Doppel is an N-glycosylated GPI-anchored protein: expression in testis and ectopic production in the brains of Prnp super0/0 mice predisposed to Purkinje cell loss

Silverman, Gregory L.; Qin, Ken; Moore, Richard C.; Yang, Ying; Mastrangelo, Peter; Tremblay, Patrick; Prusiner, Stanley B.; Cohen, Fred E.; Westaway, David

doi:10.1074/jbc.m003888200

Cited by 94 publications

(107 citation statements)

References 20 publications

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“…These mice show selective death of Purkinje Cells (PCs) and subsequent progressive cerebellar-associated ataxia (Katamine et al, 1998;Sakaguchi et al, 1996). The Rcm0 line (Moore et al, 1999;Silverman et al, 2000) displays a large deletion in the Prnp gene and unexpectedly overexpresses a homolog PrP c protein, called Doppel (Dpl, from the German double).…”

Section: Other Prp C Mutant Mice: the Puzzle Of Functions Increasesmentioning

confidence: 99%

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

Nicolas

Gavı́n

Rı́o

2009

Brain Research Reviews

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Section: Other Prp C Mutant Mice: the Puzzle Of Functions Increasesmentioning

confidence: 99%

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

Nicolas

Gavı́n

Rı́o

2009

Brain Research Reviews

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“…For example, ataxia and loss of Purkinje cells in aged mice have been observed in some Prnp −/− mouse strains where, in addition to the Prnp open reading frame (ORF), 5′ flanking genomic sequences were also deleted, which results in exon skipping between Prnp and Prnd (located 16 kb downstream of the murine gene Prnp and encoding Dpl protein) [23][24][25][26][27] . The cerebellar symptoms in these Prnp −/− mouse strains were suggested to be caused by ectopic expression of Dpl protein in brain 28 .…”

Section: Competing Interests Statementmentioning

confidence: 99%

Production of cattle lacking prion protein

et al. 2006

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Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP C , such as PrP BSE in bovine spongiform encephalopathy (BSE) in cattle and PrP CJD in Creutzfeldt-Jakob disease (CJD) in humans 1 . Disruption of PrP C expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities [2][3][4][5] . However, the impact of ablating PrP C function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP C -deficient cattle produced by a sequential gene-targeting system 6 . At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification 7 . PrP C -deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.To generate PrP C -deficient (PRNP −/− ) cattle, we transfected a male Holstein primary fetal fibroblast line 6594 with first and second knockout (KO) vectors (pBPrP(H)KOneo and pBPrP (H)KOpuro vectors) 6 to sequentially disrupt the two alleles of PRNP. PRNP −/− fetal cell lines were established at 40-60 d of gestation and three of the PRNP −/− fetal cell lines (5211, 5232 and 4296) were recloned to produce calves (Table 1 and Fig. 1a). To verify that the calves possess the PRNP −/− genotype, we collected ear biopsies and established fibroblast cell lines for genotyping. Genotyping was done by genomic PCR specific to each gene targeting event 6 (primer pairs: neoF7 × neoR7 and puroF14 × puroR14, Fig. 1b COMPETING INTERESTS STATEMENTThe authors declare competing financial interests (see the Nature Biotechnology website for details).Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Fig. 1c) and confirmed the disruption of PRNP-specific mRNA expression in PRNP −/− calves. For protein expression analysis, we performed PrP-specific western blot analyses on fibroblasts (Fig. 1d), peripheral blood lymphocytes (Fig. 1e) and brain stem (Fig. 1f) from wild-type and PRNP −/− calves using the mouse anti-bovine PrP monoclonal antibody F89. We detected PrP-specific bands in the wild-type calves, whereas no reaction was observed in PRNP −/− calves and negative control mouse fibroblasts. These data clearly demonstrate that the PRNP gene is functionally inactivated in the PRNP −/− calves.PRNP −/− cattle were monitored for growth and general health status from birth to 20 months of age. Mean birth weight was 46 kg and average daily gain was 0.91 kg/d to 10 months. Both values were in the normal range for Holstein bulls. Serum chemistry was evaluated at 6 months of age and compared with published reference ranges. All the values for PRNP −/− calves (n = 12) were well within the reference range (Supplementary Table 1) and obvious abnormalities w...

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“…To account for toxic properties of internallydeleted PrP C causing cerebellar degeneration and the lack of an overt phenotype in Prnp 0/0 mice, a model has been proposed wherein PrP C docks to a hypothetical membrane protein L PrP to initiate intracellular signals that maintain cell viability, with a second hypothetical molecule π supplying a PrP-like function when PrP C is removed by genetic deletion (18). In terms of known PrP-like molecules, Doppel is barely expressed in the CNS in wild-type mice (35,36) and can be excluded from consideration. However, because Sho is expressed in the CNS (21 and present study) and has a number of PrP C -like biochemical properties (21,25,37), it is considered as a candidate for π.…”

Section: Activities Needed To Maintain Cell Viability In the Adult Cnmentioning

confidence: 99%

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP ^C -deficiency

Daude¹,

Wohlgemuth²,

Brown

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The Sprn gene encodes Shadoo (Sho), a glycoprotein with biochemical properties similar to the unstructured region of cellular prion protein (PrP C ). Sho has been considered a candidate for the hypothetical π protein that supplies a PrP C -like function to maintain the viability of Prnp 0/0 mice lacking the PrP C protein. To understand these relationships more clearly we probed the cell biology of Sho and created knockout mice. Besides full-length and a “C1” C-terminal fragment, we describe a 6-kDa N-terminal Sho neuropeptide, “N1,” which is present in membrane-enriched subcellular fractions of wild-type mice. Sprn null alleles were produced that delete all protein coding sequences yet spare the Mtg 1 gene transcription unit that overlaps the Sprn 3′ UTR; the resulting mice bred to homozygosity were viable and fertile, although Sprn 0/0 mice maintained in two genetic backgrounds weighed less than wild-type mice. Lack of Sho protein did not affect prion incubation time. Contrasting with lethality reported for knockdown of expression in Prnp 0/0 embryos using lentiviruses targeted against the Sprn 3′ UTR, we established that double-knockout mice deficient in both Sho and PrP C are fertile and viable up to 690 d of age. Our data reduce the impetus for equating Sho with the notional π protein and are not readily reconciled with hypotheses wherein expression of PrP C and Sho are both required for completion of embryogenesis. Alternatively, and in accord with some reports for PrP C , we infer that Sho’s activity will prove germane to the maintenance of neuronal viability in postnatal life.

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Doppel is an N-glycosylated GPI-anchored protein: expression in testis and ectopic production in the brains of Prnp super0/0 mice predisposed to Purkinje cell loss

Cited by 94 publications

References 20 publications

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

Production of cattle lacking prion protein

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP ^C -deficiency

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Doppel is an N-glycosylated GPI-anchored protein: expression in testis and ectopic production in the brains of Prnp super0/0 mice predisposed to Purkinje cell loss

Cited by 94 publications

References 20 publications

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

Production of cattle lacking prion protein

Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP C -deficiency

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Product

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Knockout of the prion protein (PrP)-like Sprn gene does not produce embryonic lethality in combination with PrP ^C -deficiency