• lysosome;
  • macrophage;
  • prion;
  • proteasome


  1. Top of page

It has been reported that macrophages degrade infectious forms of prion protein (PrPSc). In order to investigate the mechanisms underlying PrPSc degradation in macrophages, the effects of lysosomal and proteasomal inhibitors on macrophage cell lines which were incubated with scrapie-affected brain homogenate were studied. PrPSc degradation was inhibited in the presence of both proteasomal and lysosomal inhibitors. Indirect fluorescence assays to determine the cellular localization of PrPSc were undertaken. PrPSc colocalized with the lysosomal membrane protein Lamp-1 and ubiquitin, a protein that is related to the proteasome. The present data indicate that macrophages might degrade PrPSc via the lysosomal and proteasomal pathways.

List of Abbreviations: 

bovine spongiform encephalopathy


gut-associated lymphoid tissue


indirect fluorescence assay


monoclonal antibody


polyclonal antibody


proteinase K


the host-encoded cellular prion proteins


a pathogenic isoform of the PrPC


the PrPSc form that is resistant to limited proteolysis


transmissible spongiform encephalopathies


variant Creutzfeldt-Jakob disease

Transmissible spongiform encephalopathies, often referred to as prion diseases, are progressive neurodegenerative disorders that affect humans and animals. The recent appearance of vCJD has raised public health concerns that BSE may be transmissible to humans across the species barrier via dietary exposure to BSE-contaminated foods (1). TSEs are caused by a pathogenic isoform of the PrPC, termed PrPSc, which accumulates in TSE-affected tissues as relatively PK resistant aggregates. The PrPSc form which is resistant to limited proteolysis is referred to as PrP-res. Following oral exposure, TSE agents firstly accumulate within GALT. Macrophages within gut tissue may play an important role as an initial host responder against PrPSc. It has recently been reported that macrophages are able to degrade PrPSc, and reduce infectivity (2, 3). These results suggest that immune cells may be involved in the clearance of PrPSc. Although antigens generally enter the lysosomal compartment of phagocytic cells rapidly, eukaryotic cells also contain proteasomal pathways which are able to degrade both abnormal endogenous and exogenous proteins (4). PrPC can be degraded by the proteasome system (5). Definition of the cellular pathways and molecular mechanisms involved in the early response against TSE infection may help in identifying important processes that influence disease susceptibility. In the present study, in order to analyze the degradation mechanisms of PrPSc in macrophages, the effects of lysosomal and proteasomal inhibitors on PrPSc degradation were investigated using mouse macrophage cell lines Raw and J774, and the prion strains of Obihiro and Chandler. The localization of PrPSc in macrophage cells was also investigated by IFA and confocal microscopy.

The mouse macrophage cell lines Raw and J774 were maintained in RPMI 1640 medium supplemented with 10% inactivated FCS, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 2.5 mg/mL of amphotericin B. Terminal stage brain homogenates from mice with scrapie of the Obihiro (6) and Chandler strains (7) were prepared in culture medium. The macrophage cells were then incubated with 0.025% of this brain homogenate for 2 hr, washed, and cultured with or without inhibitors for 0–48 hr. To determine the PrP-res degradation pathway, the lysosomal and proteasomal inhibitors on macrophages incubated with the Obihiro and Chandler strains were used. MG132 and ALLN were used as proteasomal inhibitors, and leupeptin and E64-d as lysosomal inhibitors, as outlined previously (2, 8, 9). With the exception of MG132 (BIOMOL, Exeter, UK), all inhibitors were purchased from Sigma (St Louis, MO, USA). Cells were collected with the supernatant to prevent loss of PrPSc from the culture medium. The collected supernatant and cells were then centrifuged at 2150 g for 10 min, and the pellets lysed and treated with PK as previously described (10), with some minor modifications. Briefly, after PK digestion the cell lysates were incubated with 0.3% sodium phosphotungstic acid at 37°C for 30 min (11), and then centrifuged at 15,500 g for 30 min to concentrate the PrP-res. The resulting samples were subjected to Western blot analysis. Briefly, the protein samples were separated on 12% polyacrylamide gels and electroblotted onto Immobilon-P membrane (Millipore, Bedford, MA, USA). Immunodetection was then carried out using anti-PrP mAb 44B1 (12) and peroxidase-conjugated anti-mouse antibody (GE Healthcare, Buckinghamshire, UK). Immunodetection was carried out using enhanced chemiluminescence (ECL kit; GE Healthcare) and quantified using a LAS-4000 lumino-image analyzer (Fujifilm, Tokyo, Japan).

The Obihiro and Chandler strains were found to have different PrP-res dynamics (Fig. 1). With the Obihiro strain (Fig. 1a), in both Raw and J774 cell lines there was a significant time-dependent reduction in PrP-res protein intensity as reported previously (12). The intensity of the PrP-res protein band decreased from 0.4 to 0.18 at 24 hr (Fig. 1aC). With the Chandler strain (Fig. 1b), PrP-res increased transiently and then decreased in a time-dependent manner in both Raw and J774 cell lines (Fig. 1bA). The intensity of PrP-res decreased from 0.5 to 0.4 at 48 hr (Fig. 1bC). Attempts were made to persistently infect Raw and J774 cells with prions by incubating them with 0.5% of brain homogenates of the Obihiro and Chandler strains. After 2 hr of incubation, followed by cell wash and medium change, PrP-res were examined over 10 passages. PrP-res of Obihiro and Chandler strains decreased in both cell lines, disappearing within 10 passages (data not shown). Although the PrP-res dynamics differed between the Obihiro and Chandler strains, neither of them became persistently infected with murine macrophages. In this study, the precise mechanisms underlying the transient increase in macrophages in the Chandler strain were not clarified, however, it is well established that the Chandler strain is able to persistently infect N2a-3 cells, while the Obihiro strain cannot.


Figure 1. Immunoblotting of PrP-res degradation in murine macrophages. (a) Brain homogenates of the Obihiro strain were added to the (A) Raw and (B) J774 cell lines. (b) Brain homogenates of Chandler strain were added to the (A) Raw and (B) J774 cell lines. (C) The presented ratios were calculated by comparing the signal intensity at 0 hr and the indicated times. Results are presented as the mean ± standard deviation from three independent assays.

Download figure to PowerPoint

With the Obihiro strain, the presence of MG132 and E64-d did not result in a reduction in protein intensity over the 24 hr incubation period (Fig. 2aB, E and G). PrP-res intensity was reduced in the presence of ALLN and leupeptin, however the reduction was much less than that without inhibitor (Fig. 2aC, D and G). At 48 hr, immunoblotting demonstrated prominent effects of the inhibitors on PrP-res degradation (Fig. 2aF). In the absence of inhibitors, only low intensities of PrP-res were observed, whereas the samples incubated with MG132, ALLN, leupeptin and E64-d showed a similar intensity to the 0 hr sample.


Figure 2. Inhibition of PrP-res degradation in macrophages by proteasomal and lysosomal inhibitors. Brain homogenates from the (a) Obihiro and (b) Chandler strains were added to the Raw cell line. After 2 hr incubation, cells were cultured for a further 0, 3, 6, 12, 24 or 48 hr (A) without inhibitor, or with (B) MG132, (C) ALLN, (D) leupeptin or (E) E64-d. (F) 48 hr incubation. (G) The presented ratios were calculated by comparing the signal intensity at 0 hr with that at 3, 6, 12 and 24 hr. Each bar shows 0-, 3-, 6-, 12- and 24-hr incubations from the left, respectively. Results are presented as the average ± standard deviation from three independent assays.

Download figure to PowerPoint

With the Chandler strain, although the inhibitory effects on PrP-res degradation were masked by an increase in PrP-res (Fig. 2b A-E and G), prominent inhibitory effects on Chandler strain PrP-res degradation in macrophages were found at 48 hr (Fig. 2bF). Whereas protein intensity was undetectable in the absence of inhibitor at 48 hr, the MG132- and ALLN-treated samples showed a similar intensity to the 0 hr sample. The leupeptin- and E64-d-treated samples demonstrated an increase in intensity. The extent of PrP-res increase in the presence of lysosomal inhibitor was similar to that in the absence of inhibitor.

It has been previously reported that the PrPSc degradation pathway in phagocytic cells occurs through the lysosomal pathway (4). Even though abnormal proteins and PrPC are known to be degraded in the proteasomal pathway, the present data suggest that this pathway contributes to PrPSc degradation in macrophages. The results of the inhibitor assay in the Chandler strain suggest that apparent PrP-res increase or decrease might be influenced by the balance of PrP-res degradation and propagation. MG132 and ALLN treatment may inhibit PrP-res replication in the initial stages of Chandler strain prion infection. A longer incubation time, such as 72 hr, might help to clarify PrP-res dynamics including PrP-res replication, however, the influence of inhibitors on cell viability is a problem. In the present study, the same results were found in the J774 cell line with both Obihiro and Chandler strains (data not shown).

In order to confirm the reduction in PrP-res intensities observed on the immunoblotting, and to determine the cellular localization of PrPSc, IFA was performed. Elimination of detectable PrPC is necessary in order to specifically detect PrPSc using IFA. It has recently been reported that a combination of guanidine treatment and administration of an anti-PrP mAb132 results in a decrease in amounts PrPC to background levels (14). Raw cells cultured on chamber slides were incubated with 0.025% mouse scrapie brain homogenate for 2 hr. The cells were then fixed with 4% paraformaldehyde in 4% sucrose, permeabilized with 0.1% saponin and treated with 5 M guanidine thiocyanate (14). They were then blocked with 5% FCS, and incubated with anti-PrP mAb 132 (12) and anti-Lamp-1 mAb 1D4B, or anti-ubiquitin polyclonal antibody FL-76 (Santa Cruz Biotechnology, Santa Cruz, DA, USA). The cells were then incubated with anti-mouse alexa488, anti-rat alexa546 or anti-rabbit alexa594 secondary antibodies (Invitrogen, Eugene, Oregon, USA). Finally, the cells were mounted with 90% glycerol and analyzed using a LSM510 confocal microscope (Carl Zeiss, Oberkochen, Germany).

Degradation of PrPSc in macrophages was observed microscopically (Fig. 3). The amounts of PrPSc decreased and finally disappeared altogether when the Obihiro strain was used (Fig. 3a). In contrast, PrPSc formed numerous small aggregates and exhibited stronger fluorescence when the Chandler strain was used (Fig. 3b, 6 hr). Although the decrease and increase dynamics of the Obihiro and Chandler strain were demonstrated by both biochemical and fluorescent results, the time courses were different. With the Chandler strain, by 24 hr the PrPSc intensity as shown by IFA had decreased more than that shown by immunoblotting. This discrepancy might have been caused by differences in the detection antibodies and denaturation methods for PrPSc. At 3 hr, Obihiro (Fig. 4a) and Chandler (data not shown) strain PrPSc partially colocalized with the lysosomal membrane protein marker Lamp-1, and ubiquitin (Fig. 4a). In the presence of MG132 or leupeptin, PrP-res was not degraded, even after 24 hr incubation (Fig. 4b). PrPSc incubated with MG132 and leupeptin was found to colocalize with either Lamp-1 (Fig. 4b) or ubiquitin (data not shown). Lamp-1 is a membrane glycoprotein which is found primarily in late endosomes and lysosomes and is thought to protect these membranes from acidic, proteolytic vacuole contents (15). The partial colocalization of PrPSc and Lamp-1-containing compartments suggest that PrPSc degradation occurs partially through lysosomal pathway. Ubiquitin is thought to function in the clearing of abnormal, foreign or improperly folded proteins by targeting them for degradation by the 26S proteasome. The proteasome is unable to efficiently degrade the aggregated protein (16), thus a relationship between the lysosomal pathway and PrPSc is necessary. How PrPSc enters the cytosol region and results in proteasome-system activation remains unknown. However, cross presentation of exogenous antigens has been reported, and the possibility of export from the lysosome to the intracellular matrix has been discussed (17).


Figure 3. The PrPSc detected by IFA as time course. Raw cells were incubated with Obihiro (a) or Chandler (b) strain-affected brain homogenates. The panels show 0, 3, 6, and 24 hr after infection. The top panels show differential interference contrast. DIC, differential interference contrast.

Download figure to PowerPoint


Figure 4. Cellular localization of PrPSc in macrophage. Raw cells were incubated with Obihiro strain-affected brain homogenates. (a) Colocalization of PrPSc, Lamp-1 (lysosomal membrane marker) and ubiquitin (proteasomal pathway related protein) at 3 hr. (b) The cells were incubated for 24 hr without (left column), or with MG132 (middle column) or leupeptin (right column). Colocalization of PrPSc and Lamp-1 is shown. The scale bar indicates 10 μm. DIC, differential interference contrast; Ub, ubiquitin.

Download figure to PowerPoint

In conclusion, in macrophages PrPSc appears to be degraded through the lysosomal and proteasomal pathways. The exact molecular mechanisms underlying these pathways are not yet fully defined. Further studies are required to determine how PrPSc is degraded in the cellular trafficking and re-cycling systems. The role of the autophagy system that serves as an alternative route for cellular protein clearance must also be taken into consideration when defining the PrPSc degradation system (18). Although cellular localization in fixed cells was investigated in the present study, performance of live cell imaging of PrPSc may prove useful for determination of the exact degradation and proliferation mechanisms.


  1. Top of page

This work was supported by Grant-in-Aid for JSPS Fellows (no. 20-1751).


  1. Top of page
  • 1
    Collinge J., Sidle K.C., Meads J., Ironside J., Hill A.F. (1996) Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature 383: 68590.
  • 2
    Luhr K.M., Nordstrom E.K., Low P., Ljunggren H.G., Taraboulos A., Kristensson K. (2004) Scrapie protein degradation by cysteine proteases in CD11c+ dendritic cells and GT1-1 neuronal cells. J Virol 78: 477682.
  • 3
    Sassa Y., Inoshima Y., Ishiguro N. (2009) Bovine macrophage degradation of scrapie and BSE PrPSc. Vet Immunol Immunopathol 133: 339.
  • 4
    Luhr K.M., Wallin R.P., Ljunggren H.G., Low P., Taraboulos A. & Kristensson K. (2002). Processing and degradation of exogenous prion protein by CD11c+ myeloid dendritic cells in vitro. J Virol 76: 12,25964.
  • 5
    Ma J.Y., Lindquist S. (2001) Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc Natl Acad Sci USA 98: 14,95560.
  • 6
    Shinagawa M., Munekata E., Doi S., Takahashi K., Goto H., Sato G. (1986) Immunoreactibity of a synthetic pentadecapeptide corresponding to the N-terminal region of the scrapie prion protein. J Gen Virol 67: 174550.
  • 7
    Clarke MC and Haig DA (1970). Evidence for the multiplication of scrapie agent in cell culture. Nature 225: 100101.
  • 8
    Yedidia Y., Horonchik L., Tzaban S., Yanai A., Taraboulos A. (2001) Proteasomes and ubiquitin are involved in turnover of the wild-type prion protein. EMBO J 20: 538391.
  • 9
    Doh-ura K., Iwaki T., Caughey B. (2000) Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol 74: 48947.
  • 10
    Kim C.L., Karino A., Ishiguro N., Shinagawa M., Sato M., Horiuchi M. (2004) Cell-surface retention of PrPC by anti-PrP antibody prevents protease-resistant PrP formation. J Gen Virol 85: 347382.
  • 11
    Wadsworth J.D., Joiner S., Hill A.F., Campbell T.A., Desbruslais M., Luthert P.J., Collinge J. (2001) Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 358: 17180.
  • 12
    Kim C.L., Umetani A., Matsui T., Ishiguro N., Shinagawa M., Horiuchi M. (2004) Antigenic characterization of an abnormal isoform of prion protein using a new disease panel of monoclonal antibodies. Virology 320: 4051.
  • 13
    Rybner-Barnier C., Jacquemot C., Cuche C., Dore G., Majlessi L., Gabellec M., Moris A., Schwarts O., Santo J., Cumano A., Leclerc C., Lazarini F. (2006) Processing of the bovine spongiform encephalopathy-specific prion protein by dendritic cells. J Virol 80: 465663.
  • 14
    Nakamitsu S., Kurokawa A., Yamasaki T., Uryu M., Hasebe R. Horiuchi M. (2009) Cell density-dependent increase in the level of protease-resistant PrP in prion-infected neuro2a mouse neuroblastoma cells. J Gen Virol 91: 1539.
  • 15
    Rabinowitz S., Horstmann H., Gordon S., Griffiths G. (1992) Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. J Cell Biol 116: 95112.
  • 16
    Verhoef L.G., Lindsten K., Masucci M.G., Dantuma N.P. (2002) Aggregate formation inhibits proteasomal degradation of polyglutamine proteins. Hum Mol Genet 11: 2689700.
  • 17
    Beauvillain C., Delneste Y., Scotet M., Peres A., Gascan H., Guermonprez P., Barnaba V., Jeannin P. (2007) Neutrophils efficiently cross-prime naive T cells in vivo. Blood 110: 296573.
  • 18
    Heiseke A., Aguib Y., Schaetzl H.M. (2009) Autophagy, prion infection and their mutual interactions. Curr Issues Mol Biol 12: 8798.