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British Medical Bulletin 66:43-60 (2003)
© 2003 The British Council

PrP knock-out and PrP transgenic mice in prion research

C Weissmann and E Flechsig

MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, London, UK
Institut für Virologie und Immunbiologie, Würzburg, Germany


   Abstract
Top
 Abstract
Generation and properties of...
 Transgenesis and gene...
 Effect of PrP mutations...
 Pathological mouse phenotypes...
 Ectopic expression of PrP...
 Concluding remarks
 Acknowledgements
 References
 
Spongiform encephalopathies such as scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jacob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome (GSS) in humans is caused by a transmissible agent designated prion. The ‘protein only’ hypothesis proposes that the prion consists partly or entirely of a conformational isoform of the normal host protein PrPC, designated PrP*1 and that the abnormal conformer, when introduced into the organism, causes the conversion of PrPC into a likeness of itself. PrP* may be congruent with PrPSc, a protease-resistant, aggregated conformer of PrP that accumulates mainly in brain of almost all prion-infected organisms.

PrPC consists of a flexible N-terminal half, comprising Cu2+-binding octapeptide repeats, and a globular domain consisting of three {alpha}-helices, one short antiparallel ß-sheet and a single disulphide bond2. It is anchored at the outer cell-surface by a glycosyl phosphatidylinositol (GPI) tail and is present in almost all tissues, however, mainly in brain3.

Compelling linkage between the prion and PrP was established by biochemical and genetic data4–8 and led to the prediction that animals devoid of PrP should be resistant to experimental scrapie and fail to propagate infectivity. This prediction was indeed borne out, adding substantial support to the ‘protein only’ hypothesis. In addition, the availability of PrP knock-out mice provided an approach to carry out reverse genetics9 on PrP, both in regard to prion disease and to its physiological role.


   Generation and properties of mice devoid of PrP
Top
 Abstract
 Generation and properties of...
Transgenesis and gene...
 Effect of PrP mutations...
 Pathological mouse phenotypes...
 Ectopic expression of PrP...
 Concluding remarks
 Acknowledgements
 References
 
PrP is encoded by a single-copy gene10 that comprises three exons, with the entire reading frame contained in the third exon (Fig. 1b). Several lines of mice devoid of PrPC have been generated by homologous recombination in embryonic stem cells, using either of two strategies. The ‘conservative strategy’ involves disruptive modifications restricted to the open reading frame (ORF)11,12. Mice homozygous for the inactivated gene (such as Prnpo/o [Zürich I] or Prnp-/- [Edinburgh]) develop normally, show no striking pathology and are resistant to prion infection.



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  		Fig. 1 Knock-out strategies and their consequences. (a) Various strategies used to target Prnp by homologous recombination. The black boxes represent PrP ORFs; white boxes, non-coding Prnp regions; grey boxes, inserted sequences; dotted line, deleted regions; neo, neomycin phosphotransferase; HPRT, hypoxanthine phosphoribosyltransferase; loxP (black arrowhead), a 34-bp recombination site from phage P1. (Reprinted, with permission, from Rossi et al, 2001 (copyright EMBO Journal) (b) Coding and non-coding exons of Prnp, Prnd and intergenic exons of unknown function (c) Exon skipping leads to expression of Doppel under the direction of the Prnp promoter. Deletion of the splice receptor site upstream of the third Prnp exon entails the formation of several chimeric mRNAs comprising the first two exons of Prnp, which are spliced directly or indirectly to the Dpl-encoding exon14. (d) Comparison of domains of Dpl with full-length PrP and with PrP lacking residues 32 to 134. (b–d) Reprinted, with permission, from Weissmann and Aguzzi56 (copyright Science).

 
PrP knock-out by the ‘radical strategy’ involves deletion of not only the reading frame, but also of its flanking regions, in particular the splice acceptor site of the third exon (Fig. 1a)13. This type of PrP knock-out mouse also develops normally, but exhibits severe ataxia and Purkinje cell loss in later life13–16.

The phenotype of PrP knock-out mice
 Prnpo/o (Zürich I) or Prnp-/- (Edinburgh) mice develop and reproduce normally11,12; ageing mice show demyelination in the peripheral nervous system, albeit without clinical symptoms17. Behavioural studies revealed no significant differences to wild-type mice11,18 except for alterations in circadian activity and sleep rhythms19,20. Electrophysiological studies showed that GABA-A receptor-mediated fast inhibition was weakened, long-term potentiation was impaired21–24, and Ca2+-activated K+ currents were disrupted in some cells25,26. Biochemical changes reported for Zürich-I-type knock-out mice suggest impairment of enzymatic activity required for anti-oxidant defence27–29.

In contrast, mice with extensive deletions in the Prnp gene (Prnp-/- [Nagasaki], Rcm0, Prnp-/- [Zürich II]) exhibit severe ataxia and Purkinje cell loss in later life13–16. Because this phenotype was abolished by introduction of a PrP transgene, it was attributed17 to the absence of PrPC. However, because cerebellar symptoms were not observed in the Zürich I and Edinburgh knock-out lines, they were likely caused not by the absence of PrP but rather by the deletion of sequences flanking the PrP ORF30. The discovery of Prnd and the ectopic expression of its product, Doppel (Dpl), in the brains of all three ataxic PrP knockout lines, Nagasaki, Zürich II and Rcm0, led to the resolution of this conundrum14.

Dpl is an N-glycosylated, GPI-anchored protein15,31 normally expressed in many tissues but not in post-natal brain14. Dpl and PrP show about 25% sequence similarity14 and share similar globular domains15,31,32. However, Dpl lacks a counterpart to the flexible amino-terminal segment of PrP (Fig. 1d). Its physiological function is still unknown, but its absence causes male sterility in mice33.

Dpl is encoded by Prnd, located 16 kb downstream of Prnp14. In brain of wild-type mice, with Prnd transcription under control of its own promoter, Dpl is not detected. As consequence of the radical PrP gene deletion strategy, the acceptor splice site of the third Prnp exon is lost, causing exon skipping and formation of chimeric transcripts that place Prnd transcription under control of the Prnp promoter (Fig. 1c)14,34. Time to appearance of disease is inversely correlated with the expression level of Dpl in brain16, and the phenotype is rescued by co-expression of PrPC16,17. Thus, ectopic expression of Dpl in the absence of PrPC, rather than absence of PrPC itself causes Purkinje cell loss in Nagasaki-type PrP knock-out mice.

How does Dpl cause damage in the brain? It has been proposed that overexpression of Dpl entrains an increase of haem oxygenase 1 and both neuronal and inducible NO synthase, causing oxidative stress deleterious to sensitive neurons; this effect would be counteracted by the antioxidant properties of PrPC35. Another explanation is discussed below, in connection with the pathogenic properties of an amino-proximally truncated PrP that resembles Dpl in some respects14,15.

PrP knock-out mice are resistant to scrapie
 Prnp+/+ mice inoculated intracerebrally with mouse-adapted scrapie prions develop clinical symptoms at about 160 days and die about 10 days later (Fig. 2A). In contrast, Prnpo/o mice remained free of clinical symptoms for at least 2 years and showed no scrapie-specific pathology as late as 57 weeks after inoculation36. Moreover, Prnpo/o [Zürich I] mice challenged with scrapie prions failed to propagate prions in brain and spleen, whereas prion levels in brain and spleen of Prnp+/+ mice increased to about 8.6 and 6.9 log LD50 units/ml, respectively, by 20 weeks’ post-infection36. Rarely, low-level infectivity was detected in the brains of PrP knock-out mice after intracerebral inoculation, perhaps due to residual inoculum or, less likely, to contamination37,38.



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  		Fig. 2 Survival, prion titres and PrPSc in brains of scrapie-inoculated mice. (A) Wild-type and (B) Prnpo/+ mice at various times after inoculation with mouse prions. Although both types of mice have similar levels of prions and PrPSc at 20 weeks after infection, the wild-type mice succumb within the following 8 weeks, while Prnpo/+ mice survive for another 30 weeks or longer without clinical symptoms. (Reprinted, with permission, from Büeler et al39 (copyright Molecular Medicine).

 
Properties of mice hemizygous for the Prnpo allele
 Mice carrying a single Prnp allele (Prnpo/+ mice) had prolonged incubation times of about 290 days to appearance of disease, as compared to 170 days in the case of Prnp+/+ mice. The Prnpo/+ mice harboured high levels of infectivity and PrPSc by 140 days after inoculation, as did wild-type controls, but survived thereafter for at least another 140 days without showing severe clinical symptoms39. PrP gene dosage affects the timing of disease, but not the final pathology. It has been suggested that the pathological processes must extend to a so-called ‘clinical target area’ before disease and death ensue40. In several instances, prion disease leads to death without substantial accumulation or even detectable levels of PrPSc in the brain41–45, and discrete changes in the postulated target area, which might be in the brain stem or upper spinal cord44, have not been routinely searched for. It is of practical interest that under certain conditions clinically healthy mice can harbour high levels of infectivity for long periods of time46–50, because it suggests that apparently healthy cattle or humans could contain infectious agent, a point to consider when animal or human tissues are used for preparing pharmaceuticals or grafts, or blood is transfused.


   Transgenesis and gene replacement
Top
 Abstract
 Generation and properties of...
 Transgenesis and gene...
Effect of PrP mutations...
 Pathological mouse phenotypes...
 Ectopic expression of PrP...
 Concluding remarks
 Acknowledgements
 References
 
Modifications of the genome may be achieved by transgenesis (i.e. insertion of cloned genes) or by in situ gene replacement using homologous recombination in embryonic stem cells. Gene insertion is usually based on nuclear injection into a one-cell embryo of a DNA segment containing the ORF, which may be mutated in the coding region or placed under the control of a foreign promoter in order to target expression to specific tissues. The DNA segment usually concatenates prior to integration, resulting in random insertion of multiple gene copies into one or few sites of the genome; therefore, each resulting transgenic mouse line is unique. Depending on the site of insertion, expression of the transgene can be silenced or modulated by neighbouring regulatory elements51,52.

In situ gene replacement is based on homologous recombination and allows modification of a resident gene in a variety of fashions. The normal gene copy number is preserved and the modified gene remains in its physiological environment and is not be influenced by alien enhancers or position-dependent silencing. A double replacement gene targeting strategy was used to introduce point mutations into the Prnp locus of a murine embryonic stem (ES) cell line lacking hypoxanthine phosphoribosyl transferase (HPRT). In the first step, the entire PrP ORF was replaced with an HPRT minigene by homologous recombination, and successfully targeted clones were isolated by selection in medium lacking HAT (hypoxanthine, adenine, thymidine) and containing gancyclovir, which is lethal for cells lacking HPRT. In the second step the HPRT minigene was replaced with a mutated PrP coding sequence, again by homologous recombination. Cells devoid of HPRT, selected by virtue of their resistance to 6-thioguanine, contained the mutated Prnp allele. Selected ES cell clones were injected into blastocysts and mice carrying the modified gene were obtained by appropriate breeding45,53–55.

Transgene vectors for PrP expression
 When a certain phenotype is generated by gene ablation – in this case resistance to scrapie – it is important to show that this is indeed the consequence of the targeted modification and not to some unintended event, such as obliteration of an enhancer governing another gene or disruption of an unidentified reading frame. The most effective, albeit not infallible (see below and Weissmann & Aguzzi56) strategy to link a phenotype to the ablation of a specific protein is to abrogate the effect of the deletion by introducing into the knock-out animal a cDNA encoding the protein in question.

It is desirable to reproduce the expression pattern of the wild-type gene as closely as possible but, as is the case for most mammalian genes, the location of all elements required for regulation of tissue-specific expression is not known. The 40-kb mouse cosmid I/InJ, derived from the murine Prnpb allele contains 6 kb of upstream sequence, the three exons and two introns, and approximately 18 kb of 3' downstream sequence that include Prnd14,57. Because such large cosmid vectors are laborious to work with, the ‘half-genomic PrP’ expression vector58 was constructed by deleting the large intron 2 and all but 2.2 kb of 3' flanking sequence. Thus, the half genomic PrP vector contains about 6 kb Prnpb promoter sequence controlling expression of PrP encoded by the Prnpa locus58 and lacks Prnd. The expression pattern elicited by the half-genomic construct is similar to that in cosmid-transgenic mice, except that cerebellar Purkinje cells express neither PrP nor PrP mRNA at detectable levels16,58. Perhaps the half-genomic construct lacks a Purkinje-cell-specific enhancer, which could be located in the large intron or in the distal part of the 3' non-coding region, both of which are absent in the half-genomic construct.

Restoration of susceptibility of Prnpo/o mice to prions by PrP transgenes
 Two lines of mice transgenic for the half-genomic Prnp construct, expressing PrP at about 3–4 and 6–7 times the level of wild-type mice, were challenged with mouse prions. As shown in Figure 2A, they succumbed to disease even more rapidly than wild-type CD-1 mice58, confirming that the incubation times are inversely related to PrP expression levels59.

These experiments support the conclusion that the scrapie-resistant phenotype of the Zürich I PrP knock-out mice is indeed due to ablation of PrP. However, it is worth emphasising the pitfalls that may beset the interpretation of knock-out experiments. In the case of the Nagasaki-type PrP knock-out mice described above, extensive deletions of the PrP-encoding exon gave rise to an ataxic phenotype that was reversed by introduction of an intact PrP-expressing transgene, the classical experiment correlating a phenotype with ablation of a gene. Nonetheless, in that case the conclusion was erroneous, because the ataxic phenotype did not result from the deletion of the PrP ORF but from the incidental up-regulation of a deleterious gene product whose pathogenicity was offset by wild-type PrPC56.


   Effect of PrP mutations on health and prion disease in the mouse
Top
 Abstract
 Generation and properties of...
 Transgenesis and gene...
 Effect of PrP mutations...
Pathological mouse phenotypes...
 Ectopic expression of PrP...
 Concluding remarks
 Acknowledgements
 References
 
The ability to reconstitute susceptibility to scrapie in mice devoid of PrP or to replace the wild-type gene by modified counterparts paved the way to the analysis of various consequences of modified PrP expression.

Delineation of PrP regions dispensable for susceptibility to prion disease
 Treatment of scrapie prion preparations with protease cleaves off about 60 amino-terminal residues of PrPSc but does not abrogate infectivity. To ascertain whether amino-terminally truncated PrPC could serve as substrate for the conversion to PrPSc and sustain susceptibility to scrapie in the mouse, transgenic Prnpo/o [Zürich I] mice expressing PrP that retained the signal sequence but had an amino-proximal deletion were challenged with scrapie prions. PrP knock-out mice overexpressing PrP with deletions from position 32 to 80 ({Delta}32–80)58 or to 93 ({Delta}32–93)60 yielded normal mice that, after intracerebral inoculation with scrapie prions, developed disease, propagated prions and exhibited protease-resistant, truncated PrPSc, albeit, in the case of the deletion to residue 93, with longer incubation times and with lower levels of infectivity and PrPSc44. PrP with a deletion extending to position 106 was unable to restore susceptibility to prions (E Flechsig, I Hegyi, A Aguzzi & C Weissmann, unpublished data).

Thus, at least 60 residues of the amino-proximal region of mature PrPC are expendable; they include not only the segment that is cleaved off PrPSc by proteinase K, but also the entire octarepeat region. This is remarkable because amplification of the octarepeat number is associated with familial CJD and GSS61,62. In addition to a partial deletion of the flexible tail ({Delta}23–88), the first {alpha}-helix ({Delta}141–176) could also be removed without abrogating its capacity to restore prion susceptibility to Prnpo/o mice. The resulting ‘PrP106’ contains only 106 amino acids, compared to the 208 residues in full-length PrP (Figure 3C)63.




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  		Fig. 3 Susceptibility to scrapie of PrP knock-out mice carrying various transgenes encoding full-length or truncated PrP. (A) Prnpo/o mice were rendered transgenic for Prnp genes. tga19/+ mice had 3–4 times the normal PrPC level; tga20/+ mice had 6–7 times the normal level. Data from Fischer et al58.(B) Prnpo/o and Prnpo/+ mice with hamster PrP transgenes were inoculated with the Sc237 isolate of hamster prions. Arrow: one animal died spontaneously without scrapie symptoms and one was killed because of a tumour. Mice with a mouse PrP allele are completely resistant to hamster prions while the presence of hamster PrP in a PrP knock-out mouse renders them very susceptible. The additional presence of a mouse PrP allele reduces susceptibility to hamster prions, as evidenced by increased incubation times. Modified from Büeler et al36. (C) Susceptibility to scrapie prions of Prnpo/o mice expressing various Prnp transgenes. {alpha}, 3F4-tag; *relative to wild-type. References: (a) Fischer et al58; (b) Flechsig et al44; (c) E Flechsig, I Hegyi, A Aguzzi, C Weissmann, unpublished results; (d) Supattapone et al63.

 
PrP mutations affecting the ‘species barrier’
 In many cases, prions originating in one species fail to elicit disease in a different species or do so only inefficiently and/or after a long incubation time. This phenomenon is attributed to a ‘species barrier’64; however, at least in the case of mice inoculated intracerebrally with hamster prions, PrPSc and infectivity may accumulate in the brain despite the absence of clinical symptoms46,47, so that the barrier is not absolute. In many cases, introduction into mice of PrP transgenes containing all or part of the PrP sequence of the prion donor overcomes the species barrier. Thus, Prnp+/+ mice transgenic for hamster PrP transgenes are susceptible to both mouse and hamster prions59,65 while Prnpo/o mice containing Syrian hamster PrP transgenes are susceptible to hamster but not mouse-derived prions; interestingly, the presence of a mouse PrP allele diminished the susceptibility to hamster prions (Fig. 2B)36. Similarly, Prnpo/o mice expressing human transgenes were susceptible to human sCJD prions, while Prnp+/+ mice with the same transgene cluster were as resistant to human prions as wild-type mice66,67. Surprisingly, transgenic mice overexpressing human PrP are less susceptible to human variant CJD (vCJD) than wild-type mice68. Prnpo/o mice transgenic for bovine PrP genes are susceptible to bovine69,70 as well as to vCJD and sheep scrapie prions, while mice containing ovine PrnpVRQ or PrnpARQ transgenes showed vastly decreased incubation times for sheep scrapie prions as compared to wild-type mice71,72.

Inasmuch as a decrease in incubation time is viewed as a lowering of the ‘species barrier’, it is noteworthy that this can be achieved by mutations in murine PrP that do not increase the similarity to the PrP of the prion donor73. Polymorphisms in the PrP gene have been shown to alter incubation time and susceptibility to prion disease in mice54, sheep74, and man75,76, but the mechanism by which this is achieved is not known. Certain polymorphisms of the ovine PrP gene confer resistance to scrapie, raising the possibility of preventing sheep scrapie by breeding resistance alleles into susceptible sheep flocks77.


   Pathological mouse phenotypes elicited by PrP mutations
Top
 Abstract
 Generation and properties of...
 Transgenesis and gene...
 Effect of PrP mutations...
 Pathological mouse phenotypes...
Ectopic expression of PrP...
 Concluding remarks
 Acknowledgements
 References
 
Unexpectedly, various PrP mutations have given rise to pathological phenotypes when overexpressed in mice. For example, Prnpo/o mice expressing PrP with deletions of the flexible tail extending to amino acid 121 or 134 developed severe ataxia and apoptosis of the cerebellar granule cell layer as early as 1–3 months of age [Plate VII(A)]60. Neurons in the cortex and elsewhere expressed truncated PrP at similar levels as granule cells but did not undergo cell death, arguing against an unspecific toxic effect. Strikingly, the pathological phenotype was completely abolished by the introduction of a single wild-type Prnp allele, although the level of truncated PrP remained unchanged, exceeding that of the wild-type counterpart. When the truncated PrP was specifically targeted to Purkinje cells of Prnpo/o [Zürich I] mice using the L7 promoter, ataxia and Purkinje cell degeneration developed, while the cerebellar granule layer remained unaffected [E Flechsig et al, EMBO J 2003 (In press); Plate VII(B)]. The phenotype resembled that observed in the three lines of Nagasaki-type PrP knock-out mice with up-regulation of Dpl in brain, which was also abrogated by PrP13,16,17. Because the overall structure of Dpl is remarkably similar to that of the globular domain of PrP lacking the flexible N-terminus, the mechanism of pathogenesis might be the same in both cases14,56. The results have been explained by a model in which truncated PrPC acts as dominant negative inhibitor of a functional homologue of PrPC, with both competing for the same putative PrPC ligand60.




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  		Plate VI Dpl is required for late stages of spermatogenesis. Histological analysis of wild-type and Prndneo/neo testes (A,B). Arrows in A indicate Dpl immunoreactivity. (C,D) Bright field images of spermatozoa isolated from Prnd+/+ (C) and Prndneo/neo mice (D). (E,F) Spermatozoa isolated from Prnd+/+ (E) and Prndneo/neo mice (F) were stained with mitotracker to detect mitochondria (in green) and with the DNA stain Hoechst (in blue).

 
Mice expressing PrP with the octarepeats (PrP{Delta}23–88) deleted or even, in addition, lacking {alpha}-helix-1 and ß-sheet-2 ({Delta}141–176, ‘PrP106’) remained healthy78. However, deletion of either {alpha}-helix-2 ({Delta}177–202) or {alpha}-helix-3 ({Delta}201–217) strongly affected correct PrP folding and caused a lethal illness resembling neuronal storage disease78.

In cultured cells, mature PrPC is subject to retrograde transport to the cytosol and degradation by proteasomes. Accumulation of even small amounts of cytosolic PrP was strongly neurotoxic to cultured cells79. Wild-type mice transgenic for murine PrP23–230, which promotes accumulation of cytosolic PrP, developed severe ataxia, with cerebellar degeneration and gliosis80.

In an attempt to generate spontaneous prion disease in transgenic mice, Scott et al substituted Ala by Val at positions 113, 115 and 118 of PrP (‘AV3’) to promote de novo ß-sheet formation in the flexible tail81. Founders indeed developed a fatal neurological disorder82; however, this was not transmissible and no protease-resistant PrP was detected81.

Transgenic mouse models of inherited prion diseases
 More than 20 mutations of the PrP gene have been identified in families suffering from inherited human prion diseases. It has been speculated that mutations in the PrP gene give rise to an unstable PrPC protein that can spontaneously convert into the abnormal conformer, PrPSc, whereas sporadic forms of the disease are attributed to rare spontaneous transitions of PrPC into PrPSc or to somatic mutations in the PrP. Studies on thermodynamic stability of the C-terminal domain of PrP with those mutations do not generally support the concept of mutation-induced PrPC destabilisation83,84. Mice transgenic for mutant PrPs associated with familial human prion diseases have failed to develop transmissible disease, with the possible exception of PrPP101L.

Mice overexpressing murine PrPP101L (the counterpart of human PrPP102L linked to familial GSS) 8-fold, on a Prnpo/o background, spontaneously developed neurodegeneration at 140 days of age, but little, if any, PrPSc could be detected85. The disease could not be transmitted to wild-type mice, but only to transgenic mice that expressed the same mutation at low levels (2-fold) and developed neurodegeneration only late in life, if at all85–87. It was suggested that the amino acid difference constituted a barrier to transmission to wild-type mice. However, human P102L-linked GSS can be transmitted to both monkeys and wild-type mice88,89. It has been argued that the P101L mutation may be an important susceptibility factor rather than a direct cause of GSS90.

Mice expressing PrP with the counterparts of the inherited CJD-linked mutations at T183A or E199K on a PrP null background did not develop any pathological signs85,91. Expression of a mouse PrP version of a nine-octapeptide insertion associated with ‘prion dementia’ in humans92 produced a slowly progressive cerebellar disorder and progressive myopathy93. Because the disease was not transmissible it qualifies as a ‘proteinopathy’ rather than a ‘prion disease’.

Thus, the human familial prion diseases have not been modelled successfully in the mouse.

Transmissible versus non-transmissible PrP-linked disease
 Many mutant forms of murine PrP are pathogenic, mostly or perhaps only when overexpressed. It is not surprising that neurons accumulating abnormal forms of protein may suffer damage. With the possible exception85 of the neurodegeneration associated with highly overexpressed PrPP101L, none of the PrP-linked diseases in mice were claimed to be transmissible. It is, therefore, appropriate to distinguish between ‘prion diseases’, which are transmissible, and ‘non-transmissible conformational diseases’, or ‘non-transmissible proteinopathies’ which are not88.

In the case of human ‘prion diseases’, transmission to experimental animals has been achieved for some, but not all, familial cases88. Doubtless, in any particular instance, lack of transmission may be due to inadequate recipients, but in view of the results with mice, it is possible that at least some of the familial human spongiform encephalopathies may be truly non-transmissible conformational diseases and, therefore, in view of the definition of prions as transmissible agents, not ‘prion diseases’.


   Ectopic expression of PrP transgenes
Top
 Abstract
 Generation and properties of...
 Transgenesis and gene...
 Effect of PrP mutations...
 Pathological mouse phenotypes...
 Ectopic expression of PrP...
Concluding remarks
 Acknowledgements
 References
 
Several questions regarding the cell specificity of prion propagation have been addressed by generating mice that express PrP in only one type of tissue or organ, rather than almost ubiquitously, as in wild-type mice. This was achieved by introducing the PrP ORF linked to tissue- or organ-specific promoters into PrP knock-out mice.

Expression of PrP controlled by the astrocyte-specific glial fibrillary acid protein (GFAP) promoter in PrP knock-out mice rendered the animals susceptible to prions and led to clinical disease and prion propagation. It would thus seem that astrocytes are competent for prion replication. Interestingly, the neuropathology exhibited by these transgenic mice is quite similar to that found in scrapie-infected wild-type mice50.

In most mouse scrapie models, infectivity appears in the spleen within days after intracerebral inoculation and rises slowly throughout the life-time of the mouse, whereas infectivity in the brain starts rising a few weeks after inoculation and reaches titres two orders of magnitude higher than in the spleen36,94,95. In spleen, PrPSc is found mainly in follicular dendritic cells (FDCs)96,97. Fractionation of scrapie-infected spleen showed that infectivity was associated with B- and T-lymphocytes and with the FDC-containing stromal fraction, but not with neutrophils. Surprisingly, no infectivity was detected in circulating lymphocytes98. This raised the question as to whether B- and T-cells were able to propagate prions or whether they acquired them from another source. Transgenic mice overexpressing PrP under the control of the T-cell-specific lck promoter99 showed high levels of PrP in T-cells, both in thymus and spleen but not in brain. Intraperitoneal inoculation led to no symptoms and no infectivity was detected in spleen, thymus or brain up to one year after inoculation100. Transgenic mice overexpressing PrP under control of the B-cell-specific CD19 promoter had 10–20-fold higher PrP levels on B-cells compared to wild-type mice101, yet no pathology or prion propagation was observed after intraperitoneal inoculation with scrapie prions. Thus, the presence of PrP does not suffice to support prion replication; maybe location within a particular plasma membrane region is required102 and/or other components are necessary. Moreover, it follows that prions associated with splenic T- and B-lymphocytes must stem from another source, almost certainly FDCs103,104.

To determine whether spread of prions from the periphery to the CNS is dependent on hematogenous or lymphoreticular tissue (LRS), PrP knock-out mice transgenic for hamster PrP under control of the neuron-specific enolase (NSE) promoter, which express PrP in brain and nerves but not in spleen, lymph nodes or bone marrow, were inoculated with hamster prions orally or intraperitoneally. In both cases, scrapie disease ensued with the same incubation time as in mice expressing hamster PrP under the control of the PrP promoter. Thus, at least after peripheral exposure to high doses of 263K hamster prions, PrPC-expressing LRS was not required for prion amplification or transport105.


   Concluding remarks
Top
 Abstract
 Generation and properties of...
 Transgenesis and gene...
 Effect of PrP mutations...
 Pathological mouse phenotypes...
 Ectopic expression of PrP...
 Concluding remarks
Acknowledgements
 References
 
Research on transmissible spongiform encephalopathies went through three phases. The first was dominated by clinical and biological research, including the recognition of transmissibility and the unusual nature of the transmissible agent. In the second phase, biochemistry and recombinant DNA technology led to the isolation of PrPSc from scrapie-infected brain, to the recognition that it is a conformational isomer of the naturally occurring, host-encoded PrPC and to the reformulation of the ‘protein only’ hypothesis. The deepest insights into the central role of PrP in the spongiform encephalopathies, or prion diseases as they came to be called, were made possible by the transgenic and knock-out technologies described above.


   Acknowledgements
Top
 Abstract
 Generation and properties of...
 Transgenesis and gene...
 Effect of PrP mutations...
 Pathological mouse phenotypes...
 Ectopic expression of PrP...
 Concluding remarks
 Acknowledgements
References
 
The Zürich/London prion groups are indebted to M Aguet, A Behrens, T Blättler, H Blüthmann (Hoffmann LaRoche, Basel), HR Büeler, S Brandner, A Cozzio, M Fischer, M Glatzel, I Hegyi, MA Klein, Y Kobayashi, H-P Lipp (Anatomisches Institut, Universität Zürich), F Montrasio, D Rossi, T Rülicke (Biologisches Zentrallabor, Universitätsspital Zürich), M Prinz, A Raeber, A Sailer, S Sakaguchi, D Shmerling, and P Schwarz for their valuable contributions.


   Footnotes
 
Correspondence to: Prof C Weissmann, MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, Queen Square, London WC1N 3BG, UK


   References
Top
 Abstract
 Generation and properties of...
 Transgenesis and gene...
 Effect of PrP mutations...
 Pathological mouse phenotypes...
 Ectopic expression of PrP...
 Concluding remarks
 Acknowledgements
 References
 

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