IEC-18 cells showed the highest PrPC expression among all the cell lines (Fig. 1b). When compared to the cell responses to PrPSc (Fig. 1), the expression of PrPC in neural and intestinal cells may reflect their ability to accumulate PrPSc.
Table 1

Responses of cell lines to prion exposure

Incubation period

Strain

Characteristics

Neural cells

Immune cells

Intestinal cells

Fibroblasts

N2a-3

GT1-7

TR6Bc1

P388-D1(IL-1)

J588L

P1.HTR

IEC-18

NIH3T3

Short incubation (up to 5 days)

Chandler

Relative PrPC expression

+++

++

+++

±

±

±

++++

+

PrPSc degradation

+

+

+

+

Accumulation of PrPSc

+

+

+

+

Long Incubation (up to 28 days)

Chandler

Early transient PrPSc accumulation

±

±

+

±

+

PrPSc degradation

+

+

+

+

+

+

Late-stage propagation of PrPSc

+

+

+

+

+

+

±

±

Establishment of persistent infection

+

+

+

PrPSc transfer at co-culture

+

+

+

+

+

+

+

Obihiro

Early transient PrPSc accumulation

NT

NT

+

±

±

±

+

PrPSc degradation

NT

NT

+

±

+

+

Late-stage propagation of PrPSc

NT

NT

+

+

±

+

+

+

Establishment of persistent infection

NT

NT

+

PrPSc transfer at co-culture

NT

NT

+

+

+

+

++++, very high; +++, high; ++, moderate to high; +, moderate; ±, low; −, negative

NT, not tested

Characterization of late responses to PrPSc exposure during prolonged incubation

In vivo, several cell types may live longer than 5 days, and the disease progresses slowly, so we next monitored cell responses to Chandler-PrPSc exposure for 28 days using the same eight cell lines. Cells were exposed to 1 mg of Chandler-infected brain homogenate for 24 h, rinsed, and continuously incubated for 28 days without passage. However, they were supplemented with 1.5 ml of fresh medium every 2 days. Dishes of treated cells were harvested each day until the 28th day and treated with PK. Western blotting revealed that six cell lines (TR6Bc1, P1.HTR, P388-D1(IL-1), J588L, IEC-18 and NIH-3T3) showed similar patterns of biphasic response to PrPSc. (Fig. 2a and b; Table 1). In the first phase, a gradual disappearance of PrPSc occurred, which in some of the cell lines (P1.HTR, P388-D1(IL-1) and IEC-18) was preceded by a transient early increase in the level of PrPSc and in others (TR6Bc1, J588L and NIH-3T3) was not. In the second phase, all of these six cell lines showed reappearance and gradual accumulation of the PrPSc bands at the terminal stage of incubation. N2a-3 and GT1-7 neural cells were the only cells that constantly maintained the same pattern of PrPSc accumulation until day 18 of the experiment (data not shown).
https://static-content.springer.com/image/art%3A10.1007%2Fs00705-012-1540-3/MediaObjects/705_2012_1540_Fig2_HTML.gif
Fig. 2

Kinetics of PrPSc accumulation in cell lines during prolonged incubation after exposure to the Chandler and Obihiro strains. (a) Western blotting results of two representative cell lines treated with Chandler PrPSc. (b) Graphical representation of PrPSc accumulation in the six cell lines exposed to Chandler PrPSc. (c) Western blotting results of two representative cell lines treated with Obihiro PrPSc. (d) Graphical representation of PrPSc accumulation in six cell lines exposed to Obihiro PrPSc. The lanes of each gel show PK-treated PrPSc from infected cells on the indicated day post-exposure. The monoclonal antibody 31C6 was used for detection of PrPSc. The images were analyzed using Image-J software, and the relative amount of PrPSc is expressed in pixels

Late cellular accumulation of PrPSc in relation to scrapie strain

To determine whether the PrPSc cell responses were specific for the Chandler strain, we repeated the experiment using the Obihiro strain with the six cell lines that showed the first phase of PrPSc degradation. Cells were treated in the same way using 1 mg of Obihiro-infected brain homogenate from a mouse at the terminal stage of the disease. TR6Bc1, IEC-18 and NIH-3T3 cell lines showed similar results to those obtained with the Chandler strain, showing the two phases of response (Fig. 2c and d). However, the P1.HTR, P388-D1(IL-1) and J588L cell lines were found to maintain large amounts of PrPSc throughout the incubation period, without a degradation phase (Fig. 2d). These results show that accumulation of PrPSc at the late stage of incubation may not be specific to Chandler strain.

Transfer of PrPSc from infected cells to neural cells after prolonged incubation

To investigate the potential cell-to-cell transfer of accumulated PrPSc to neural cells, we co-cultured PrPSc-loaded cells with neural cells in vitro. Cells at the terminal stage of incubation were co-cultured with an equal number of N2a-3/EGFP cells, which were then split for up to 15 times with concurrent addition of G148 to kill the donor cells. The transfer of PrPSc from all of the donor cell lines except P1.HTR to the N2a-3/EGFP recipient cells was successful (Fig. 3 and Table 1). After a number of passages, PrPSc accumulated in N2a-3 cells as shown in the representative western blots (Fig. 3). Obihiro-infected cells were also co-cultured with N2a-3/EGFP, and the transfer of PrPSc was successful (Table 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs00705-012-1540-3/MediaObjects/705_2012_1540_Fig3_HTML.gif
Fig. 3

Representative western blot of transfer of infection to N2a3/EGFP cells after co-culture with infected cells at the terminal stage of a 28-day incubation. Left, passage of the N2a3/EGFP cell line shows successful transfer of Chandler-PrPSc from TR6Bc1 cells. Right, successful transfer of Obihiro-PrPSc to N2a3/EGFP cells passaged after co-culture with infected IEC-18 cells

Establishing persistent PrPSc infection in cell lines after prolonged infection

After the end of the incubation period, one dish of each PrPSc-infected cell line was subjected to 15 successive passages to test the ability of the cells to establish a persistent PrPSc infection. Cell lines with the ability to establish a persistent PrPSc infection are those that self-replicate the PrPSc continuously when split into new dishes a number of times. However, cell lines that lose their PrPSc load permanently by the 15th split are considered unable to establish persistent PrPSc infection. Three Chandler-infected cell lines (N2a-3, GT1-7 and TR6Bc1) became persistently infected after 2, 5 and 7 passages, respectively.

Discussion

In this study, we investigated the potential role of a variety of cell types in the biological processing, maintenance and transfer of PrPSc to neural cells. Early and late responses to PrPSc were investigated in cell lines of several origins. The short-term incubation of cell lines with PrPSc revealed two main types of cellular responses: the accumulation and degradation of PrPSc. Consistent with previous reports [3, 24, 25], N2a-3 and GT1-7 neural cells supported the accumulation of PrPSc. The P338D1(IL-1) macrophage and IEC-18 intestinal cell lines also accumulated PrPSc for a few days, but this later proved to be transient. The second type of response was the clearance of the endocytosed PrPSc, which was mostly found in the immune cell lines but at variable rates. These variations in the cellular responses to PrPSc suggest that the normal level of PrPC expression and the dynamic equilibrium between synthesis and degradation of PrPSc in infected cells together determine the fate of PrPSc in the infected cell [32]. However, these may not be the only factors affecting the cell responses to prion infection. Other micro-environmental and as yet unidentified factors may be involved [11].

We investigated the cellular responses to PrPSc exposure over a prolonged incubation period because prion diseases generally require long incubation periods to reach the brain and cause symptoms in vivo. Accumulation of PrPSc in the terminal stages of incubation was found in all cell types tested in this experiment by long-term incubation after a 24-h exposure to Chandler PrPSc. When Obihiro-infected brain homogenate was used in the same experiment, similarly late accumulation of PrPSc was found in all of the cell lines, but with no preceding degradation phase in three of them. There are two possible explanations for this late accumulation of PrPSc. The first is the presence of a subpopulation of cells that accumulate an undetectable level of PrPSc. In this case, the observed increase in PrPSc could be a result of the continuous division of these cell subpopulations, which may also be important for establishing persistent infection [11, 33, 34]. This theory may be applicable to the cell lines in which continuous multiplication was observed throughout the time course of incubation. The second possible explanation is that there may be a gradual inhibition of the cell’s degradation systems. This may be related to PrPSc itself, which has been reported to impair the cellular 20S proteasome system [35, 36].

The development of a persistently PrPSc-infected cell line was examined by continuously splitting cells at the terminal stage of incubation. Persistent PrPSc infection was established in three cells lines (N2a-3, GT1-7 and TR6Bc1). N2a-3 neuroblastoma and GT1-7 hippocampus cells have been shown previously to develop persistent infection [3, 23]. Here, we introduce a new neural cell line (TR6Bc1) that can also become persistently infected with either the Chandler or the Obihiro strain of PrPSc when incubated with infected brain homogenate for 28 days.

Late-stage PrPSc accumulation or persistent infection may increase the potential transfer of infection from PrPSc-loaded cells to adjacent cells or neurons in vivo. Moreover, the slow rate of PrPSc degradation with detectable PrPSc for up to 15 days in some cells may also increase the likelihood that PrPSc is presented to other cells. These findings give insight into the later stages of prion invasion to lymphoreticular, intestinal and neural cells and suggest that a wider range of cell types may be involved in disease development. Lymphoreticular and intestinal cells are subject to uptake and transfer exogenous agents, including prions. Consistent with this, we have previously shown that the absorption of various exogenous particles, including recombinant mouse prion protein, from the small intestinal lumen occurs in Peyer’s patches, and the transport of these particles via blood, mesenteric lymph nodes and liver was also observed [37]. It is also possible that, following uptake of PrPSc, immune cells move inside the body and make contact with nerve endings, thereby transferring PrPSc to neural cells.

The transfer of infection from PrPSc-loaded cells was investigated by co-culturing neural cells with PrPSc-treated cells at the terminal stage of incubation. Several cell lines were found to transfer infection, confirming the hypothesis that different cell types may play a role in the transfer of PrPSc to the neural pathway. The transfer of PrPSc may have occurred via direct cell-to-cell contact, close contact with dendrites, or a direct link to neurons via tunneling nanotubes (TNT), as described previously [12, 38]. Taken together, these results suggest that the accumulation of PrPSc in neural recipient cells may be in vitro evidence of a possible involvement of various cell types, including immune and intestinal cells as well as peripheral neural cells, in the maintenance, accumulation, and transfer of PrPSc to the CNS in vivo. Some of these cells may act as reservoirs for PrPSc particles until they have the opportunity to transfer them to a neural cell, which in turn, transfers them to another neural cell until the target organ is reached. Further study may be required to investigate the possible application of these results in the control of the disease by breaking or interfering with the cycle of PrPSc transfer among these cell types.

Acknowledgment

This work was supported by a grant (22380165) from the Ministry of Education, Culture, Sports, Science, and Technology, and by a grant from the Ministry of Health, Labour and Welfare of Japan.

Copyright information

© Springer-Verlag Wien 2012