Role of Plasminogen in Propagation of Scrapie
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病菌学杂志 2005年第17期
Istituto di Ricerche Farmacologiche "Mario Negri," Milano
Istituto Nazionale Neurologico "Carlo Besta," Milano
Consorzio "Mario Negri Sud," Santa Maria Imbaro, Chieti
Dulbecco Telethon Institute, Milano, Italy
ABSTRACT
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 (PrPSc). 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, PrPSc 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.
INTRODUCTION
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 (PrPC) to a scrapie disease-specific species (PrPSc) (13). In the acquired forms, the effectiveness of preventive and therapeutic strategies depends on the possibility of interfering with the initial pathways through which PrPSc enters into the body and is conveyed to the brain. Unveiling the routes of propagation and spread of PrPSc 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 PrPSc, but not to PrPC. However, at variance from those authors, Ellis et al. (3) reported that plasminogen binds to PrPC and showed that its copper content was a critical parameter in this interaction. Those authors also demonstrated that the complex between PrPC and plasminogen accelerates by 200 times the formation of plasmin by tissue plasminogen activator. More recently, Kornblatt et al. (9) reported that PrPC, 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 PrPC. No information is available on PrPSc 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 PrPSc 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 PrPSc in the brain and spleen were reduced in the early phases of disease.
MATERIALS AND METHODS
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).
PrPSc burden quantification. A quantitative evaluation of PrPSc 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).
RESULTS AND DISCUSSION
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.
Analysis of PrPSc in the brain. To determine whether the absence of plasminogen also affected the rate of PrPSc accumulation in the brain of i.p.-infected mice, the amount of the protease-resistant fraction of PrPSc 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 PrPSc 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 PrPSc 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 PrPSc 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 PrPSc accumulation. In any case, once the disease progressed and mice began to die of scrapie infection, the differences between groups in PrPSc accumulation, spongiform changes, and astrogliosis were no longer apparent (Fig. 3g through i).
Analysis of PrPSc in the spleen. Following peripheral experimental challenge, PrPSc replicates and accumulates in secondary lymphoid tissues before spreading to the central nervous system (13). To determine whether the absence of plasminogen affected PrPSc 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 PrPres 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).
As in the brain, once the disease progressed, the differences among groups in PrPSc 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 PrPSc to cell proteins could unveil the mechanism of transport of PrPSc. Previous in vitro studies showed that plasminogen binds to PrPC and PrPSc through kringle domains in a cooperative manner (14). This binding could result in sequestration or degradation of both PrPC and PrPSc, leading to a reduction of prion propagation; it could also result in transport of PrPSc, 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 PrPSc 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 PrPSc 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.
ACKNOWLEDGMENTS
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).
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Ryou, C., S. B. Prusiner, and G. Legname. 2003. Cooperative binding of dominant-negative prion protein to kringle domains. J. Mol. Biol. 329:323-333.
Tagliavini, F., G. Forloni, L. Colombo, G. Rossi, L. Girola, B. Canciani, N. Angeretti, L. Giampaolo, E. Peressini, T. Awan, L. De Gioia, E. Ragg, O. Bugiani, and M. Salmona. 2000. Tetracycline affects abnormal properties of synthetic PrP peptides and PrP(Sc) in vitro. J. Mol. Biol. 300:1309-1322.
Tagliavini, F., R. A. McArthur, B. Canciani, G. Giaccone, M. Porro, M. Bugiani, P. M. Lievens, O. Bugiani, E. Peri, P. Dall'Ara, M. Rocchi, G. Poli, G. Forloni, T. Bandiera, M. Varasi, A. Suarato, P. Cassutti, M. A. Cervini, J. Lansen, M. Salmona, and C. Post. 1997. Effectiveness of anthracycline against experimental prion disease in Syrian hamster. Science 276:1119-1122.(Mario Salmona, Raffaella )
Istituto Nazionale Neurologico "Carlo Besta," Milano
Consorzio "Mario Negri Sud," Santa Maria Imbaro, Chieti
Dulbecco Telethon Institute, Milano, Italy
ABSTRACT
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 (PrPSc). 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, PrPSc 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.
INTRODUCTION
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 (PrPC) to a scrapie disease-specific species (PrPSc) (13). In the acquired forms, the effectiveness of preventive and therapeutic strategies depends on the possibility of interfering with the initial pathways through which PrPSc enters into the body and is conveyed to the brain. Unveiling the routes of propagation and spread of PrPSc 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 PrPSc, but not to PrPC. However, at variance from those authors, Ellis et al. (3) reported that plasminogen binds to PrPC and showed that its copper content was a critical parameter in this interaction. Those authors also demonstrated that the complex between PrPC and plasminogen accelerates by 200 times the formation of plasmin by tissue plasminogen activator. More recently, Kornblatt et al. (9) reported that PrPC, 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 PrPC. No information is available on PrPSc 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 PrPSc 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 PrPSc in the brain and spleen were reduced in the early phases of disease.
MATERIALS AND METHODS
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).
PrPSc burden quantification. A quantitative evaluation of PrPSc 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).
RESULTS AND DISCUSSION
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.
Analysis of PrPSc in the brain. To determine whether the absence of plasminogen also affected the rate of PrPSc accumulation in the brain of i.p.-infected mice, the amount of the protease-resistant fraction of PrPSc 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 PrPSc 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 PrPSc 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 PrPSc 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 PrPSc accumulation. In any case, once the disease progressed and mice began to die of scrapie infection, the differences between groups in PrPSc accumulation, spongiform changes, and astrogliosis were no longer apparent (Fig. 3g through i).
Analysis of PrPSc in the spleen. Following peripheral experimental challenge, PrPSc replicates and accumulates in secondary lymphoid tissues before spreading to the central nervous system (13). To determine whether the absence of plasminogen affected PrPSc 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 PrPres 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).
As in the brain, once the disease progressed, the differences among groups in PrPSc 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 PrPSc to cell proteins could unveil the mechanism of transport of PrPSc. Previous in vitro studies showed that plasminogen binds to PrPC and PrPSc through kringle domains in a cooperative manner (14). This binding could result in sequestration or degradation of both PrPC and PrPSc, leading to a reduction of prion propagation; it could also result in transport of PrPSc, 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 PrPSc 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 PrPSc 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.
ACKNOWLEDGMENTS
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).
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