Transgenic models of the transmissible spongiform encephalopathies

Jean C. Manson and Nadia L. Tuzi

The transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases that are also transmissible. The PrP protein is central to the disease process and has been hypothesised to be the infectious agent. Polymorphisms in the PrP gene have been linked to the incubation time of TSE disease, and mutations in the human PrP gene have been suggested to result in genetic TSE disease. Several PrP transgenic models have been developed. These models express PrP from different species, with and without PrP mutations, and at different levels of gene expression. This article discusses the contribution of these transgenic models to our present understanding of the TSE diseases.

Expert Reviews in Molecular Medicine © Cambridge University Press ISSN 1462-3994

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The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative diseases that are also infectious. These diseases include scrapie of sheep and goats, which was first described over 200 years ago, bovine spongiform encephalopathy (BSE) in cattle, and a number of human forms of the disease such as Creutzfelt–Jakob disease (CJD), Gerstmann–Straussler–Scheinker syndrome (GSS), Kuru and fatal familial insomnia.

Although TSE diseases can be efficiently transmitted within a species, the transmission from one species to another usually involves a species barrier and results in long incubation times and low susceptibility (Ref. 1). However, despite this, BSE, which was first described in 1986 and reached epidemic proportions in the UK by 1988, has now been shown to have been transmitted to humans in the form of variant CJD (vCJD) (Refs 2, 3).

In order to address both the normal function of the PrP protein and its precise role in the TSEs, a number of transgenic models have been produced and studied over the past 12 years. These models are described in this review and the contribution of each model to our current understanding of the TSEs is presented and discussed. The review begins with a brief discussion of our current understanding of the structure of PrP and the pathogenesis of TSEs.

Structure and function of PrP
The murine PrP gene consists of three exons with the open reading frame residing in the third exon (Fig. 1a; fig001jme), and the messenger RNA (mRNA) (consisting of exons 1, 2 and 3) encodes a protein of approximately 250 amino acid residues. The mRNA from the human PrP gene, PRNP, has not been shown to include an exon 2 sequence, although a sequence homologous to murine exon 2 is present within the human gene (Ref. 4). PrP possesses a signal peptide, two potential sites for N-linked glycosylation, a single disulphide bond, and a glycosylphosphatidylinositol (GPI) anchor at its C-terminus (Fig. 1b; fig001jme). The structure of PrP is highly conserved among mammalian species, suggesting an important role for PrP in cell metabolism. PrP is present in high levels in the brain and at lower levels in other tissues. In the brain, it is found in both neuronal and non-neuronal cells (Refs 5, 6). The location of PrP on the cell membrane, attached via a GPI anchor, has led to suggestions that it may act as a cell receptor or as an adhesion molecule maintaining the architecture of the nervous system. However, although the involvement of PrP in the pathology of the TSEs is well documented, the normal function of PrP is not known.

Polymorphisms encoding Met or Val at amino acid position 129 and Gly or Lys at position 219 (Fig. 1c; fig001jme) of human PrP are associated with the incubation time of, and susceptibility to, human TSEs (Refs 7, 8). Amino acid differences in PrP have also been linked to different incubation times of disease in mice (Ref. 9) and sheep (Ref. 10) following exposure to TSE infectivity. In addition, a number of point mutations and insertions in the human PrP gene (Fig. 1c; fig001jme) appear to lead spontaneously to TSE disease without the addition of an infectious agent (Refs 11, 12). The mechanism by which these polymorphisms alter disease susceptibility or incubation time has not been defined but it has been suggested that the human mutations may destabilise the three-dimensional structure of PrP, making it more likely to convert to the disease-associated PrP^Sc isoform. Alternative hypotheses predict that the mutations in PrP make the individual more susceptible to a TSE infectious agent (Ref. 13).

Pathogenesis of TSEs
The pathological features of the TSE diseases include vacuolation in the central nervous system (CNS), loss of neurons and astrogliosis (increase in size and number of astrocytes). However, the underlying mechanism leading to this pathology is not yet understood. Another major pathological feature of these diseases is the abnormal deposition of the host-encoded PrP in the CNS and other tissues of the infected individual. In unaffected individuals, PrP is a protease-sensitive cell-surface glycoprotein (PrP^C) anchored in the membrane by a GPI anchor (Ref. 14). During TSE disease, PrP accumulates in and around cells in protease-resistant aggregates of PrP^Sc. The amount and distribution of PrP^Sc in the brain of an infected individual is dependent on both the strain of TSE agent and on host genetic factors (Ref. 15).

It was originally suggested in 1967 that the infectious agent in the TSEs could be a protein (Ref. 16). This was later expanded upon and gave rise to the prion (protein-only) hypothesis (Ref. 17). This hypothesis now suggests that the TSEs are attributable to a conformational change in PrP that results in a change from a predominantly a-helical form (PrP^C) to a b-sheet form (PrP^Sc) (Ref. 18). PrP^Sc itself is proposed to be the infectious agent and the prion hypothesis predicts that PrP^Sc can propagate its own conversion by acting as a template or a seed (Ref. 19), allowing further conversion of PrP^C to PrP^Sc to occur. If PrP^Sc is the infectious agent, it is proposed that the strain characteristics of each of the TSE agents would be maintained through structural differences (Ref. 20) or post-translational modifications, such as glycosylation, in PrP. Alternatively, the conversion of PrP^C to PrP^Sc may be secondary to the infectious process that is initiated by an additional molecule such as a nucleic acid component that specifies the disease phenotype and strain characteristics associated with TSE disease (Refs 21, 22). While strains of TSE agent are more readily explained by a nucleic acid component, no such molecule has yet been identified.

Results from transgenic models of TSEs
Transgenic approaches
Several transgenic models for the TSEs have been generated by microinjection of a DNA construct containing the PrP gene of interest into the male pronucleus of a fertilised mouse egg (Fig. 2a; fig002jme). The injected eggs are transferred into the oviduct of pseudopregnant mice, and pups resulting from this are screened for the presence of the PrP transgene, usually by using the polymerase chain reaction (PCR). This approach generates transgenic mice in which the transgene is integrated randomly into the murine genome. Although the expression level and distribution of PrP cannot be controlled with this transgenic approach, its use has yielded many interesting and informative TSE models.

In an alternative approach, transgenic mice carrying modifications of the endogenous murine PrP gene have been produced by gene targeting (Fig. 2b; fig002jme). This method can be used to introduce mutations into the PrP gene or to delete or replace parts of the gene. The major advantage of this method is that the altered gene is in the correct genomic location and under the control of the elements that normally regulate the expression of the PrP gene. Any alteration in the TSE disease in these animals can therefore be attributed directly to the introduced PrP mutation. Because the only difference in the transgenic mice compared with wild-type mice is the targeted mutation, it is possible to perform experiments with the appropriate controls. Furthermore, this technology allows the comparison of different transgenic mice generated using the same approach.

PrP^C is essential for TSE disease
In order to investigate both the normal function of PrP and its role in the TSEs, a number of different lines of mice have been produced by gene targeting in which the production of PrP has been ablated (Refs 23, 24, 25, 26). Each line of PrP null mice has been produced using a different targeting vector, resulting in deletions of different amounts of the PrP gene (Fig. 3; fig003jme). Mice with no PrP have been shown to be resistant to TSE disease when inoculated with a number of strains of agent that are known to cause TSE disease in mice possessing a functional PrP gene (Refs 27, 28). In the absence of PrP, there is no evidence to suggest that the infectious agent can replicate (Ref. 27), although retention of infectivity for prolonged periods has been observed in the PrP null mice (Ref. 29). These findings clearly implicate PrP as central to the disease process but do not provide proof that PrP is the infectious agent.

Determining the normal function of PrP
The first two lines of PrP null mice to be produced (NPU Prnp^-/- and Zurich Prnp^-/-; Fig. 3; fig003jme) did not show any overt phenotypic abnormalities (Refs 23, 24). By contrast, the third and fourth lines of PrP null mice (Japan Prnp^-/- and ICM Prnp^-/-; Fig. 3; fig003jme) showed an ataxic phenotype that developed at 63–70 weeks (Refs 26, 30). Subsequent analysis of these lines of mice has suggested that this phenotype may not be due to the absence of PrP but rather to the overproduction of a protein known as Doppel (Dpl). This protein is the product of a neighbouring gene, Prnd, and shows approximately 25% identity to all known PrPs (Ref. 30). The more extensive deletions in the PrP gene in the third and fourth lines have apparently resulted in the overexpression of the gene that encodes Dpl. The possible involvement of Dpl in TSEs is currently under investigation; however, there is no evidence for disease association between polymorphisms in the human PRND gene and human TSEs (Ref. 31).

Natural routes of infection of the TSEs usually occur through the peripheral tissues of the animal rather than directly through the CNS. To investigate this in more detail, PrP null mice were grafted with brain material from wild-type mice or transgenic mice overexpressing murine PrP^C. After inoculation with a TSE agent via CNS and peripheral routes, the transfer of infectivity from the periphery to the CNS was found to be dependent on cells expressing PrP in the periphery (Ref. 37). The precise mechanisms of transport from the periphery to the CNS might differ for different strains of TSEs (Refs 38, 39, 40). Since replication and transfer of infectivity occurs, often over many years, before the neurodegeneration of the CNS, the peripheral route might be an important area for therapeutic intervention in these diseases. Transgenic models that allow for temporal and tissue specificity of PrP expression might enable these mechanisms to be established and the potential for therapeutic intervention to be investigated.

Expression levels of PrP alter the disease incubation time
Transgenic models have been produced in which PrP genes are over- or underexpressed, and these models have demonstrated a relationship between TSE incubation time and the expression level of the PrP gene. Overexpression of a hamster PrP gene in transgenic mice has shown that high levels of PrP lead to shorter incubation times than those of wild-type mice following inoculation with a hamster strain of scrapie (Table 1; tab001jme; Ref. 41). Overexpression of a murine PrP gene also demonstrated shortening of incubation time, following inoculation with a murine scrapie strain (Table 1; tab001jme; Ref. 42).

A similar relationship between PrP expression level and incubation time has been demonstrated in mice that have decreased levels of PrP. During the production of PrP null mice, heterozygous animals were produced with only one functional copy of the murine PrP gene Prnp. The level of PrP in these mice is lower than that in wild-type mice. When inoculated with a number of different strains of TSE, these animals consistently have incubation times that are longer than those of their wild-type littermates (Table 1; tab001jme; Refs 27, 28).

These models clearly demonstrate that there is a relationship between PrP expression level and the incubation time of TSE diseases. However, altered susceptibility or incubation time resulting from different expression levels of PrP has not been demonstrated in the naturally occurring TSE diseases. Nevertheless, the possibility is currently under investigation through studies examining the sequences that control PrP gene expression (e.g. promoter and 3' untranslated regions) and the polymorphic variants of these sequences that might alter the expression of PrP.

Overexpression of PrP and spontaneous neurological disease
Overexpression of a number of PrP transgenes has been shown to lead to development of disease in the CNS. Transgenic mice expressing high levels of the murine PrP gene with a proline to leucine mutation at amino acid 101 develop neurodegeneration, spongiform changes in the brain and astrogliosis (Ref. 43). Overexpression of a wild-type murine PrP gene in transgenic mice has also been shown to lead to the development of a lethal neurological disease, involving spongiform changes in the brain and muscle degeneration (Ref. 44). In addition, several of these spontaneous neurological diseases are also reported to be transmissible, since a neurological disease developed when mice were inoculated with brain material from the transgenic mice that had the spontaneous neurological disease (Refs 43, 44).

Whether the overexpression of PrP in transgenic mice reflects features of the natural TSEs in other species or whether it results in clinical artifacts that do not accurately mimic the disease process has yet to be established. Some of the PrP transgenes that have been used to express PrP in transgenic mice also include the recently described Prnd gene encoding Dpl (Refs 43, 44). Since Dpl overexpression has been postulated to lead to ataxia in mice (Ref. 30), it might be partly responsible for some of the neurological disease developing spontaneously in these animals. However, this requires further investigation.

Polymorphisms in PrP alter the disease incubation time
Allelic forms of murine PrP differing at residues 108 and 189 have been shown to be closely associated with the gene Sinc/Prni, which controls survival time of mice exposed to scrapie (Ref. 45). Sinc comprises two alleles, s7 and p7, which programme short and prolonged incubation times, respectively (Ref. 46). Gene targeting was used to establish whether Sinc, Prni and Prnp are the same gene. The PrP gene associated with the Sinc s7 allele (Leu108; Thr189) was altered by gene targeting in embryonic stem (ES) cells to that associated with a Sinc p7 mouse (Pro108; Val189) (Ref. 9). The gene-targeted mice inoculated with a mouse-passaged BSE strain (i.e. brain material sub-passaged in mice from a mouse previously inoculated with cattle brain infected with BSE) showed a dramatic alteration in incubation time (113 days) when compared with their wild-type littermates (244 days). The transgenic mice produced in these experiments were identical to the wild-type mice, apart from the amino acid 108 and 189 alterations introduced into the PrP gene. Therefore, the difference in incubation time could be attributed entirely to these polymorphic differences. It was thus established that Sinc, Prni and Prnp were indeed the same gene.

Allelic forms of PrP are also associated with altered incubation time or susceptibility in other species. Polymorphisms at positions encoding amino acids 129 and 219 are associated with human TSEs (Refs 7, 8), and polymorphisms at positions encoding amino acids 136, 154 and 171 are associated with TSE susceptibility in sheep (Ref. 10). Transgenic models overexpressing PrP genes encoding the two polymorphisms at position 129 (Met129 and Val129) have been produced to study human TSE disease. In one study, transgenic mice with a PrP transgene composed entirely of human PRNP and with the Val129 polymorphism were no more susceptible to human TSEs than were non-transgenic mice when the endogenous murine PrP was also present. However, if the endogenous murine PrP gene was removed by crossing the transgenic mice onto a PrP null background, these mice became susceptible to several human TSEs (Ref. 47). By contrast, a second study using the same line of transgenic mice showed increased susceptibility of the transgenic mice to CJD in the presence of the endogenous murine PrP gene (Ref. 48). These disparities might be explained on the basis of differences in human TSE strains used in the studies, although this remains to be established. PrP transgenes consisting of a chimaeric gene composed of mouse and human PrP DNA (with murine sequence coding for the C-terminus of PrP) were also produced. These transgenic mice were susceptible to human TSEs regardless of whether the endogenous murine PrP was present or not. These experiments have led to the suggestion that an additional component, referred to as protein X, binds at the C-terminus of PrP and is required for infectivity of TSEs. The researchers speculate that this macromolecule might act as a molecular chaperone for PrP^Sc (Ref. 47).

The species of PrP influences susceptibility
In vitro assay systems (Ref. 49) and mouse models (Ref. 50) have suggested that homology between host PrP^C and the PrP^Sc associated with infectivity facilitates the infectious process in the host. Thus, by introducing an appropriate PrP gene into transgenic mice, the species barrier can be overcome. This was demonstrated using transgenic mice expressing a hamster PrP gene. These were found to be susceptible to a hamster strain of scrapie, in contrast to wild-type mice, which did not succumb to disease with this agent (Ref. 41). However, apparently incompatible PrP sequences can also result in disease transmission. BSE has been passed through many species with different PrP sequences (e.g. mice and sheep) and yet has retained its strain identity (Ref. 51). This phenomenon has also recently been demonstrated in transgenic animals overexpressing a bovine PrP transgene. These mice are not only susceptible to BSE but are also susceptible to vCJD and sheep scrapie (Ref. 52). Thus, although these results have demonstrated that PrP is at least partly responsible for the species barrier, it is also clear that factors other than the primary sequence of PrP are also involved in transmission between species.

PrP mutations and human TSEs
While polymorphisms in PrP have been demonstrated to alter the incubation time of TSEs following exposure to an infectious agent, several point mutations, deletions and insertions in the human PrP gene appear to lead directly to spontaneous genetic disease. Many human TSEs linked to mutations in the PrP gene have also been shown to be transmissible to rodents and primates (Ref. 53). It has been hypothesised that these mutations alter the structure or processing of the protein, resulting in a destabilisation of the PrP molecule (Refs 54, 55, 56). This in turn might make it more likely for PrP^C to convert to PrP^Sc without any requirement for an exogenous infectious agent.

Transgenic models have been produced to investigate several of these human TSEs that are linked to PrP mutations. The Leu102 mutation in human PrP has been shown to be closely linked to GSS in family studies by lod score analysis (Ref. 57). The equivalent mutation (Leu101) was introduced into a murine PrP transgene and mice were generated with 64 copies of the transgene (Ref. 43). These mice produced eightfold more PrP protein than wild-type mice produced and developed a spontaneous neurological disease. It was later reported that some mice expressing a level of the transgene equivalent to the endogenous murine PrP also developed a neurological disease (Ref. 58). The neurological disease that developed in the overexpressing Leu101 mice included spongiform change, astrogliosis and amyloid plaques that were shown to contain PrP, although further analysis demonstrated that the PrP was not protease-resistant PrP (i.e. not PrP^Sc). Moreover, this spontaneous disease could be transmitted to transgenic mice expressing the Leu101 transgene and to hamsters (Ref. 43). This model thus suggests that the Leu102 mutation might indeed result in genetic disease that is initiated by an unstable PrP.

In contrast to these results, a gene-targeted model of the Leu101 mutation was recently developed and mice homozygous or heterozygous for this mutation showed no sign of spontaneous neurological disease up to 899 days of age (Ref. 13). Furthermore, no disease could be transmitted from these mice to either transgenic or non-transgenic mice. However, it was demonstrated that the introduction of a single amino acid alteration into the endogenous murine PrP gene altered the susceptibility of the transgenic mice (homozygous for Leu at amino acid 101; 101LL) to TSE infectivity (Table 2; tab002jme). This model therefore provides no evidence for a spontaneous neurological disease associated with the Leu101 mutation, but does provide evidence that this mutation alters susceptibility of the mice to TSE infectivity (Ref. 13). In addition, this model provides no evidence that the Leu101 mutation leads to an inherently unstable molecule, since Leu101 transgenic mice, identical in every other respect to the wild-type mice, can have longer incubation times than the wild-type mice when infected with some strains of TSE (Ref. 13). It has therefore not yet been definitively established whether the human TSEs associated with mutations in the PrP gene represent genetic disease or altered genetic susceptibility to TSE infectivity.

Other transgenic models have also been produced that overexpress PrP transgenes with mutations associated with human TSEs (Refs 59, 60). One of these mutations, Ala117Val, has been demonstrated to be associated with a transmembrane form of PrP. This has been detected in both transgenic mice and in a patient with the Ala117Val mutation (Ref. 61), neither of which have been shown to transmit TSE. This has led to the hypothesis that the transmissibility of TSE diseases might be due to PrP^Sc, but that the neurodegeneration might be caused by a transmembrane PrP that cannot itself transmit disease (Ref. 62).

Future transgenic models for TSE research
The transgenic models of the TSEs produced to date have provided a wealth of information on these diseases. In particular, these studies have shown that PrP is central to the disease process and that the amount of PrP controls incubation time. Furthermore, polymorphisms in PrP have been demonstrated to control incubation time in mice, and models have been developed for examining polymorphisms in other species. It has also been established that the species barrier is, at least in part, controlled by PrP, and it has been shown that a single amino acid alteration in PrP can dramatically alter incubation time.

Despite these advances, many fundamental questions still remain to be answered. Is PrP the infectious agent? How are strains of agent generated and maintained? How does infectivity reach the CNS from the peripheral tissues and can interference in this process block the onset of disease? These questions are being addressed with the transgenic models currently available, but also with the generation of new models in which gene targeting is being used to introduce specific mutations into the endogenous murine PrP gene to examine susceptibility, incubation times and strains of agent. The species of PrP can also be altered through gene targeting to develop models with no barrier to TSE agents from other species. In addition, models are being developed that will allow the expression of PrP at different time points in specific tissues (Fig. 4; fig004jme). This might be achievable using systems that allow regions of DNA, flanked by recognition tags, to be excised by transgenes that express enzymes capable of such a function. Therefore, by controlling the expression of the excision enzyme, using tissue-specific promoters to drive enzyme expression and/or drug-induced activation of the enzyme, expression of the gene of interest (e.g. PrP) can be controlled, in a temporal and/or spatial manner. Such models should allow the pathogenic events leading to neurodengeneration in the CNS to be identified and the potential for therapeutic intervention in these diseases to be investigated.

Conclusions
The work described in this review has shown that transgenic mouse models have already been invaluable in helping us achieve a better understanding of the TSE diseases. They have allowed us to determine that PrP is essential for the TSE diseases to occur and that differences in PrP sequence can affect incubation periods. However, it has also highlighted that many questions still remain unanswered. Without doubt, the present and future transgenic models will continue to generate information that will lead to much better knowledge of this fascinating group of diseases. This in turn will enable us to begin to develop potential therapeutic interventions with the aim of treating and/or preventing TSE infection.

Acknowledgements and funding
We thank Patricia McBride (Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, UK) and Robert Somerville (Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, UK) for reviewing this article. The Institute for Animal Health, the Medical Research Council, and the Biotechnology and Biological Sciences Research Council provided funding.

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