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Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

2001; Springer Nature; Volume: 20; Issue: 4 Linguagem: Inglês

10.1093/emboj/20.4.694

1460-2075

Daniela Rossi,

RNA Research and Splicing

Article15 February 2001free access Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain Daniela Rossi Daniela Rossi MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary's, London, W2 1PG UK Search for more papers by this author Antonio Cozzio Antonio Cozzio Present address: Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Eckhard Flechsig Eckhard Flechsig MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary's, London, W2 1PG UK Search for more papers by this author Michael A. Klein Michael A. Klein Institut für Neuropathologie, 8091 Zürich, Switzerland Search for more papers by this author Thomas Rülicke Thomas Rülicke Biologisches Zentrallabor, Universitätsspital Zürich, 8091 Zürich, Switzerland Search for more papers by this author Adriano Aguzzi Adriano Aguzzi Institut für Neuropathologie, 8091 Zürich, Switzerland Search for more papers by this author Charles Weissmann Corresponding Author Charles Weissmann MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary's, London, W2 1PG UK Search for more papers by this author Daniela Rossi Daniela Rossi MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary's, London, W2 1PG UK Search for more papers by this author Antonio Cozzio Antonio Cozzio Present address: Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305 USA Search for more papers by this author Eckhard Flechsig Eckhard Flechsig MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary's, London, W2 1PG UK Search for more papers by this author Michael A. Klein Michael A. Klein Institut für Neuropathologie, 8091 Zürich, Switzerland Search for more papers by this author Thomas Rülicke Thomas Rülicke Biologisches Zentrallabor, Universitätsspital Zürich, 8091 Zürich, Switzerland Search for more papers by this author Adriano Aguzzi Adriano Aguzzi Institut für Neuropathologie, 8091 Zürich, Switzerland Search for more papers by this author Charles Weissmann Corresponding Author Charles Weissmann MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary's, London, W2 1PG UK Search for more papers by this author Author Information Daniela Rossi1, Antonio Cozzio2, Eckhard Flechsig1, Michael A. Klein3, Thomas Rülicke4, Adriano Aguzzi3 and Charles Weissmann 1 1MRC Prion Unit/Neurogenetics, Imperial College School of Medicine at St Mary's, London, W2 1PG UK 2Present address: Department of Pathology, Stanford University School of Medicine, Stanford, CA, 94305 USA 3Institut für Neuropathologie, 8091 Zürich, Switzerland 4Biologisches Zentrallabor, Universitätsspital Zürich, 8091 Zürich, Switzerland ‡D.Rossi and A.Cozzio contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:694-702https://doi.org/10.1093/emboj/20.4.694 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info PrP knockout mice in which only the open reading frame was disrupted (‘Zürich I’) remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, Prnd. A new PrP knockout line, ‘Zürich II’, with a 2.9 kb Prnp deletion, developed this phenotype at ∼10 months (50% morbidity). A single Prnp allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a Prnd-containing cosmid expressed Dpl at twice the level and became ataxic ∼5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated. Introduction PrP, the prion protein, plays a central role in the pathogenesis of transmissible spongiform encephalopathies such as scrapie or BSE. The normal form of PrP, designated PrPC, is encoded by a single-copy gene (Basler et al., 1986) and is expressed in the brain of healthy and prion-infected organisms to about the same extent (Chesebro et al., 1985; Oesch et al., 1985). Several PrP knockout lines have been generated that differ in the design of the gene disruption (Figure 1). The first two lines described, hereafter called Prnp0/0 Zürich I and Prnp−/− Edinburgh, developed and reproduced normally (Büeler et al., 1992; Manson et al., 1994). In Prnp0/0 Zürich I mice, PrP codons 4–187 (72% of altogether 254 codons) were replaced by a neomycin (neo) cassette. Mice homozygous for the disrupted gene expressed transcripts containing the neo and the residual Prnp sequence in the brain, but no PrPC-containing protein was detected. In the Prnp−/− Edinburgh mice the PrP gene was disrupted by the insertion of a neo cassette into a unique KpnI site following residue 93 of the PrP open reading frame (ORF). No PrP mRNA or PrP-related protein was detected in the brain of homozygous Prnp−/− Edinburgh animals. Figure 1.Strategies used to create various PrP knockout lines. The black boxes represent PrP ORF; white boxes, non-coding Prnp regions; grey boxes, sequences inserted into the gene; dotted line, deleted regions of Prnp. neo, neomycin phosphotransferase; HPRT, hypoxanthine phosphoribosyltransferase; loxP (black arrowhead), a 34 bp recombination site from phage P1. Download figure Download PowerPoint In a third PrP knockout line, here designated Prnp−/− Nagasaki, a 2.1 kb genomic DNA segment comprising 0.9 kb of intron 2, 10 bp of 5′ non-coding region, the entire PrP ORF and 0.45 kb of the 3′ non-coding region, was replaced with a neo cassette (Sakaguchi et al., 1996). These mice developed normally but exhibited severe ataxia and Purkinje cell loss in later life (Sakaguchi et al., 1996) as well as demyelination of peripheral nerves (Nishida et al., 1999). Because this phenotype was abolished by introduction of a PrP transgene (Nishida et al., 1999) it was concluded that both ataxia and peripheral nerve degeneration were due to the absence of PrP. A fourth line, Rcm0, was cited in Moore et al. (1999) and Silverman et al. (2000) as resembling the Nagasaki line with respect to the extensive PrP gene deletion and the ataxic syndrome. Recently a gene, Prnd, was found 16 kb downstream of the murine PrP gene, which comprises an ORF encoding a 179-residue protein (Moore et al., 1999). The predicted protein, which was named Doppel (Dpl), has ∼25% identity with the C-terminal two-thirds of PrP. Prnd-derived mRNA is expressed from a promoter upstream of exon 1 of Prnd at relatively high levels in testis and heart but at very low levels in brain of wild-type mice. However, in Nagasaki and Rcm0 mice, but not in Zürich I mice, Prnd-specific RNAs were present at relatively high levels in brain. These transcripts originate at the Prnp promoter, run beyond the Prnd ORF and are processed by one or more splicing events that link the 3′ end of the second PrP exon directly or indirectly to the Dpl-encoding exon (Moore et al., 1999; Li et al., 2000). Dpl has been identified as an N-glycosylated, GPI-linked protein in testes of normal, and in brain of Rcm0 PrP knockout, mice carrying the extensive PrP gene deletion (Silverman et al., 2000). The cerebellar syndrome was therefore attributed to the ectopic expression of Dpl in the brain (Moore et al., 1999; Li et al., 2000). Here, we describe the generation of a further PrP knockout line, hereafter called Prnp−/− Zürich II, in which the PrP-encoding exon and its flanking regions were replaced by a loxP site. The homozygous mice, like their Prnp−/− Nagasaki counterparts, developed progressive ataxia and age-dependent Purkinje cell loss starting at 5–6 months, with half the mice affected by ∼10 months, and showed ectopic expression of two Prnd-derived mRNAs and Dpl in brain. A single wild-type PrP allele fully corrected the deleterious phenotype in agreement with previous reports (Sakaguchi et al., 1996; Nishida et al., 1999). The F1 offspring of a cross between the Zürich I and Zürich II lines showed partial complementation, in that they remained healthy 6 months longer than the Zürich II mice, despite the absence of a PrP ORF. The level of Prnd-derived mRNAs and Dpl in brain was half that in Zürich II mice. Introduction into Zürich II mice of a cosmid comprising both the Prnd gene and a Prnp locus lacking the PrP ORF resulted in accelerated development of the ataxic phenotype and was associated with increased levels of Prnd-derived transcripts and Dpl in brain. These results suggest that the allele with deletions extending beyond the PrP ORF is pathogenic in a dose-dependent fashion and that this pathogenicity is due to ectopic expression in brain of Dpl (Moore et al., 1999; Li et al., 2000) rather than to the absence of PrP. N-proximally truncated PrP, which resembles Dpl, also causes ataxia that, as in the case of ectopic Dpl expression, is counteracted by full-length PrP (Shmerling et al., 1998). Results Mice carrying a deletion of the PrP ORF and its flanking sequences develop a cerebellar syndrome We generated a PrP-deficient mouse line by gene targeting in embryonic stem cells, as described in Materials and methods and in Figure 2A. In this mouse the wild-type PrP allele was replaced by the Prnp lox1 allele (to be referred to hereafter as Prnp− Zürich II allele), in which 0.26 kb of intron 2, 10 bp of the 5′ flanking sequence, the entire ORF, the 3′ non-coding region of exon 3 and 0.6 kb of 3′ adjacent sequence were replaced by a 34 bp loxP sequence. Figure 2.Strategy for production of Prnp−/− Zürich II knockout mice and Cos-tTA transgenic mice. (A) Deletion of PrP exon 3 and its flanking regions. The targeting vector PrP lox3, containing the loxP-flanked positive–negative selection cassette HSV-tk/PGK-neo and a loxP site downstream of exon 3, was introduced by homologous recombination. Cre-mediated recombination yielded either allele I, in which only the selection cassette was deleted, or II, where both the selection cassette and exon 3 and its flanking regions were removed. Probes (X/N; Δ) and PCR primers (ΔUp3; TK; P5; Nco; 3′loxP3) used for detection of homologous recombinants, Cre-deleted alleles and for northern blot hybridizations are indicated. Xh, XhoI; Xb, XbaI; Nc, NcoI; Bs, BstEII; Ba, BamHI; K, KpnI; No, NotI. (B) Cos-tTA was derived from cos6.I/LnJ-4, which contains the Prnp and Prnd loci, by replacing the PrP ORF in exon 3 by the tTA ORF. Black boxes, Prnp exons; white box, tTA ORF; hatched boxes, intergene exons; dark grey boxes, Prnd non-coding regions; light grey box, Dpl ORF. tTA and Dpl are probes used for Southern and northern analyses; TA-2 and 3′Mfe are primers used for PCR analysis of tail DNA. (C) Southern blot of tail DNA derived from the F1 progeny of a chimeric founder that had Prnp lox2 (see AI), Prnp lox1 (see AII) and wild-type alleles in the germline. Ten micrograms of DNA were digested with XbaI and hybridized with probe X/N. (D) Northern blot of total brain RNA (10 μg) from wild-type, Zürich I (ZH I) and Zürich II (ZH II) knockout mice, hybridized with probe Δ (Büeler et al., 1992) and, after stripping, with GAPDH probe, as described in Materials and methods. (E) Western blot of total brain homogenate (60 μg total protein) of wild-type, ZH I and ZH II mice. PrP was detected with monoclonal antibody 6H4. Download figure Download PowerPoint Figure 3.Development of cerebellar syndrome in ageing Prnp−/− Zürich II mice. (A) Footprints of wild-type (Prnp+/+), Zürich I (Prnp0/0 ZHI) and Zürich II (Prnp−/− ZHII) mice at 11–12 months of age. The hind feet were dipped in ink and the animals prompted to walk on filter paper. Zürich II mice are unable to walk straight and show a generally reduced step length compared with Zürich I and wild-type animals. (B) Time-course of the cerebellar symptoms (intention tremor and trembling gait) in Zürich II (ZH II) mice (n = 63), F1 of Zürich I × Zürich II (ZH I/ZH II) mice (n = 13), Zürich II mice transgenic for cosmid Cos-tTA (Cos-tTA/ZH II) (n = 23) or a Tga20 allele (Tga20/ZH II) (n = 17) and Zürich II mice containing a single wild-type allele (Prnp+/− ZH II) (n = 3 for observation over 20 months; n = 19 for observation over 7–8 months). Download figure Download PowerPoint Mice carrying the Prnp− Zürich II allele were bred to homozygosity; hemizygous Zürich II (Prnp−/+) and homozygous Zürich II (Prnp−/−) mice were born in the ratio expected from Mendelian inheritance. In brains of Prnp−/− Zürich II mice no PrP-specific mRNA was found (Figure 2D), nor could PrP be detected by immunoblotting (Figure 2E) or immunohistochemistry (Figure 4B). Figure 4.Histological and histochemical analysis of cerebellar cortex of wild-type and PrP knockout mice at different ages. (A) The top row shows parasagittal sections of wild-type, Zürich I (ZH I), Zürich II (ZH II) and F1 progeny of Zürich I × Zürich II (ZH I/II) mice stained with HE. Scale bar, 1 mm. The middle row shows cerebellar cortical sections stained for calbindin (specific for Purkinje cells). Wild-type and ZH I mice show an intact Purkinje cell layer at 62 and 80 weeks, whereas ZH II mice present extensive cell loss at 63 weeks. ZH I/II mice show progressive Purkinje cell loss from 63 to 95 weeks. Scale bar, 70 μm. The sections were stained on different occasions, resulting in different intensities. The bottom row shows GFAP staining of the cerebellar cortex with slight astrocytosis in ZH II and ZH I/II mice at the latest time points. Scale bar, 70 μm. (B) Immunohistochemical staining for PrP reveals high PrP levels in Purkinje cells and lower levels in the granule cell and molecular layers of wild-type mice. No PrP is detected in ZH I and ZH II tissue. Tga20/ZH II mice show high PrP levels in the molecular and granule cell layers, but not in Purkinje cells and their dendritic trees. Scale bar, 25 μm. Download figure Download PowerPoint Prnp−/− Zürich II mice were normal until ∼6 months of age, when they began to develop intention tremor and trembling gait (Figure 3A); this phenotype, which was taken as criterion for diagnosis of the cerebellar syndrome, was present in 50% of the mice by 10 months, and in 100% by 13 months (n = 63) (Figure 3B). Progression of the disease led to hypotony of the hind limbs and impaired equilibrium on a moving surface (Crawley and Paylor, 1997) as compared with wild-type animals (P.Valenti and A.Cozzio, data not shown). The hindlegs seemed more affected than the forelegs. Histological analysis revealed a severe age-dependent loss of Purkinje cells in Prnp−/− Zürich II mice (Figure 4A), which was most prominent in the vermis regions I–VIII (Figure 5), the simple lobule, crus I and II ansiform lobules, paramedian lobule and copula pyramis, while vermis regions IX and X were less affected (data not shown). The average cell loss in vermis I–VIII was ∼40 and 80% at 30 and 63 weeks, respectively. Concomitant with the gaps in the monolayer of Purkinje cells, the thickness of the molecular layer in these areas was reduced, probably due to the loss of the dendritic trees of the Purkinje cells. Importantly, the granular cell layer as the major afferent source of the Purkinje cells showed no alterations that could be held responsible for secondary damage of their target cells. Figure 5.Purkinje cell counts in PrP null and wild-type mice. Purkinje cells were counted in parasagittal sections stained with calbindin. Each bar represents the average cell count per mm in vermis I–VIII. The cell numbers in Zürich II (ZH II) mice were reduced as compared with Zürich I (ZH I) mice and wild-type mice at 30 weeks (P < 0.1) and strongly reduced at 63 weeks (P < 0.05). In ZH II mice transgenic for the tTA cosmid (Cos-tTA/ZH II), reduction was already apparent at 6 weeks (P < 0.1). In F1 offspring of a Zürich I × Zürich II cross (ZH I/II) and in Zürich II mice transgenic for a Tga20 allele (Tga20/ZH II), reduction in cell numbers, as compared with wild type, only became significant after 90–95 weeks (P < 0.05). The statistical significance of Purkinje cell loss was analysed separately for each genotype by non-parametric statistics (Kruskal–Wallis test, followed by the Mann–Whitney U-test). The age at which mice were killed and the number of mice examined are indicated at the bottom of the figure. Download figure Download PowerPoint Prnp0/0 Zürich I mice showed no ataxia or Purkinje cell loss up to at least 80 weeks (Figures 4 and 5). Mice homozygous for the Prnp lox2 allele (in which the PrP reading frame and surrounding regions were flanked by loxP sequences) were derived from the same founder as the Prnp−/− Zürich II mice, as explained in Materials and methods. These animals also remained healthy for at least 57 weeks and showed no Purkinje cell loss (data not shown). Northern analysis of 5 μg of poly(A)+ mRNA from Zürich II mouse brains showed two Prnd-specific RNAs of ∼4.3 and 2.7 kb, with about the same intensity as the bands from the same amount of poly(A)+ mRNA from testis, which were slightly smaller, 3.9 and 2.2 kb (Figure 6A). The molecular weights are similar to those determined by Li et al. (2000), 3.4 and 2.2 kb, but considerably higher than those reported by Moore et al. (1999), 2.7 kb and 1.7 kb for both brain and testis, perhaps because of discrepancies in the molecular weight determinations. No Prnd-specific bands were detected in wild-type brain. Prnp0/0 Zürich I brain showed a single weak band at ∼3.4 kb, with ∼1/20th the intensity of the sum of the two bands in Zürich II brain. Its origin was not investigated further, but the transcripts may reflect synthesis from the Prnd promoter (Moore et al., 1999). Figure 6.Analysis of Dpl mRNA and protein levels in testis of wild-type and in brain of wild-type and PrP knockout mice. (A) Northern blot analysis was performed on poly(A)+ mRNA using a labelled Dpl ORF probe. After stripping, the membrane was re-hybridized with a GAPDH probe. For quantification in brain (bottom panel), the value resulting from the sum of both Dpl transcripts was normalized relative to GAPDH. (B) Western blot analysis was performed on tissue homogenates as described in Materials and methods. Strain designations are as in Figure 5. Download figure Download PowerPoint Western analysis using a polyclonal antibody against recombinant Dpl (Figure 6B) reveals a strong Dpl band in brain of Zürich II mice, almost as intense as in testes of wild-type mice, but no detectable Dpl in Zürich I or wild-type brain (less than ∼5% of that in Zürich II brain). F1 offspring of a Zürich I × Zürich II cross have a mitigated ataxic phenotype Five breeding pairs (Prnp0/0 Zürich I × Prnp−/− Zürich II) yielded 33 F1 offspring with a compound heterozygote Prnp0/− Zürich I/II genotype; 13 of these were evaluated over the long term (Figure 3B). They remained healthy until 12 months of age and only then began exhibiting the same cerebellar symptoms as those seen in Prnp−/− Zürich II mice at 5–6 months. After 17 months, all F1 mice showed symptoms (Figure 3B). Histological analysis revealed that at 6 weeks both homozygous Prnp−/− Zürich II and compound heterozygous Prnp0/− Zürich I/II mice showed a normal laminar arrangement (Figure 5). At 30 weeks of age, the compound heterozygotes showed no statistically significant cell loss (average of vermis I–VIII, −15%), and at 63 weeks, when Prnp−/− Zürich II mice had lost 80% of their Purkinje cells, the heterozygotes showed an average loss of only 30%. At 95 weeks, ∼70% of the Purkinje cells were lost (Figures 4A and 5), with the exception of vermis IX–X where the loss was only ∼25% (data not shown). Offspring of intercrosses of compound heterozygous Prnp0/− Zürich I/II mice showed co-segregation of the cerebellar phenotype with the Zürich II allele. Among 28 offspring, six mice were homozygous Prnp0/0 Zürich I, nine were homozygous Prnp−/− Zürich II and 13 animals were compound heterozygous Zürich I/II. At 30 weeks of age, all seven homozygous Zürich II mice observed showed the cerebellar phenotype; two mice were killed for histological examination and showed ∼40% Purkinje cell loss (data not shown). The six Zürich I mice remained healthy for up to at least 48 weeks. As expected, the two Prnd-specific RNA bands (Figure 6A) as well as the Dpl protein bands (Figure 6B) in Zürich I/Zürich II brains had about half the intensity of those in Zürich II brain. A cosmid expressing Prnd-derived mRNAs but not PrP mRNA in brain of Zürich II mice causes accelerated appearance of the ataxic syndrome The DNA segment contained in Prnpb cosmid cos6.I/LnJ-4 extends from 19 kb upstream to 19 kb downstream of the PrP ORF and comprises the Prnd locus (Westaway et al., 1994; Moore et al., 1999). In connection with a different project, the PrP ORF was replaced with the 900 bp tet-dependent transactivator ORF (Gossen and Bujard, 1992; Furth et al., 1994; Schultze et al., 1996). Nuclear injection yielded founders transgenic for this modified cosmid, Cos-tTA, (A.Cozzio, D.Rossi and C.Weissmann, unpublished data). In order to determine whether an allele containing the flanking regions of the third PrP exon but not the PrP ORF could abrogate the ataxic syndrome, the cosmid transgene was bred into Zürich II mice. Unexpectedly, mice homozygous for the Zürich II (Prnp−) allele and containing Cos-tTA showed a much accelerated course of the disease: onset of ataxia was at ∼19 weeks (50% morbidity) (Figure 3B) and Purkinje cell loss in vermis I–VIII was 40 and 70% at 6 and 30 weeks, respectively (Figure 5). Expression of tTA at a 4-fold higher level in mice transgenic for a similar vector [mouse line Prnp-tTA/F959 (Tremblay et al., 1998)], which, however, does not contain the Prnd locus, has no deleterious effects up to at least 10 months of age (A.Servadio and D.Rossi, unpublished results). For reasons that are not immediately clear, brains of Zürich II mice containing the Prnd cosmid showed about twice the level of chimeric Prnd-derived mRNAs (Figure 6A) and Dpl protein (Figure 6B) compared with Zürich II brain. Maybe the replacement of the PrP ORF by a prokaryotic sequence impairs splicing to the acceptor site of the third exon (Lavigueur et al., 1993; Zandberg et al., 1995; Chiara et al., 1996; Dominski and Kole, 1996). Thus, there is again an inverse correlation between the time to appearance of ataxic symptoms and the level of Prnd-derived mRNAs and Dpl. Abrogation of the effects of the Zürich II allele by a single Prnp wild-type allele or a PrP-encoding transgene cluster Hemizygous Prnp−/+ Zürich II mice exhibited no cerebellar symptoms up to at least 100 weeks (Figure 3B). Because, as described above, a single Zürich II allele causes disease at ∼68 weeks (50% morbidity), we conclude that a single Prnp+ allele can abrogate the deleterious effects of the Prnp− Zürich II allele for practically the lifespan of the mouse. In a further experiment, Prnp−/− Zürich II mice were crossed with Tga20 mice. These mice carry multiple PrP transgenes devoid of the large intron on a Zürich I PrP knockout background and express PrP in most areas of the brain with the distinct exception of Purkinje cells (Fischer et al., 1996). The F1 progeny were back-crossed with Zürich II mice to eliminate the Zürich I allele. None of the resulting offspring presented clinical symptoms by 90 weeks of age (Figure 3B). There was no loss of Purkinje cells at 63 weeks and 40% loss at 90 weeks (Figure 5), showing that expression of PrP strongly mitigated the cerebellar phenotype even when it was not expressed in Purkinje cells at a discernible level (Fischer et al., 1996) (Figure 4B). Discussion Five independent PrP knockout mouse lines have been reported. Three of these show cerebellar symptoms and loss of Purkinje cells on ageing, namely the Prnp−/− Nagasaki mice (Sakaguchi et al., 1996), the Rcm0 mice of Moore et al. (Moore et al., 1999; Silverman et al., 2000) and the Prnp−/− Zürich II mice (this paper), while the Prnp−/− Edinburgh (Manson et al., 1994) and the Prnp0/0 Zürich I mice (Büeler et al., 1992) do not. The strategies used to abolish PrP differed in an important respect: in the lines remaining healthy, PrP expression was abrogated either by placing an insert within the PrP coding region (the Edinburgh mice) or by replacing the coding region between codons 3 and 188 by a neo cassette. In contrast, the Nagasaki, Rcm0 and Zürich II lines were generated by deleting not only the ORF, but also 5′ flanking sequences extending into the second intron and 3′ non-coding sequences. As shown in Figure 1, the three lines have in common the loss of 270 bp upstream of the PrP reading frame and of 450 bp downstream. Whilst the deleted sequences in the Nagasaki mice were replaced by a neo cassette, which, at least in some cases, causes an abnormal phenotype (Fiering et al., 1995), those in the Zürich II mice were replaced by a 34 bp loxP sequence, which is not known to cause deleterious effects. Although the cerebellar phenotype resulting from extended deletions in the PrP gene can be rescued by a wild-type PrP allele, it is clearly not caused by the absence of PrP, because the Zürich I and the Edinburgh mice remain healthy despite their lack of PrP (Weissmann, 1996). In the Edinburgh mice no PrP-specific mRNA was detected, so that significant levels of any fusion protein containing PrP sequences are unlikely. In the Zürich I mice the neo cassette was inserted between the third and the 188th codon of the PrP sequence; although its coding and 3′ non-coding sequence were in-frame with the 67 residual PrP codons, there were two termination codons in between, which would preclude read-through. The relevant DNA segment from the Zürich I mice currently in use was resequenced and the presence of the termination codons confirmed (data not shown). Therefore, the presence of a PrP fragment or a fusion product is not responsible for maintaining the normal phenotype in either of the two lines. The fact that the knockout lines showing the cerebellar phenotype lack sequences flanking the PrP ORF suggested that critical information was partly or entirely located in these regions. The pathological phenotype could come about either by loss of function, if these flanking regions controlled the formation or encoded part or all of some essential protein or RNA, or by gain of function, if the extended deletion resulted in the production of a deleterious product. The finding that introduction of a wild-type Prnp allele, either by breeding or as a transgene, abrogated the ataxic phenotype could be accommodated by either explanation: because the Prnp allele contains the flanking sequences, it could supply the missing function conjectured by the loss-of-function hypothesis. Alternatively, within the framework of the gain-of-function hypothesis, PrP might overcome the pathogenic effect of a postulated deleterious product. The discovery of Prnd, the gene encoding Dpl, and its expression in the brains of Zürich II, Nagasaki and Rcm0 mice, suggested that the PrP knockout alleles in these animals give rise to a deleterious product. Analysis of brain-derived cDNAs indicated that in wild-type mice Dpl mRNA is very weakly expressed, mainly from a promoter upstream of exon 1 of Prnd, whilst the strong expression in Nagasaki and Rcm0 mice is due to chimeric RNAs that originate at the Prnp promoter, run all the way across and past the Prnd ORF and are processed by one or more splicing events that link the 3′ end of the second PrP exon directly or indirectly to the Dpl-encoding exon (Moore et al., 1999; Li et al., 2000). This intergenic splicing, which was also detected by PCR at very low levels in wild-type mice, is greatly enhanced in the ataxic mice because the splice acceptor site upstream of the PrP-encoding exon (Figure 1) is deleted, thus diverting the splice to a downstream acceptor site. Prnd-specific mRNA was expressed undiminished in brain of Nagasaki mice ‘cured’ by the introduction of a PrP-expressing transgene (Nishida et al., 1999). The hypothesis that expression of Dpl in brain is responsible for the ataxic syndrome is supported not only by the fact that in Zürich I knockout mice containing a single Zürich II allele onset of ataxia and Purkinje cell degeneration is retarded, but also by the finding that Dpl expression in the brain at twice the level of that in Zürich II mice, found in mice transgenic for a cosmid devoid of the PrP ORF but containing Prnd, accelerates appearance of the symptoms. Why should overexpression of Dpl cause ataxia and concurrent overexpression of PrP restore normal function? Shmerling et al. (1998) found that introduction into Zürich I Prnp0/0 mice of an amino-proximally truncated transgene encoding PrP devoid of the octa repeats and the conserved 112–126 region (PrPΔ32–134) leads to ataxia and degeneration of the cerebellar granule cell layer within weeks of birth. Moreover, introduction of a single wild-type PrP allele prevented the disease. They proposed that PrP interacts with a ligand to elicit an essential signal and that a conjectured PrP-like molecule with lower binding affinity can fulfil the same function in the absence of PrP. According to this hypothesis, in PrP knockout mice the truncated PrP could interact with the ligand, displacing the PrP-like molecule, without, however, eliciting the survival signal. If PrP has the higher affinity for the ligand, it would displace its truncated counterpart and restore function. Because Dpl resembles the t