Healthy goats naturally devoid of prion protein
Prion diseases such as scrapie in small ruminants, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in man, are fatal neurodegenerative disorders. These diseases result from the accumulation of misfolded conformers of the host-encoded prion protein (PrP) in the central nervous system. To date naturally-occurring PrP free animals have not been reported. Here we describe healthy non-transgenic animals, Norwegian Dairy Goats, lacking prion protein due to a nonsense mutation early in the gene. These animals are predicted to be resistant to prion disease and will be valuable for research and for production of prion-free products.
KeywordsPrion Protein Prion Disease Bovine Spongiform Encephalopathy Scrapie Classical Scrapie
Introduction, methods and results
Transmissible spongiform encephalopathies (TSE) or prion diseases such as Creutzfeldt-Jakob disease (CJD) in man, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats are invariably fatal diseases. These diseases may be inherited (mutations in the prion protein gene), sporadic (unknown cause) or acquired (transmission of infectious agent). They are characterized by the accumulation of conformational isoforms of the normal cellular prion protein (PrP) in the central nervous system. According to the “protein-only” hypothesis, the partly protease-resistant, misfolded PrP, designated PrPSc, is the disease agent. Prion diseases are entirely dependent on endogenous PrP expression. For instance, PrP gene knockout mice are resistant to prion disease, and further studies of transgenic mice have shown that the level of PrP expression is inversely correlated with the incubation time[4, 5]. In addition, knockdown of the PrP expression by RNAi rescues early neuronal dysfunction and prolongs survival time in mice with prion disease.
As with sheep, goats are susceptible to both classical[7, 8, 9, 10, 11] and atypical[10, 12, 13, 14] scrapie. One goat has also been identified as infected with BSE. Classical scrapie in goat has been reported in the USA, Canada and many European countries, and its prevalence is particularly high in Cyprus (accounting for 85% of all cases in the EU between 2002 and 2007). As described in many other species, the goat PrP gene (PRNP) is polymorphic and some variants have been associated with resistance to the disease[10, 11, 16, 17, 18].
The present study is based on the discovery, in the indigenous goat breed Norwegian Dairy Goat originating from the north-European Landrace, of a new variant of the PRNP displaying a premature stop codon. Additional goats were subsequently screened for PRNP polymorphisms and one goat homozygous for the new variant was analysed for the presence of PrP. Animal experiments described in this paper have been subjected to stringent ethical evaluation and were carried out in compliance with institutional and national regulations.
Genomic DNA was extracted from brain or blood samples using DNeasy 96 blood and tissue kit (Qiagen) according to the suppliers’ protocol. A fragment covering the coding region of the goat PRNP was amplified by PCR with M13 tailed primers GoatF1: M13-21-CAGTCATTCATTATGCTGCAGACTT (this work) and BilG2-R: M13rev-CTATCCTACTATGAGAAAAATGAG and analysed by sequencing with Big Dye Primer chemistry (Applied Biosystems, Foster City, USA).
Whilst experimental production of PrP gene knockouts of cattle and goats[21, 22] has been described, this is the first description of apparently healthy non-transgenic animals lacking PrP due to a naturally-occurring mutation. Whether the 32Stop haplotype is unique to Norwegian Dairy Goats is not known. Several PrP polymorphisms have been described in goats[10, 11, 17], but only one animal heterozygous for a nonsense mutation late in the goat PRNP, at codon 163, has been reported.
The 32Stop goats are unique natural tools for investigating how the PrP may be involved in both physiological and pathological processes. PrP is a cell surface protein largely expressed within the nervous system and is highly conserved across mammalian species. However, the natural function of the protein remains elusive. Similar to transgenic PrP gene knockout animals[1, 20, 21], the identified goats homozygous for the 32Stop haplotype lack PrP, yet are viable and appear normal. This suggests that the protein is not involved in vital physiological processes or that its absence can be compensated for by other mechanisms. In disease-related processes PrP might be both positively and negatively involved. Studies of transgenic animals suggest that PrP has a role in tissue repair and protection against cellular stress[1, 23]. Recent reports also indicate that PrP is involved in mediating Aβ-induced cytotoxicity in Alzheimer’s disease[24, 25, 26].
In conclusion, this is the first description of naturally-occurring mammals lacking PrP. Goats lacking PrP differ from the valuable PrP knockout mice in many aspects and might provide a supplementary model for investigating the function of the protein. Additionally, goats resistant to prion disease will be valuable not only for breeding but also for production of prion-free materials.
We thank Agnes Klouman and the staff at the Animal Production Experimental Centre and Thor Blichfeldt and the staff at the Norwegian Association of Sheep and Goat Breeders for providing blood and DNA samples for genotyping of goats and Torleiv Løken, Department of Production Animal Sciences, Norwegian School of Veterinary Science for the clinical assessment. We are grateful to Torfinn Moldal and Magne Kaldhusdal, Norwegian Veterinary Institute, for assistance with the necropsy, the laboratory staff at the Norwegian Veterinary Institute and the Norwegian School of Veterinary Science for performing the protein analyses and Simon Hardy and Alison Coulthard for critical reading of the manuscript.
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