Protease-resistant PrP deposition in brain and non-central nervous system tissues of a murine model of bovine spongiform encephalopathy

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1 Journal of General Virology (I 996), 77, 194 I-I 946. Printed in Great Britain Protease-resistant PrP deposition in brain and non-central nervous system tissues of a murine model of bovine spongiform encephalopathy C. F. Farquhar, I J. Dornan, I R. C. Moore, I R. A. SomervilleJ A. M. TunstalV and J. Hope I Institute for Animal Health, BBSRC & MRC Neuropathogenesis Unit, West Mains Road, Edinburgh EH9 3JF, UK 2 Department of Biological Sciences, Napier University, 219 Colinton Road, Edinburgh EH14 1DJ, UK Infectivity within the central nervous system has been demonstrated by the transmission of bovine spongiform encephalopathy (BSE) from affected cattle to inbred laboratory mice. Sedimentable, protease-resistant PrP (PrP so) has also been extracted from BSE-affected cattle brain. Both infectivity and PrP s~ have been reported in the lymphoreticular tissues of sheep and mice clinically and preclinically affected with scrapie. Neither infectivity nor PrP sc has yet been detected in nonneural tissues of naturally occurring, clinical cases of BSE in cattle. We have used a murine model of BSE (301V isolate in VM/Dk mice) to investigate when and where PrP sc accumulates. PrP sc was detected both in brain and in extraneural sites prior to the onset of clinical symptoms. This murine BSE model differs, however, in four important aspects from our previously published findings for murine scrapie models: (a) PrP s~ was found relatively late into the incubation period; (b) after intracerebral inoculation, PrP s~ was found in brain before it was found in other tissues; (c) no PrP sc was found in most of the spleens from clinically affected animals after intracerebral inoculation; and (d) even after intraperitoneal infection, PrP sc was detected in brain first. Introduction Bovine spongiform encephalopathy (BSE) is a member of the transmissible spongiform encephalopathies (TSEs), which include ovine scrapie and human Creutzfeldt-Jakob disease (CJD). Epidemiological evidence suggests that the BSE outbreak in the UK was caused by the contamination of highprotein feed concentrates with meat and bone meal from TSEinfected ruminant carcases. Infection is therefore by a peripheral route (Wilesmith et al., I991). In contrast with scrapie (Zlotnik, 1962), there is little variation in the central nervous system (CNS) pathology in cattle affected with BSE (Wells et al., 1992). Primary transmissions from cattle into laboratory mice (Fraser et al., 1988, 1992) have given remarkably uniform incubation periods and pathology, indicative of a single, or major, infecting strain (Bruce et ai., 1994a, b). Many strains of scrapie have been described, distinguished by their characteristic incubation period and pathology in mice of defined Sinc genotype (Dickinson et al., 1968; Dickinson & Meikle, 1971; Bruce eta]., 1991). The Sinc gene, with two alleles s7 and p7, is the major l Author for correspondence: C. F, Farquhar. 1 Fax t , Christine'Farquhar@BBSRC'AC'UK determinant of incubation period length in mice (Dickinson e~ al., 1968), None of the scrapie strains characterized to date produce incubation periods or pathology which resemble those obtained from BSE transmissions into mice (Fraser et al., 1992; Bruce et a]., 1994a, b). There is no practical ante-mortem diagnostic test for the animal TSE diseases. No infecting organism, exogenous nucleic acid or host immune response to infection has been identified. However, a proportion of a host-encoded sialoglycoprotein, PrP, aggregates and accumulates in the brains of all animals affected by the causative agents of TSEs (Diringer et ai., 1983; McKinley et al., 1983 ; Bolton et ai., 1985 ; Oesch et al., 1985). PrP has been shown by gene ablation to be essential for TSE disease development and agent replication (B6eler et al., 1993). PrP se can be distinguished from the normal cellular isoform, PrP c, by its sedimentation properties after detergent extraction with N-lauroylsarcosine and its relative resistance to hydrolysis with proteinase K (PK) in non-denaturing conditions (McKinley et ai., 1983; Bolton et al., 1985; Hope et ai., 1986, 1988a; Meyer et al., 1986). PrP se aggregates have been identified in detergent extracts of brain from cattle terminally affected with BSE (Hope et al, 1988 b). Identification of PrP se by immunoblotting of brain can be used to validate a clinical or histopathoiogicai diagnosis of TSE disease and to identify SGM 94

2 preclinical cases, post-mortem (Czub et al., 1986; Rubenstein et al., 1986, 1987; Farquhar et al., 1989, 1994a, b; Ikegami et al., 1991; Mohri et ai., 1992). PrP se has also been identified in extracts of non-cns tissues from TSE-infected rodents by immunoblotting (Shinagawa et al., 1986; Doi et al., 1988; Kitamoto et al., 1989; Farquhar et al., 1990, 1994a; Rubenstein eta]., 1991; Race & Ernst, 1992). Although PrP se has been found in the spleen and lymph nodes from clinically and preclinically scrapie infected sheep (Ikegami et al., 1991; Mohri et al., 1992; Race et al., 1992; Onodera et al,, 1993; Farquhar eta]., 1994b), there is no evidence to date for the accumulation of PrP se in the peripheral tissues of cattle terminally affected with BSE (Mohri et al., 1992; Farquhar et al., 1994 b) or in cases of CJD (Kitamoto et al., 1989). In addition, all attempts at transmission from a large number of non-cns tissues from clinical BSE cases have failed (Middleton & Barlow, 1993; Fraser & Foster, 1994). In murine scrapie models the sensitivity of PrP se detection by immunoblotting of brain extracts is of the order of 100 times greater than for brain extracts from sheep with scrapie, using the polyclonal antiserum 1B3 (Farquhar et al., 1989, 1994a, b). The mouse Sinc genotype, the scrapie strain, and its route of inoculation and dose, determine when and where PrP se is found in the peripheral tissues of individual mice (Farquhar et al., 1994a, b). We now report a sequential immunoblotting study performed to identify when and where PrP se accumulates in a BSE-derived murine model, 301V isolate in Sinc pv (VM/Dk) mice. Methods Murine BSE model. 301V is a mouse passaged isolate derived from a Holstein-Friesian cow terminally affected with BSE (Bruce et al., 1994a). These experiments were conducted with animals infected with the fourth to sixth passage through Sinc p7 (VM/Dk) mice. Mice were injected intracerebrally (i.c.) or intraperitoneally (i.p.) with 0"02 ml of a 1% (w/v) brain homogenate from a VM/Dk mouse terminally affected with 301V from the previous passage. Animals were sacrificed at selected time-points throughout incubation by cervical dislocation and tissues collected into liquid nitrogen prior to storage at - 70 C. Brain, spleen, pancreas, thymus, submaxillary salivary glands and a pool of subcutaneous and cervical lymph nodes were collected from each animal and from age-matched control animals injected with normal brain homogenate. Preparation and identification of PrP s~. Tissues from individual animals were weighed and homogenized. PrP s was sedimented and digested with PK by a method developed for peripheral tissues and previously described (Farquhar et al., 1994a). The same technique was also used for brain samples for comparability, although the recovery of PrP s from brain using the 'peripheral method" is an order of magnitude less than with the CNS extraction method (Hope et al., 1986, 1988a; Farquhar et al., 1994a), which is not appropriate for non-cns tissues. Samples, in two lanes representing 50 % of the total tissue weight without PK treatment and 50% after PK treatment, were run on SDS--PAGE, electroblotted onto nitrocellulose, and immunostained with a polyclona[ anti-mouse PrP (1B3) at a dilution of I in 1000 as previously described (Farquhar et ~1., 1989, 1994a). Samples were not run on a weight equivalent, but on a whole organ basis because of the wide variation in peripheral tissue weights during pathogenesis in murine scrapie (Outram, 1972; Carp et al., 1984) and BSE models (C. F. Farquhar, unpublished). Results were determined by the presence or absence of immunoreactive PrP s on the immunoblot lanes of samples treated with PK. Results Tissue weights during 301V infection After i.c. infection, spleen, lymph node and pancreas weights decreased suddenly after the onset of clinical symptoms. After i.p. infection, the total weight of pooled lymph nodes decreased over the last 20% of the incubation time, other tissues being unaffected (data not shown). However, animals inoculated by the i.p. route were sacrificed before the late clinical stage when weight loss often occurs as a result of dysfunctional eating and drinking behaviour (Outram, 1972). PrP s deposition in SirJc p7 mice infected with 301V PrP se was identified in brain, spleen, lymph nodes, pancreas and submaxillary salivary glands but not in thymus. It exhibited a four-band pattern, between molecular mass values 20 and 35 kda by SDS--PAGE, prior to PK digestion, and a three-band pattern, between molecular mass values 20 and 30 kda, after PK digestion. For all tissues the first signal obtained was very faint, increasing in strength over the time-course of the experiments. This immunoreactive profile was the same as that of PrP s from the murine scrapie models identified with the same antiserum (Farquhar ef al., 1994a, b). No PrP se was found in samples taken within the week after injection (week 0), by either route, when PrP se from residual inoculum might have been detectable. No protease-resistant PrP se was detected in any of the tissues from control animals inoculated with normal brain homogenate (data not shown). PrP s deposition after intracerebral infection After i.c. inoculation the mean incubation was 16 weeks (SE 0"2, range ). PrP se was detected first in brain. It was identified in all brain extracts from 11 weeks post-inoculation (p.i.) (Table 1). This represented approximately three-quarters of the duration of the mean i.c. incubation period and was a month before the onset of overt disease. PrP se was first identified in a brain sample from an animal sacrificed at 5 weeks p.i., when other samples were negative, while no PrP se was found in the brains of two animals sacrificed at 9 weeks p.i. The range over which PrP se was first seen approximates to the range in incubation periods recorded for these experiments. PrP se was detected in only one out of six spleen samples, but in all of the eight lymph node samples, from clinically affected animals (13-19 weeks p.i.). Four of these positive lymph node samples came from animals with no detectable spleen PrP se. PrP se was identified in all of the lymph node extracts from individual, late-stage preclinical (11-13 weeks p.i.) animals, but in only three out of the five spleen extracts. There was only one example, out of 18 mice tested, where PrP se was found in a non-neural tissue without also being 94;

3 !iiiiiiiiiiiiiiiiiiiii iiiii iiii i i i i iiii iiii i!ii iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii Table 1. PrP s~ in VM mice inoculated intracerebrally with 301V Table 2. PrP sc in VM mice inoculated intraperitoneally with 301V No. of animals in which PrP sc was detected/ no. of animals tested Weeks Lymph Salivary p.i. Brain Spleen nodes Pancreas glands Thymus 0 0/2 0/2 ND ND 0/2 0/2 2 0/1 0/2 ND 0/1 0/1 0/1 3 0/2 0/2 ND ND ND O/I 4 0/4 0/3 0/2 0/2 o/1.o 5 I/5 0/3 0/3 0/1 0/1 ND 6 3/4 0/3 0/3 ND 0/2 0/1 7 I/I ND ND ND ND ND 8 1/2 0/2 0/2 0/1 0/1 0/2 9 0/2 0/2 0/2 1/2 0/1 ND 10 4/4 0/2 0/4 1/3 1/2 0/2 11 2/2 3/4 2/2 1/2 1/1 0/1 13" 1/1 o/1 1/1 1/1 o/i 0/2 14* ND 0/I I/1 1/1 0/1 0/2 16' 1/1 1/3 2/2 1/1 1/1 0/1 17t 1/1 0/1 1/1 ND ND ND 19. 3/3 0/I 3/3 2/2 0/1 ND * 50% of the animals in this group displayed clinical signs. -I" 100% of the animals in those groups displayed clinical signs. ND, Not done. found in the brain. In this individual, sacrificed at 9 weeks p.i., PrP se was detected in the pancreas extract but not in the spleen and lymph node extracts. In another individual, PrP se was found in the pancreas, submaxillary salivary glands and brain but not in the spleen or lymph nodes. Throughout the latestage preclinical and clinical phases of disease the presence of PrP se in pancreas samples was, after brain and lymph node samples, the most reliable indicator of disease status. PrP se was found in three of the 16 submaxillary salivary gland extracts tested, the first at 10 weeks p.i., but was found in only one of the three samples from clinically affected animals. No PrP se was identified in any of the I5 thymus extracts examined, which included samples from both latestage preclinical and clinical animals. PrP sc deposition after intraperitoneal infection After i.p. inoculation the mean incubation period was 28 weeks (SE 2"0, range ). By this route PrP se was also detected first in a brain sample, at 8 weeks p.i. (Table 2). This is approximately 3 weeks later than after i.c. injection. The incubation period range was much larger than expected after i.p. inoculation and some of this variability was reflected in the PrP se results. For example, no PrP se was identified in brain samples from animals culled at 14 and 19 weeks p.i. In subsequent passages of 301V i.p. the wide range of incubation periods has been confirmed. No. of animals in which PrP sc was detected/ no. of animals tested Weeks Lymph Salivary p.i. Brain Spleen nodes Pancreas glands Thymus 0 0/2 o/2 N~ 0/2 ~ 0/1 2 0/1 ND ND ND ND ND 3 0/2 ND ND ND ND ND 4 0/2 0/1 ND ND ND ND 5 0/2 0/2 0/1 ND ND ND 6 0/2 0/2 ND ND ND 0/1 7 ND 0/1 0/1 0/1 ND ND s I/2 0/2 0/2 0/1 o/z ~ 10 2/2 0/3 I/3 2/3 I/3 0/1 11 ~ 0/2 1/2 N~ 0/I 0/2 12 2/2 2/2 2/2 2/2 0/1 ND 13 2/2 1/2 2/2 0/I ND ND I4 o/2 o/2 o/1 o/1 o/i ~ I5 3/3 2/3 2/3 2/3 i/2 ND I6 I/1 2/2 2/2 2/2 2/2 ND 17 I/1 2/2 I/2 0/2 0/1 NO i8 Ill Oll ~o 19 0/1 2/2 2/2 2/2 1/1 ND 21 2/2 1/2 2/2 2/2 ND ND 24* 1/1 I/1 2/2 ND ND ND * One of the group displayed clinical signs. ND, Not done. By I0 weeks p.i. PrP se was also found in lymph nodes, pancreas and submaxillary salivary gland extracts, similar timing for the latter tissues as after i.c. injection, although the lymph node PrP se was detected earlier than after the i.c. route. Another 2 weeks elapsed before PrP se could be detected in spleen extracts. After i.p. injection the interval between PrP se being found in brain and in non-neural tissues was shorter than after i.c. inoculation. In addition, peripheral tissues, especially spleen, from an individual with PrP se detectable in the brain, were more likely to be positive for PrP se after i.p. injection. PrP se was identified in almost all of the brain, spleen, lymph node and pancreas extracts from approximately 40 % into the incubation time. As was the case after i.c. infection, the detection of PrP se in submaxillary salivary gland extracts was much less consistent than for other tissues. No PrP se was detected in thymus from five samples taken during the 11 weeks after infection. Discussion The 30IV isolate in Sinc p7 mice is the shortest of all the conventional (non-transgenic) murine TSE models. There are several differences in the timing and tissue distribution of PrP se accumulation in this BSE model from those of the murine 94.~

4 scrapie models previously reported by our group (Farquhar et al., I994a, b). In this model, PrP se is found first in the brain after i.c. infection and there is a delay of approximately 1 month before PrP se accumulation is seen in non-neural tissues and of 6 weeks before it is found in the spleen and lymph nodes. In contrast, in the scrapie models infected by this route, accumulation of PrP se is found first in non-neural tissues (Farquhar et al., 1994a, b). The early detection of PrP so in the scrapie models correlates with the early rise in infectivity in the lymphoreticular system (LRS) before neuroinvasion (Eklund et ai., 1967; Fraser & Dickinson, 1970, I978; Kimberlin & Walker, 1979). This occurs even after i.c. infection as most of the inoculum is dispersed immediately into the periphery (Millson et al., 1979). As this must also be the case for the 301V inoculum, the inability to find PrP se in non-neural tissues relatively early in such a short incubation model implies either that this isolate does not replicate immediately within the peripheral tissues or that its replication does not result in the deposition of PrP se, as previously defined, within those tissues. Although we do not at present understand the relationship between PrP e, PrP se and infectivity the simplest models predict co-localization of infectivity and PrP se. The time at which PrP se can be detected in the murine scrapie models cannot be predicted from the incubation period length, since it is affected by such factors as the strain of agent, host Sinc genotype, and route and dose of infection (Farquhar e~ al., 1994 a). Despite this, there is a correlation with incubation period in that PrP se is found later in those scrapie models with longer incubation periods (Farquhar et ai., 1994a). This correlation, however, does not apply to the murine BSE/301V model where PrP se accumulation in both neural and non-neural tissues is found relatively late compared with the shortest scrapie mode/ studied. In the BSE model, too, there is considerable variation in the detection of PrP se, something associated with much longer scrapie models which have larger incubation period ranges (Farquhar eta]., 1994 a). This problem is exacerbated when infection is by the i.p, route, the spread of incubation periods being considerably broader than is seen for scrapie models. PrP se is only rarely found in the spleens of clinically affected 30IV/Sinc p7 mice inoculated by the i.c. route, although it was present in the lymph nodes of the same individuals and in the spleens of animals sacrificed in the late preclinical phase. In this model therefore, again in contrast to the scrapie models, PrP se accumulation in spleen is not a reliable indicator of whether those individuals are affected. Only rarely can PrP se not be found in the spleens of mice clinically affected with experimental scrapie (Farquhar et al., 1994a). Spleens from Sinc p7 mice clinically affected after i.c. injection with 301V contain very high infectivity titres of I07 IDs0/g (Taylor, 1994); this is 1-2 logs higher than spleen titres from mice terminally affected with scrapie. However, in the BSE model, spleen weights decrease much more dramatically with advanced clinical disease than in the scrapie models. PrP se content may be below the limits of detection by immunoblotting, either as a consequence of catabolism, or cell migration or death. In the two i,p. scrapie models previously reported using the same protocols, ME7 strain in Sinc s7 and 22A strain in Sinc pv mice, PrP se is detected in non-neural tissues long before it is found in brain (Farquhar et at., 1994a). Other workers using a different scrapie isolate, and tissues pooled from several animals, have also identified PrP se in LRS extracts before those of the brain after i.p. inoculation (Doi et ai., 1988). Yet in this BSE model, PrP se is found in the CNS after i.p. infection some 2 weeks before it is found in lymph nodes, pancreas and submaxillary salivary glands and a month before accumulation is detected in the spleen. The CNS is the primary target, even after peripheral inoculation. The i.p. route only increases the reliability of spleen PrP se as an indicator for disease. The time at which PrP se is detected outside the brain appears unaltered by the original site of inoculation. Only by bioassay can it be determined whether the distribution and replication dynamics of infectivity are also unaffected by route in the 301V model. The oral route is the most likely route of infection for BSE (Wilesmith eta]., 1991). The accumulation of PrP se in the gastro-intestinal tract of mice infected orally with scrapie has not been investigated as this route is relatively inefficient (Kimberlin & Walker, 1988). However, infectivity has been found in the distal ileum of cattle challenged orally with BSE, albeit with doses much greater than would be likely to occur in cattle infected naturally (Wells et al., I994). The literature documents differences between known scrapie strains and BSE (Wells et al., 1992; Middleton & Barlow, 1993; Bruce eta]., 1994a, b; Fraser & Foster, I994). Data from PrP se immunoblotting suggests differences in pathogenesis, at least in murine models. Although the presence of PrP se in non-neural tissue extracts can be a good, early indicator of infection in some murine scrapie models, the brain is a much more reliable indicator of infection in VM mice infected with 301V. This is true even after peripheral infection, the most likely natural transmission route. R. C. Moore was supported by an undergraduate bursary from the Nuffield Foundation. This work was partially funded by the Ministry of Agriculture, Fisheries and Food. The authors wish to thank the staff of the animal facility at the Neuropatbogenesis Unit. References Bolton, D. C., Meyer, R. K. & Prusiner, S. B. (1985). Scrapie PrP is a sialoglycoprotein. Journal of Virology 53, Bruce, M. E., McConnell, I., Fraser, H. & Dickinson, A. G. (1991). The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. Journal of Genera] ViraJagy 72, Bruce, M. E., Chree, A., McConnell, I., Foster, J., Pearson, G. & Fraser, H. (1994o). Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philosophical Transactions of the Royal Society of London B 343, ,~

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