Journal of Molecular Biology
Regular articleAtaxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel1
Introduction
Prion diseases such as scrapie and Creutzfeldt-Jakob disease (CJD) are fatal neurodegenerative disorders that can manifest as infectious, sporadic, or genetic illnesses. Many lines of evidence indicate that the underlying event in these diseases is a structural transition where the normal, host-encoded protein cellular PrP (PrPC) posttranslationally adopts a conformation rich in β-sheet, designated PrPSc (Prusiner, 1998).
Although PrPC binds Cu(II) ions through histidine residues within four glycine-rich octarepeats near the N terminus, the function of PrPC remains uncertain Brown et al 1997, Hornshaw et al 1995, Pan et al 1992, Stockel et al 1998, Sulkowski 1989, Viles et al 1999. Moreover, database searches have been fruitless because PrP exhibits no significant homology to any known protein (Bamborough et al., 1996). It was anticipated that mice deficient for PrP (Prnp0/0) might develop a phenotype which would provide clues to the normal function of PrP; however, the first two Prnp0/0 lines created were viable and appeared to be phenotypically normal Bueler et al 1992, Manson et al 1994. Subsequently, it was reported that PrP deficiency resulted in alterations in circadian rhythms (Tobler et al., 1996) and electrophysiological abnormalities (Collinge et al., 1994). The significance of these electrophysiological abnormalities remains uncertain, since they could not be reproduced by some investigators Herms et al 1995, Lledo et al 1996. The lack of severe defects in these two lines of Prnp0/0 mice was ascribed to adaptation, since PrP was absent throughout embryogenesis. However, bigenic mice expressing inducible PrP transgenes that were rendered PrP deficient as adults by the administration of doxycycline have remained healthy for more than 1.5 years (Tremblay et al., 1998). This argues against the adaptation hypothesis and raises the likelihood of surrogate proteins with functions overlapping that of PrPC.
When a third line of Prnp0/0 mice was produced (Ngsk Prnp0/0), the animals developed late-onset ataxia accompanied by Purkinje cell degeneration (Sakaguchi et al., 1996). Recently, an additional independently generated Prnp0/0 line (designated Rcm0) has also developed a late-onset ataxia (Moore, 1997). It has been suggested that PrP may have a role in the long-term survival of Purkinje neurons; however, it was puzzling why Ngsk Prnp0/0 and Rcm0 Prnp0/0 mice should develop a fatal ataxia while two other lines of Prnp0/0 mice did not exhibit CNS dysfunction. In an effort to determine the role of PrP in this phenotype Ngsk Prnp0/0 mice were crossed with transgenic (Tg) mice overexpressing wild-type (wt) mouse PrP. This rescued the phenotype in Ngsk Prnp0/0 offspring expressing PrP (Nishida et al., 1999).
Since a common approach to determining the function of a gene is the analysis of related genes, we searched for PrP-related genes by hybridization; however, these studies were uninformative (Westaway & Prusiner, 1986). Vertebrate genomes contain a number of protein families sharing sequence homology and showing overlapping function (Henikoff et al., 1997), and because a number of them have been shown to be organized in clusters, we undertook sequencing of large cosmid clones containing the PrP gene (Lee et al., 1998). Studies of regions flanking the human, sheep, and mouse PrP genes in cosmid clones failed to reveal additional open reading frames (ORFs) (Lee et al., 1998). Only when the sequencing of a cosmid clone isolated from a Prnpb/b mouse (I/LnJ-4) was extended downstream of PrP was a novel PrP-like gene found.
The discovery of the doppel gene (PrnD) not only represents the first PrP-related gene, but it also provides an explanation for some otherwise perplexing observations with different lines of Prnp0/0 mice recounted above. Prnd encodes a novel 179 residue protein designated doppel (Dpl) with ∼25 % identity with all known prion proteins (PrP). The Dpl locus, Prnd, is 16 kb downstream of the PrP gene Prnp and produces two major transcripts of 1.7 and 2.7 kb, as well as unusual chimeric transcripts generated by intergenic splicing with Prnp. Like PrP, Dpl mRNA is expressed during embryogenesis but, in contrast to PrP, it is expressed at low levels in the adult CNS and at high levels in the testis. Dpl is upregulated in the CNS of the two Prnp0/0 lines that develop late-onset ataxia and Purkinje cell degeneration but not in a Prnp0/0 line that does not develop ataxia. Our findings suggest that Dpl may provoke neurodegeneration in PrP-deficient mice, an observation which may explain why some lines of Prnp0/0 mice develop cerebellar dysfunction and Purkinje cell death while others do not. The similarities between Dpl and PrP suggest that these two proteins may share some biological properties and as such, Dpl may open new avenues of investigation in prion biology.
Section snippets
A PrP-like sequence downstream of the mouse PrP gene
Previous large-scale sequencing studies of phage and cosmid molecular clones encompassing human, sheep, and the “a” allele of the mouse PrP gene, Prnpa, failed to reveal any evidence of additional coding regions, either immediately adjacent to PrP or within intronic sequences (Lee et al., 1998). However, sequencing of the mouse Prnpb cosmid clone I/LnJ-4 (Westaway et al., 1991), which extends further in a 3′ direction, and analysis with the “XGRAIL 1.3c” program (Uberbacher & Mural, 1991)
The Prnd gene
Several lines of evidence argue that Prnd is a bona fide gene. First, Prnd is transcribed to generate a variety of alternatively spliced polyadenylated mRNAs Figure 1, Figure 2, Figure 3, Figure 4. Second, an intact Dpl ORF is found in mouse, rats, and humans, suggesting selection pressure for an ability to express the corresponding protein. The third and most direct line of evidence is that antisera raised against a 21-mer peptide corresponding to the sequence of Dpl detects a protein by
Genomic clones
Cosmids and YAC clones were propagated by standard procedures and have been described (Westaway et al., 1994a). Prnp-containing BAC clones 185E12 and 321L17 (Research Genetics) were kindly provided by Dr Deborah Nagle (Millenium Pharmaceuticals) (Nagle et al., 1999).
RACE cloning of Prnd RNAs
5′ RACE “Marathon” mouse brain cDNA (Clontech) was amplified with adapter primer AP1 and the Prnd anti-sense strand primer DW112, 5′-CGGTTGGTCCACGGCGACCCGAA-3′ (0.2 μM) using a Perkin Elmer 2400 thermocycler and “touchdown” PCR
Acknowledgements
This work was supported by research grants from the National Institutes of Health to L.H. and S.B.P. and from Health Canada and the Alzheimer Association of Ontario to D.W. A fellowship from the Human Frontier Science Program supports R.C.M. We thank Hong Yao for assistance in the early phases of this work.
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