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Journal of Virology, September 2005, p. 11225-11230, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11225-11230.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of Plasminogen in Propagation of Scrapie

Mario Salmona,¹ Raffaella Capobianco,² Laura Colombo,¹ Ada De Luigi,¹ Giacomina Rossi,³ Michela Mangieri,² Giorgio Giaccone,² Elena Quaglio,^1,4 Roberto Chiesa,^1,4 Maria Benedetta Donati,³ Fabrizio Tagliavini,² and Gianluigi Forloni^1*

Istituto di Ricerche Farmacologiche "Mario Negri," Milano,¹ Istituto Nazionale Neurologico "Carlo Besta," Milano,² Consorzio "Mario Negri Sud," Santa Maria Imbaro, Chieti,³ Dulbecco Telethon Institute, Milano, Italy⁴

Received 29 December 2004/ Accepted 1 June 2005

Top Abstract Introduction Materials and Methods Results and Discussion References

 
To investigate whether plasminogen may feature in scrapie infection, we inoculated plasminogen-deficient (Plg^–/–), heterozygous plasminogen-deficient (Plg^+/–), and wild-type (Plg^+/+) mice by the intracerebral or intraperitoneal (i.p.) route with the RML scrapie strain and monitored the onset of neurological signs of disease, survival time, brain, and accumulation of scrapie disease-associated forms of the prion protein (PrP^Sc). Only after i.p. inoculation, a slight, although significant, difference in survival (P < 0.05) between Plg^–/– and Plg^+/+ mice was observed. Neuropathological examination and Western blot analysis were carried out when the first signs of disease appeared in Plg^+/+ animals (175 days after i.p. inoculation) and when mice reached the terminal stage of illness. At the onset of symptoms, PrP^Sc accumulation was higher in the brain and spleen of Plg^+/+ and Plg^+/– mice than in those of Plg^–/– mice, and these differences were paralleled by differences in the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures. Immunohistochemical analysis of the spleens before inoculation did not show any impairment of the immune system affecting follicular dendritic or lymphoid cells in Plg^–/– mice. Once the disease progressed and mice began to die of infection, differences were no longer apparent in either brains or spleens. In conclusion, our data indicate that plasminogen has no major effect on the survival of scrapie agent-infected mice.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Prion diseases are transmissible neurodegenerative disorders of humans and animals that are sporadic and either inherited or acquired (1, 13). They all have identical pathogenic mechanisms, i.e., a posttranslational modification of the prion protein from a normal cellular isoform (PrP^C) to a scrapie disease-specific species (PrP^Sc) (13). In the acquired forms, the effectiveness of preventive and therapeutic strategies depends on the possibility of interfering with the initial pathways through which PrP^Sc enters into the body and is conveyed to the brain. Unveiling the routes of propagation and spread of PrP^Sc may be particularly relevant since the emergence of the new variant of Creutzfeldt-Jakob disease that has raised the fear of an outbreak of an epidemic in human populations following exposure to bovine spongiform encephalopathy-contaminated products.

Fischer et al. (4) and Maissen et al. (11) showed that plasminogen binds selectively to PrP^Sc, but not to PrP^C. However, at variance from those authors, Ellis et al. (3) reported that plasminogen binds to PrP^C and showed that its copper content was a critical parameter in this interaction. Those authors also demonstrated that the complex between PrP^C and plasminogen accelerates by 200 times the formation of plasmin by tissue plasminogen activator. More recently, Kornblatt et al. (9) reported that PrP^C, in complex with plasminogen, also undergoes a proteolytic degradation, forming two main oligomers, although those authors did not clarify whether the cleavage was carried out by tissue plasminogen activator, plasmin, or both. M atrix-assisted laser desorption ionization mass spectrometry analysis indicated that the most abundant oligomer has a molecular mass of 13,862 Da and corresponds to the C-terminal core of PrP^C. No information is available on PrP^Sc degradation under the same experimental conditions. In light of these observations, and most importantly, in the absence of an agreement among in vitro data, we decided to determine in an in vivo experimental model if plasminogen might affect PrP^Sc propagation. To investigate this issue, we used plasminogen-deficient mice which were infected with the scrapie agent by either the intracerebral (i.c.) or intraperitoneal (i.p.) route. We found that in plasminogen-deficient mice, the survival time was slightly increased only after peripheral infection and that the accumulations of PrP^Sc in the brain and spleen were reduced in the early phases of disease.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Transmission studies. Homozygous plasminogen-deficient (Plg^–/–), heterozygous plasminogen-deficient (Plg^+/–), and wild-type (Plg^+/+) mice were provided by Peter Carmeliet (Center for Transgene Technology and Gene Therapy, Vlaams Interuniversitair Institut voor Biotechnologie, Leuven, Belgium) and were genotyped by Southern blot analysis (12).

The mouse-adapted scrapie isolate RML (8) was originally obtained from B. Caughey and R. Race (Rocky Mountain Laboratory, Hamilton, Montana) and was passaged repeatedly in CD1 mice. A 10% (wt/vol) homogenate of RML-infected CD1 mouse brain in phosphate-buffered saline was diluted to a final concentration of 1% in phosphate-buffered saline, and 50 µl (i.p.) or 25 µl (i.c.) was injected into 4- to 6-week-old mice. To monitor the appearance and development of neurological signs, mice were observed daily and were scored once a week (2, 5, 16). Three animals per group (randomly selected before inoculation) were culled for histology and PrP immunohistochemistry of brain and spleen and for biochemical analysis of brain tissue when clinical signs of disease were first apparent in Plg^+/+ mice, while the remaining mice were sacrificed at the terminal stage of the disease. Furthermore, the spleens from three each of the noninfected Plg^–/–, Plg^+/–, and Plg^+/+ mice were examined histologically and immunohistochemically with antibodies to follicular dendritic cells and B and T lymphocytes. Procedures involving animals and their care were conducted in conformity with national and international laws and policies (EEC Council Directive 86609, OJ L358, 1, 12 December 1987; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana no. 10, 18 February 1992; and Guide for the Care and Use of Laboratory Animals [11a]).

Neuropathology. At autopsy, the right cerebral hemisphere, the brain stem, and the cerebellum were dissected at standard levels, fixed in Carnoy solution, and embedded in paraplast, while the left cerebral hemisphere was frozen and stored at –80°C for Western blot analysis. Five-micrometer-thick serial sections from paraplast-embedded blocks were stained with hematoxylin-eosin or incubated with a polyclonal antibody to a synthetic peptide homologous to residues 95 to 108 of human PrP (PrP95-108, 1:800 dilution) that strongly recognizes mouse PrP, or with an antiserum against glial fibrillary acidic protein (GFAP; 1:1,000 dilution; Dako, Carpinteria, CA). Before PrP immunostaining, the sections were sequentially subjected to proteinase K digestion (10 µg/ml, 25°C, 2 min) and guanidine thiocyanate treatment (3 M, 25°C, 30 min). Immunoreactivity for GFAP was enhanced by pretreatment with a 4% formaldehyde solution. Immunoreactions were revealed by the polyclonal Envision system (Dako) using 3-3'-diaminobenzidine as the chromogen.

Spleen analysis. Spleens from noninfected mice were cut lengthwise; one half was fixed in Carnoy solution and embedded in paraplast, while the other half was frozen. Five-micrometer-thick sections from paraplast-embedded blocks were stained with hematoxylin-eosin or immunostained with rat anti-mouse CD 45R (1:1,000; Cymbus Biotechnology) and goat anti-mouse CD 3 (1:1,000; Santa Cruz Biotechnology) antibodies that recognize B and T cells, respectively. Five-micrometer-thick sections from frozen spleens were immunostained with a rat anti-mouse antibody that labels follicular dendritic cells (1:50; BD Pharmigen). Spleens from scrapie agent-infected mice were fixed in Carnoy solution and embedded in paraplast. Five-micrometer-thick sections were stained with hematoxylin-eosin or immunostained with the antibody PrP95-108 (1:100 dilution) after proteinase K digestion and guanidine thiocyanate denaturation as described previously (7).

PrP^Sc burden quantification. A quantitative evaluation of PrP^Sc burden in the spleen was carried out using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) equipped with a color video camera (Nikon; DXM 1200) and a computer-based image analysis system (Nikon; Lucia measurement, version 4.82). Lamp intensity, video camera setup, and calibration parameters were constant throughout all measurements. The total cross-sectional area of the spleen and the number and surfaces of PrP95-108-immunoreactive deposits were determined at a magnification of 2 mm by 1.6 mm (4x objective) and 820 µm by 660 µm (10x objective), respectively.

Biochemical assays. Ten percent homogenates (wt/vol) of the left cerebral hemispheres were prepared using 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate. Aliquots corresponding to 50 µg protein were analyzed by Western blotting with an anti-plasminogen antibody (Innovative Research, Plymouth, MN; 1:1,000 dilution) and the anti-actin monoclonal antibody C4 (Chemicon International, Temecula, CA; 1:10,000 dilution). To assay the proteinase K resistance of PrP, aliquots of brain homogenates were diluted to 5 mg protein/ml and incubated with proteinase K (25 to 100 µg/ml) at 37°C for 1 h. Digestion was terminated by the addition of phenylmethylsulfonyl fluoride to a final concentration of 5 mM, and PrP was analyzed by Western blotting using the monoclonal antibody SAF 75 (kindly provided by J. Grassi, CEA/Saclay, Gif sur Yvette, France; 1:600 dilution), which recognizes the region from positions 142 to 160 of mouse PrP. Immunoreactive bands were visualized by enhanced chemoluminescence (Amersham), and their average signal intensities were quantified by densitometry, as described by Tagliavini et al. (15).

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Survival of scrapie agent-infected mice. Following i.c. inoculation, no differences in incubation period, survival time, or severity of the spongiform changes were observed among Plg^–/–, Plg^+/–, and Plg^+/+ mice (Table 1). On the other hand, after i.p. inoculation, a statistically significant difference was observed only between Plg^–/– and Plg^+/+ mice (log rank test) (Table 1 and Fig. 1). However, this difference could have been underestimated due to the poor health of Plg^–/– mice, which displayed retarded postnatal growth and, shortly after inoculation, became runted, listless, and cachectic. For the same reason, it was not possible to determine clearly the onset of the disease in Plg^–/– mice, at variance with results for Plg^+/– and Plg^+/+ mice, which showed initial signs of scrapie infection at 173 ± 5 and 175 ± 3 days after i.p. inoculation, respectively.

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TABLE 1. Disease onset and survival times of Plg–/–, Plg+/–, and Plg+/+ mice following i.c. or i.p. inoculation of the RML scrapie straina

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FIG. 1. Survival time of Plg–/–, Plg+/–, and Plg+/+ mice injected i.p. with the RML scrapie strain.

Analysis of PrP^Sc in the brain. To determine whether the absence of plasminogen also affected the rate of PrP^Sc accumulation in the brain of i.p.-infected mice, the amount of the protease-resistant fraction of PrP^Sc was evaluated by Western blot analysis at different times during the course of disease. At the onset of the symptoms in the controls, 175 days after inoculation, the densitometric analysis of the PrP^Sc immunoblots showed some differences among the three conditions examined (31,023.3 ± 3,831.3 in Plg^+/+, 20,688.0 ± 4,237.6 in Plg^+/–, and 3,847.0 ± 361 in Plg^–/– mice; arbitrary units for three samples per group). Conversely, no significant differences in the amounts of PrP^Sc were found between terminally ill mice of different Plg genotypes (Fig. 2a and b). Similarly, PrP immunohistochemistry of brain sections from animals sacrificed when clinical signs of disease were first apparent in Plg^+/+ mice revealed that PrP^Sc accumulation was higher in Plg^+/+ and Plg^+/– mice than in Plg^–/– mice (Fig. 3a through c). This difference was paralleled by differences in the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures (Fig. 3d through f). The intrinsic variability at the onset of symptoms in experimental scrapie might partially justify the difference in PrP^Sc accumulation. In any case, once the disease progressed and mice began to die of scrapie infection, the differences between groups in PrP^Sc accumulation, spongiform changes, and astrogliosis were no longer apparent (Fig. 3g through i).

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FIG. 2. Western blot analysis of the protease-resistant fraction of PrPSc in Plg–/–, Plg+/–, and Plg+/+ mice at the terminal stage of disease, and quantification of the results. (a) Brain homogenates of Plg–/–, Plg+/–, and Plg+/+ mice were incubated with 100 µg/ml of proteinase K at 37°C for 1 h and analyzed by Western blotting with the anti-PrP antibody SAF 75 (upper panel). The same samples were probed with anti-plasminogen (middle panel) and anti-actin (lower panel) antibodies. (b) Values were obtained by densitometric analysis of immunoblots and are expressed as arbitrary units ± standard errors of the means for four animals.

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FIG. 3. Sections from Plg–/–, Plg+/–, and Plg+/+ brains processed by immunohistochemistry for PrPSc and GFAP. At the onset of the clinical signs of disease, PrPSc (a-c) and GFAP (d-f) immunoreactivities are higher in Plg+/+ and Plg+/– than in Plg–/– mice. Conversely, no difference between groups in PrPSc accumulation is detectable in the terminal stage of disease (g-i). The pictures are representative of the immunohistochemical results obtained in the various groups.

Analysis of PrP^Sc in the spleen. Following peripheral experimental challenge, PrP^Sc replicates and accumulates in secondary lymphoid tissues before spreading to the central nervous system (13). To determine whether the absence of plasminogen affected PrP^Sc accumulation in lymphoid tissue, the spleens from Plg^+/+, Plg^+/–, and Plg^–/– mice culled 175 days after i.p. inoculation were analyzed immunohistochemically using anti-PrP antibodies. The study showed that PrP-immunoreactive deposits were remarkably more abundant in germinal centers of Plg^+/+ and Plg^+/– mice than in those of Plg^–/– mice (Fig. 4a to d), while no differences between results from Plg^+/+ and Plg^+/– mice were observed. The morphometric study showed that the percentage of tissue area occupied by PrP^res deposits was considerably smaller in Plg^–/– mice (0.08%) than in Plg^+/+ and Plg^+/– mice (1.99% and 1.60%, respectively; n = 4) (Fig. 4e). Statistical analysis demonstrated that this difference was significant (P < 0.05, Dunnett's test).

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FIG.4. PrP immunohistochemistry of the spleens from Plg–/– and Plg+/+ mice culled at the onset of clinical signs of disease in Plg+/+ mice. The accumulation of protease-resistant PrP is higher in Plg+/+ (a and c) than in Plg–/– (b and d) mice. Magnifications, 4x (a and b) and 20x (c and d). Samples in panels c and d were counterstained with hematoxylin. In panel e, the quantifications of PrPSc burdens in the spleen at the onset of symptoms are reported. The data are expressed as the percentages of tissue area occupied by PrPres deposits and are the means ± standard errors of the means for four animals. Differences between results for Plg–/– mice and those for Plg+/+ mice are significant at a P value of <0.05 (Dunnett's test).

As in the brain, once the disease progressed, the differences among groups in PrP^Sc accumulation disappeared. To rule out the possibility that this difference could be due to impairment of the immune system affecting follicular dendritic or lymphoid cells, spleens from noninfected Plg^+/+, Plg^+/–, and Plg^–/– mice were subjected to histological and immunohistochemical examination, using antibodies that selectively label follicular dendritic cells and B and T lymphocytes. The study did not reveal any difference in number, structure, or cellular composition of the lymphatic follicles among the three groups of animals (data not shown).

Understanding the binding sites of PrP^Sc to cell proteins could unveil the mechanism of transport of PrP^Sc. Previous in vitro studies showed that plasminogen binds to PrP^C and PrP^Sc through kringle domains in a cooperative manner (14). This binding could result in sequestration or degradation of both PrP^C and PrP^Sc, leading to a reduction of prion propagation; it could also result in transport of PrP^Sc, favoring the spread and progression of the disease process. Our results support the second hypothesis, since i.p.-infected Plg^–/– mice showed a delay in the accumulation of PrP^Sc in the spleen and brain. However, the survival time of Plg^–/– mice inoculated intraperitoneally with the scrapie agent was only slightly increased compared with that of the Plg^+/+ mice. It is conceivable that other kringle-containing proteins or complement components C3 and C1q (6, 10, 14) might cooperatively contribute to conveying PrP^Sc to the brain; therefore, its propagation from the periphery to the central nervous system would have many interacting partners, which might make very difficult the possibility of interfering with this process. In conclusion, our data indicate that plasminogen does not play a pivotal role in prion infectivity propagation.

 
This research was supported by the Italian Ministry of Health (grant RF 2001.96), the Italian Ministry of University and Research (grant PRIN 2001), and the European Union (grants QLRT 2001-00283 and QLRT-2000-02353). R.C. is an assistant Telethon scientist (Dulbecco Telethon Institute).

 
* Corresponding author. Mailing address: Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milan, Italy. Phone: 39-02-39014-462. Fax: 39-02-354-6277. E-mail: forloni{at}marionegri.it.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Journal of Virology, September 2005, p. 11225-11230, Vol. 79, No. 17
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.17.11225-11230.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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