Neuropathogenesis Unit, Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK1
Sir Alastair Currie CRC Laboratories, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK2
Author for correspondence: Jean Manson. Fax +44 131 668 3872. e-mail jean.manson{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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To elucidate the role of PrP in TSE disease and the normal function of PrP, a number of lines of PrP null (PrP-/-) mice have been generated. The first two lines of PrP-/- mice produced (ZrchI and NPU) developed and reproduced normally (Bueler et al., 1992 ; Manson et al., 1994a
). However, more subtle phenotypic alterations such as altered circadian rhythm (Tobler et al., 1996
), electrophysiological defects (Collinge et al., 1994
) and alterations in copper binding and superoxide dismutase activity in the CNS (Brown et al., 1997
) have subsequently been attributed to the loss of PrP in these mice. Three further lines of PrP null mice (Nsgk, Rcm0 and Zrch II) have since been reported. These lines all present with an ataxic phenotype and Purkinje cell loss from approximately 50 to 70 weeks of age (Moore et al., 1999
; Rossi et al., 2001
; Sakaguchi et al., 1996
). A recent study investigating the cause of ataxia in these mice mapped a gene 16 kb downstream of the murine Prnp gene (PrP), termed Prnd. This gene encodes a PrP-like protein named doppel (Dpl). This gene was found to be expressed in the CNS of all PrP null mice that went on to develop the ataxic phenotype, but no Prnd expression was detected in the CNS of the ZrchI (Moore et al., 1999
) or NPU null mice (N. L. Tuzi & J. C. Manson, unpublished observation). It has been suggested that overexpression of Dpl in Prnp-/- mice causes Purkinje cell loss and ataxia and that this ataxia can be prevented by expressing wild-type PrP (Nishida et al., 1999
; Rossi et al., 2001
). Furthermore, a recent study reported that by reducing the amount of Prnd mRNA, the onset of ataxia was delayed by 6 months (Rossi et al., 2001
).
Dpl and PrP have 25% amino acid sequence identity (Moore et al., 1999
) and Dpl has been shown to be anchored to the cell surface via a GPI anchor (Silverman et al., 2000
), as is PrP (Stahl et al., 1990
). Detailed analysis of recombinant Dpl has shown that the protein is very similar to PrP both in structure and in topology (Mo et al., 2001
). However, Dpl lacks the octapeptide repeats and conserved amino acid region 106126 found in the N terminus of PrP, and thus resembles a truncated form of PrP. Indeed, transgenic mice expressing truncated versions of PrP that lack the octapeptide repeats and amino acid region 106126 (d32121 or d32134 PrP), and hence resemble Dpl, have been reported to develop spontaneous behavioural disorders at a young age, including ataxia (Shmerling et al., 1998
). It has been suggested that the structural similarity between PrP and Dpl may result in the two proteins competing for binding to the same ligand (PrPL) (Weissmann & Aguzzi, 1999
). It has also been proposed that binding of PrP to PrPL generates a survival signal; however, binding of Dpl or truncated forms of PrP to PrPL would not result in a survival signal being generated, resulting in Purkinje cell death and ataxia.
Since PrP has a central role in TSE disease, the Prnp and Prnd genes are in close proximity and the proteins share a structural similarity, it has been proposed that Dpl might also play a role in TSE disease. A number of studies have investigated whether there was any evidence to involve Dpl with altered susceptibility to TSE diseases in humans. The human Dpl gene, PRND, has been sequenced from control individuals and patients with CJD. Although four polymorphisms within the coding region of PRND have been found in two studies, there is no apparent association between these polymorphisms and human TSE diseases (Mead et al., 2000 ; Peoch et al., 2000
).
In a further study, grafts of Dpl-deficient cells were implanted into the brains of adult PrP null mice. When the grafts were inoculated with the RML isolate of TSE agent, classical signs of TSE pathology were observed in the grafts, demonstrating that the absence of Dpl in the grafts did not lead to absence of TSE disease pathology (Behrens et al., 2001 ).
However, rather than being an absolute requirement for TSE disease, Dpl may be capable of modulating the outcome of TSEs, resulting in alterations in incubation time or in the targeting or intensity of pathological lesions in the brain. In order, therefore, to investigate this hypothesis, we crossed our two inbred lines of PrP null mice which either express (Rcm0) or do not express (NPU) the Prnd gene in the CNS, with mice expressing two copies of the Prnpa[108F189V] allele (Moore et al., 1998 ) of the PrP gene (BB mice). This PrP targeted line of transgenic mice was chosen because of the relatively short incubation period observed when BB mice are challenged with the TSE agent 301V. Critically, these three lines of mice all share an identical genetic background, 129/Ola, thereby removing any effects due to non-specific genetic differences. We have inoculated these lines of mice (BORCM and BONPU) with the mouse-passaged 301V strain of BSE. We have compared the level of Prnd expression in the infected and uninfected mice of each line, to establish whether Prnd expression is altered in the CNS during TSE infection. We have also investigated whether Prnd expression in the CNS can modulate TSE disease by comparing the incubation times and pathological lesions in the CNS of the two lines of TSE infected mice.
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Methods |
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301V challenge of mice.
BONPU and BORCM mice were inoculated under halothane anaesthesia intracerebrally (i.c.) with 20 µl of a 1% brain homogenate (in PBS) prepared from brains of VM mice terminally infected with the 301V strain of TSE. A group of BB mice was also inoculated as controls for the targeting of CNS pathology with 301V. Signs of TSE illness were scored as previously described (Fraser & Dickinson, 1968 ). Incubation times were calculated as the time interval between inoculation and terminal illness. A control group of BORCM and BONPU mice were inoculated by the i.c. route with 20 µl of a 1% brain homogenate prepared from a normal, non-TSE challenged mouse brain.
Lesion profiles.
Mice were killed by cervical dislocation and their brains removed and fixed in 10% formal saline. Haematoxylin and eosin-stained coronal sections (6 µm) were scored for vacuolation on a scale of 0 to 5 in nine standard grey matter areas and three white-matter areas, as described previously (Fraser & Dickinson, 1967 , 1968
).
PrP immunocytochemistry.
Following fixation in formal saline the brains were treated with formic acid (98%) for 90 min before being placed in fresh formal saline for a minimum of 24 h. The brains were trimmed, dehydrated in alcohol and impregnated with wax in a cycle lasting approx. 7 h. Sections (6 µm) were mounted on Superfrost plus glass slides, air dried at room temperature overnight then for 2 days at 37 °C. Sections were immunostained using mouse monoclonal antibody 6H4 (Prionics AG) as described. Briefly, sections were de-waxed and rehydrated prior to autoclaving at 121 °C for 15 min followed by treatment with 98% formic acid for 5 min. Endogenous peroxidases were inhibited using methanol and 1% hydrogen peroxide. Sections were incubated with 5% normal rabbit serum prior to the addition of the primary antibody. To each test slide a 1:1000 dilution of primary antibody was added and left to incubate overnight at room temperature. The biotinylated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, USA) was added at a 1:400 dilution and bound antibody was visualized using the ABC kit (Elite) and diaminobenzidine tetrahydrochloride. Sections were counterstained lightly with haematoxylin. Normal mouse serum in place of primary antibody was used as a control. All washes were done using buffer consisting of PBS0·1% BSA.
Northern blotting.
Total RNA was isolated from terminal brains using RNAzol B, based on the guanidinium thiocyanatephenolchloroform extraction method (Chomczynski & Sacchi, 1987 ). Total RNA (50 µg) was separated on a 1·0% agaroseformaldehyde denaturing gel and transferred to Hybond-N (Amersham Pharmacia) by capillary transfer overnight. RNA was fixed to the membrane by baking at 80 °C for 2 h before probing for a Prnd transcript using a 32P-labelled 540 bp PCR fragment generated from the Dpl ORF (Moore et al., 1999
). Membranes were hybridized overnight using ULTRAhyb (Ambion) and washed according to the manufacturers instructions. A 936 bp KpnIEcoRI fragment from Prnp exon 3 was used to generate the PrP probe. Prior to probing the membrane to correct for loading, membranes were stripped by adding to 0·1% SDS heated to 100 °C and shaken until cool. The stripped membrane was hybridized as before and probed with a 32P-labelled murine B-actin probe (GenBank acc. no. BE689156, isolated with restriction enzymes EcoRI and NotI).
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Results |
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Discussion |
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Dpl is not normally expressed in the CNS of adult mice but is expressed in the periphery. Dpl mRNA was shown to be present in the gut and endothelial cells of the brain and spleen of 6-day-old mice with the highest levels being detected in testis and heart, with spleen and skeletal muscle showing lower levels and brain, kidney, liver and lung having barely detectable levels in 9-week-old mice (Li et al., 2000 ). Therefore, Dpl may have a function primarily in the periphery rather than the CNS of adult mice. It has also been reported that Ngsk Prnp-/- mice showed evidence of demyelination and axon loss in the PNS. However, whether this is due to Dpl overexpression or the absence of PrP is unclear as Zrch I Prnp-/- mice (which do not overexpress Dpl) were also reported to show demyelination of the sciatic nerve (Nishida et al., 1999
). Thus if Dpl were to influence the outcome of TSE disease it may achieve this through events in the periphery rather than in the CNS. With TSE diseases the most natural route of infection is via the periphery where events such as uptake and replication of infectivity and its transport to the CNS are likely to be controlled by a number of different factors. Experiments are currently under way to address the effect of Dpl expression in the CNS on TSE disease when mice are challenged via the peripheral route but, at present, there is no evidence for alterations in Dpl expression leading to different outcomes of TSE disease with peripheral routes of infection. However, since Prnd expression is altered in the CNS of BORCM mice, this may not result in alterations in the peripheral events of TSE disease in this line of mice. In order to address this issue we have produced a line of mice in which the Prnd gene has been ablated resulting in an absence of Prnd expression throughout the mouse. Inoculation of this line of mice with TSE strains by i.c. and peripheral routes will establish if expression of Prnd influences any aspect of TSE disease in mice. This work is currently in progress.
In summary, we have found no evidence that the expression of Prnd is altered in the terminal stages of TSE infection or that Prnd expression in the CNS influences the incubation time of TSE disease. Moreover, the expression of Prnd in the CNS does not appear to alter either the targeting or the intensity of pathological lesions in the brain of animals terminally infected with TSE disease. Thus we have demonstrated that Dpl has no apparent role to play in the TSE diseases in i.c. inoculated mice. However, experiments are currently under way to determine whether expression of Dpl can affect naturally occurring TSE diseases.
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Acknowledgments |
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References |
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Received 12 September 2001;
accepted 23 November 2001.