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British Medical Bulletin 66:35-42 (2003)
© 2003 The British Council
Physiological and pathological functions of the prion protein homologue Dpl
Mammalian Genetics Laboratory, Cancer Research UK, London, UK
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Abstract |
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Abstract
IntroductionA misfolded version of the prion protein PrPC, known as PrPSc, is the major component of scrapie infectivity, the pathological agent in transmissible spongiform encephalopathies. The Prnp gene that encodes the cellular PrPC protein was cloned almost 20 years ago, but remained without sequence or structural relatives for over a decade. Only recently a novel protein, named Doppel (Dpl), was identified, which shares significant biochemical and structural homology with PrPC. When overexpressed, Dpl is neurotoxic and causes a neurological disease. Strikingly, Dpl neurotoxicity is counteracted and prevented by PrPC. In contrast to its homologue PrPC, Dpl is dispensable for prion disease progression and for the generation of PrPSc, but Dpl appears to have an essential function in male spermatogenesis. Although Dpl research is still in its infancy, the discovery of Dpl has already solved some enigmas of prion biology and an understanding of its physiological function is emerging.
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Introduction |
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Nowadays, in the post-genome era, it takes only a few mouse clicks to determine whether there are homologous proteins in a sequenced genome. It is, therefore, from today's perspective surprising that Prnd, the gene that encodes Dpl, has remained unknown until very recently.
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The identification of Doppel |
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The generation of knock-out mice, that are deficient in a gene of interest is a standard strategy to investigate gene function. To elucidate the physiological function of PrPC, several research groups independently generated mouse lines with targeted disruptions of its coding gene Prnp. The gene targeting strategies were different, but all mutant mouse lines lacked significant regions of the Prnp open reading frame (ORF) and PrPC expression was abolished. However, two strikingly different phenotypes were observed: Zrch Prnpo/o and Edbg Prnp-/- (named after the cities of origin, Zurich and Edinburgh) showed only minor defects1,2, whereas Ngsk (Nagasaki) Prnp-/-, Zürich II and Rcm0 mice developed cerebellar Purkinje cell degeneration that caused ataxia with advancing age3–5. Although it was not realised at the time, these striking differences of the Prnp-mutant phenotypes were caused by the then unidentified PrPC homologue Dpl.
Prnd was identified rather incidentally during the sequence analysis of a cosmid containing Prnp. It was noted that directly adjacent to Prnp there is an ORF which codes for a protein with striking similarities to PrPC. The novel gene, Prnd, encodes a protein of 179 residues, which has been named Dpl (‘downstream of the Prnp locus’ or ‘doppel’ – German for ‘double’)5. The Prnd gene is located only 16 kb downstream of Prnp in the mouse genome and its sequence is evolutionarily conserved from humans to sheep and cattle, indicating an essential function of Dpl6.
The prion gene locus, Prn, which comprises Prnp and Prnd, probably arose by gene duplication. The Prn locus is also conserved in other species including humans, although the human Prnd is located 27 kb downstream of Prnp. As there is no significant sequence homology at the nucleic acid level between Prnp and Prnd, the presumed gene duplication of a proto-prion gene may be an ancient event.
The predicted Dpl protein shows 
25% identity with the C-terminal two-thirds of PrPC, but has a lower molecular weight than PrPC because it lacks the N-terminally located octameric repeats and hydrophobic region present in PrPC [Plate V(A)]. Both PrPC and Dpl are glycosylphosphatidylinositol (GPI)-anchored membrane proteins7,8. The NMR structure of recombinant mouse Dpl shows that the overall topology of Dpl is similar to PrPC. Dpl contains three 
-helices and two short ß-sheet motifs, as does PrPC. PrPC possesses one disulphide bond and there are two disulphide bridges in Dpl, one of which is a close cognate to the bond in PrPC 9.
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Dpl expression is up-regulated in some PrPC-deficient mice |
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The conundrum posed by the different phenotypes of the Prnp-mutant mice was solved when it was realised that in the brain of ataxic (Ngsk Prnp-/-, Zürich II and Rcm0), but not of healthy (Zrch Prnpo/o and Edbg Prnp-/-), Prnp-mutant mice, expression of Prnd mRNA was up-regulated (Table 1)5. An intergenic splicing event places the Dpl locus under the control of the Prnp promoter, most likely due to the deletion of the Prnp intron 2 sequence (including its splicing acceptor). This intergenic splicing event could also be detected at very low levels in wild-type mice, but was greatly enhanced by the absence of the intron 2 splice acceptor5. Whereas the Prnp promoter is strongly expressed in neuronal cells, the Prnd promoter is not; therefore, Prnd expression from the Prnp promoter results in overproduction of Dpl in the brain4,5,10. Further experiments have demonstrated an inverse correlation between the mRNA levels of Prnd and the onset of ataxia. Disease progression is accelerated by increasing Prnd levels, supporting the idea that ectopic Dpl expression, but not functional loss of PrPC, might be responsible for neuronal degeneration in ataxic Prnp-deficient mice4.
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Dpl-induced neuronal cell death is PrPC dependent |
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Ngsk and Zürich II Prnp-/- mice are rescued from the neuronal degeneration by introduction of a Prnp transgene11. Therefore, PrPC can antagonize Dpl neurotoxicity, and the absence of PrPC is necessary for Dpl to induce cell death.
Interestingly, overexpression of an N-terminally truncated transgene encoding PrPC that is devoid of the octameric repeats and the conserved 106–126 region [PrP32–135, nicknamed 
F; Plate V(A)] into Zürich I Prnpo/o mice causes ataxia and degeneration of the cerebellar granule cell layer within weeks after birth; introduction of a single intact PrPC allele prevents the disease12. It is noteworthy that the F truncation lacks PrPC regions that are also absent in Dpl, and, thus, shares some structural similarities with Dpl. Because Dpl resembles truncated PrPC, and because PrPC antagonizes both Dpl- and F-induced neuronal death, they may cause disease by similar mechanisms.
Whereas overproduction of Dpl from the Prnp promoter causes degeneration of Purkinje cells, F expression is toxic for cerebellar granule cells. Although differences in biological activity between Dpl and F cannot be excluded, it appears more likely that differences in the promoter activity directing Dpl and F expression account for the altered target cell specificity. The promoter used to express the truncated PrPC is active in granule cells but not in Purkinje cells, while the wild-type Prnp promoter that directs the ectopic expression of Dpl is active in Purkinje cells5,13. In agreement with this, targeting the truncated F to Purkinje cells causes ataxia and degeneration of Purkinje cells4.
It should be noted that both the genomic Prnp promoter and the ‘half-genomic’ PrP promoter construct used to express F are active in a variety of neural and other cell types in addition to cerebellar cells14–17. Curiously, the toxicity of Dpl and F is observed only in Purkinje and granule cells, while forebrain neurones of F mice, even at relatively old age, are unaffected. The molecular events that underlie this cell-type specificity are completely unknown.
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Molecular basis of Dpl–PrPC antagonism |
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There are currently only speculations about the mechanism of antagonistic interaction between Dpl/
F and PrPC. In the original paper describing the F phenotype, the authors proposed a competition model where PrPC interacts with a ligand and they also postulated a PrPC-like, as of then unidentified, molecule with lower binding affinity for the ligand12. Dpl may be this postulated molecule. Ligand binding to Dpl/F in the absence of PrPC would trigger an apoptotic signal; however, if PrPC has the higher affinity, it would preferentially bind and sequester the ligand and thus prevent Dpl signalling [Plate V(B)]. The hypothesized PrPC ligand may only be expressed in cerebellum, thereby providing an attractive explanation for the observed cell-type specificity of Dpl/F-induced cell death. However, only through the identification of the PrPC ligand can this theory be proven.
Alternatively, the absence of PrPC may sensitize cerebellar cells to a constitutive weak apoptotic signal elicited by Dpl and F. In agreement with a survival function for PrPC, Prnp-mutant neuronal cell lines are hypersensitive to serum withdrawal18 and PrPC was shown to possess superoxide dismutase activity which may catabolize superoxide species19. A correlation between Dpl expression and the induction of both haem oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS) was observed that is suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp-mutant mice20. Therefore Dpl expression may cause oxidative damage and cerebellar cells lacking PrPC could be unable to detoxify the produced oxidative damage. The main difference between both scenarios is that in the first case PrPC and Dpl compete for a common factor X, but in the sensitization model PrPC and Dpl act independently [Plate V(B)]. Secondly, competition could be non-cell autonomous, i.e. PrPC and Dpl could compete for a ligand being expressed on different cells, whereas a hypersensitivity of PrPC-deficient cells is most likely due to a cell-autonomous function. However, GPI-linked proteins can undergo intermembrane transfer in vivo, which could account for a ‘pseudo’-non-cell autonomous effect21. Both above-mentioned models await experimental verification.
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Function of Dpl in prion disease |
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A wealth of evidence indicates that PrPC is essential for the development of prion disease. Most importantly, mice that lack PrPC are resistant to experimental scrapie inoculation22 and all familial cases of human TSEs are characterized by Prnp mutations23. The Dpl protein resembles an N-terminally truncated PrPC protein that lacks the octamer repeats. However, the latter version of PrPC is actually capable of supporting PrPSc propagation24, which suggests that the Dpl protein might, in principle, be susceptible to conversion into ‘DplSc’. However, all experimental evidence to date argues against an essential function of Dpl in prion diseases.
The role of Dpl in prion pathogenesis has been investigated using a neural grafting paradigm. Embryonic stem cells carrying a homozygous null mutation of the Prnd locus, but a normal Prnp locus, were found to undergo normal neural differentiation, and were capable of giving rise to all neural cell lineages when transplanted into host brains. After inoculation with scrapie prions, Dpl-deficient neural grafts showed spongiosis, gliosis and unimpaired accumulation of PrPSc, and infectivity similar to that in wild-type neuroectodermal grafts25. Therefore, in neural grafts, Prnd deficiency does not prevent prion pathogenesis. It has also been investigated whether increased levels of Dpl alter prion disease progression. Transgenic overexpression of Prnd in the CNS was found to have no influence on the incubation period, vacuolar pathology or amount or distribution of PrPSc deposition in the brains of the TSE-infected mice. Doppel has, therefore, no apparent influence on the outcome of TSE disease in rodents, suggesting it is unlikely to be involved in the naturally occurring TSE diseases in other species26,27.
A possible linkage disequilibrium of Prnd alleles in human prion diseases was also investigated. Four polymorphisms in Prnd were detected, but no strong association was found between any of these polymorphisms and human prion diseases28,29. These findings further argue against an important function of Dpl in neurones during genetically determined forms of human prion disease.
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Physiological function of Dpl |
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Dpl mRNA is expressed at high levels in testis, at lower levels in other peripheral organs and, notably, at very low levels in brain of adult wild-type mice. To clarify the physiological function of the Dpl protein, mice with a homozygous targeted disruption of the Prnd gene were generated. Whereas females lacking Dpl were viable and fertile, male mice without Dpl were sterile30. Dpl protein was expressed in the centres of wild-type seminiferous tubules, but not in testis sections from Dpl-mutant mice [Plate VI(A,B)]. Both round and elongated spermatids were strongly immunoreactive for Dpl. The sterility of Prndneo/neo males was not due to abnormal mating behaviour since their sexual activity was similar to that of controls as revealed by a normal number of copulation plugs. However, the spermatozoa from mutant males showed several structural abnormalities. In Prndneo/neo spermatozoa, the sperm head was disoriented with respect to the flagellum and often the flagellum appeared to fold back towards the sperm head instead of being straight and elongated. Prndneo/neo sperm heads were also severely malformed and lacked a discernible, well-developed acrosome [Plate VI(C–F)]. The role of Dpl in acrosome function was further validated by in vitro fertilization (IVF) experiments. After IVF, the percentage of 2-cell embryos recovered in the control group was 77%, but spermatozoa isolated from Prndneo/neo males were unable to fertilize wild-type oocytes (Table 2). Spermatozoa of Prndneo/neo males were occasionally sticking to, but never penetrating, the zona pellucida; however, after the zona pellucida was partially dissected and IVF was performed, a fertilization rate of 35% was achieved by Prndneo/neo spermatozoa (Table 2). These data indicated that Prndneo/neo spermatozoa are capable of oocyte fertilization, albeit at a lower frequency than controls, but that they cannot overcome the barrier imposed by the zona pellucida30. Therefore, mice lacking Prnd identify Dpl as a critical regulator of acrosome function and male gametogenesis.
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Conclusions |
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The similarities between PrPC and Dpl in primary amino acid sequence, structure and subcellular localization have suggested related biological functions. However, the genetic evidence gathered from Prnp-deficient mice has surprisingly indicated antagonistic functions for PrPC and Dpl. The elucidation of the mechanism underlying the opposing roles of PrPC and Dpl in neurotoxicity may not only provide insights into Dpl biology, but also serve as a window into PrPC function. At present, it is not clear whether impairment of PrPC function during prion disease progression contributes to neuronal death. If that was the case, molecules through which PrPC exerts its neuroprotective function could be interesting candidates for gene therapy of prion diseases.
While Dpl is toxic to cerebellar neurons, it has an essential physiological function in male gametogenesis. In testes, PrPC is not required to protect cells from Dpl toxicity, since Prnp-mutant male mice are fertile despite high levels of Prnd expression. Since Dpl is a cell-surface molecule, it is conceivable that there is a ligand present in testes that binds to membrane-bound Dpl, thereby eliciting a signalling cascade that is essential for spermiogenesis. Whether this putative Dpl ligand is identical or dissimilar from the postulated cerebellar PrPC ligand remains to be determined.
Apart from a defect in male fertility, no other physiological defects were detectable in Prnd-deficient mice. Because of the similarity between both proteins, a possible role for PrPC and Dpl during development could be masked by functional redundancy. To address this, it is necessary to generate mice that lack Prnp and Prnd, and to study whether the lack of both PrPC and Dpl results in an exacerbation of the mutant phenotypes. These double-mutant mice might finally reveal the true physiological function of PrPC and Dpl, and pave the way for the long-awaited understanding of the functions of these proteins.
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Footnotes |
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Correspondence to: Dr Axel Behrens, Mammalian Genetics Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
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