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Test the hypothesis that scrapie resistance in 171QR heterozygous sheep is associated with predominance of Prnp 171R allele transcripts at sites of preclinical PrPsc accumulation. <P>
There are two objectives. First, correlate the known cell specificity of PrPsc accumulation with the cell specificity of PrPc production in the alimentary tract of Suffolk sheep with defined PrnP genotypes. Second, measure Prnp mRNA concentration and determine the use of polymorphic Prnp alleles at alimentary tract accumulation sites of PrPsc in Suffolk sheep with Prnp polymorphism at position 171.
NON-TECHNICAL SUMMARY: Scrapie is endemic in the United States and threatens the viability of the sheep industry and sustainability of international markets for the entire ruminant livestock industry because of the link between sheep scrapie and bovine spongiform encephalopathy. The study will investigate natural genetic resistance to sheep srapie by determining whether or not resistance is due to differential PrP gene expression.
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APPROACH: Studies in Prnp gene ablated mice have demonstrated that the ability to generate PrPc is required for PrPsc accumulation and susceptibility to experimental prion infection. In order to investigate allele specific effects of Prnp polymorphism on disease susceptibility it is necessary to identify tissue subcompartments and cells that express PrPc. Follicular dendritic cells (FDC) in gastrointestinal tract related lymphoid tissue are sites of preclinical PrPsc accumulation in ovine scrapie. The first objective is to show that follicular dendritic cells produce host cellular prion protein (as a prerequisite for PrPSC accumulation), rather than accumulate PrPSC trafficking to gut-associated lymphoid tissue from other anatomical sites. Double label indirect immunofluorescence will be developed for detection of normal cellular prion and cellular markers in gut tissue. Frozen sections from alimentary tract and alimentary lymphoid tissue will be obtained from normal, adult Suffolk sheep, genotyped QQ (n=3), QR (n=3) and RR (n=3) at position 171. Specific staining will be observed using conventional fluorescence microscopy employing filters for simultaneous detection of different fluorescent labels. Data will be recorded as positive or negative. Genetic resistance to Scrapie is linked to allelic polymorphism of the gene Prnp. Hence, information on the use of Prnp alleles in cells critical for disease progression deriving from this specific aim should provide insight on mechanisms that lead to failure of preclinical PrPsc accumulation and result in disease resistance. Since American Suffolk sheep show little variability at codons 112, 136 and 154 of the Prnp, susceptibility to Scrapie in this breed is determined by codon 171. In analogy to the situation in familial CJD, preferential use of the 171R allele may render 171QR heterozygous sheep resistant to infection. In particular, relative paucity of convertible PrP in cells capable of amplifying PrPsc at peripheral accumulation sites may reduce the probability of a conversion event. If, however findings of this study confirm equivalent PrP mRNA levels in sheep polymorphic for Prnp at position 171, it must be concluded that resistance is not the effect of allelic use (presence of 171Q PrPc) but rather due to a dominant inhibitive effect of PrPc171R protein over PrPc 171Q protein when exposed to the disease isoform of PrPSC. Real time PCR will be employed for the second objective to measure allele specific prion mRNA transcripts. Real time PCR probes will be designed to differentiate nucleic acid differences at the polymorphic site at codon 171. Homogeneity at known polymorphic loci other than codon 171 will be determined since findings in ovine Scrapie indicate that several loci can influence disease susceptibility and the character of disease. Since the level of Prnp transcription may vary among individual sheep, age groups, tissues, cell types and allelic variants, pilot experiments will be run to determine the homogeneity of Prnp mRNA expression (total and allelic) within experimental groups and permit selection of group sizes using statistical power calculations.