A functional genomic approach to understanding the molecular pathogenesis of sheep scrapie

This project tested the hypothesis that: 1). there are changes in relative levels of gene transcripts occurring in the CNS and FDC at specific times after scrapie infection in sheep; 2). TSE-induced changes to host cell gene expression are related to the clinical manifestation of scrapie disease; 3). the differential susceptibility of the distinct genotypes to scrapie-induced disease is related to differential gene expression in the CNS and FDC. These hypotheses will be tested by microarray technology using a non-redundant bovine brain EST library augmented by genes from a sheep FDC cDNA library, from TSE infected animals. We will identify and quantify changes in CNS and FDC gene expression related to scrapie disease progression, in homozygous sensitive VRQ/VRQ, heterozygous VRQ/ARQ, and homozygous resistant ARR/ARR New Zealand Cheviot sheep.

Scrapie is a fatal degenerative disease of the brain of sheep and is closely related to BSE (mad cow disease) and human CJD. The nature of the infectious agent is controversial. It is clearly not a bacterium like meningitis and it may be a special type of virus (like measles) but the most commonly held view is that these diseases are a result of a change of structure of a common nerve cell protein from its normal form to the disease form, PrPC to PrPSc. Indeed, the susceptibility to scrapie is strongly linked to the genetics of the PrP gene (Prnp genotype); different genotypes have distinct incubation periods or are resistant. Infection results in the accumulation of PrPSc fibrils in the brain - similar to the accumulation of amyloid in Alzheimer's disease. An understanding of how changes in PrP structure and accumulation of fibrils affect nerve and other brain cells is thought to be crucial for early diagnosis and therapeutic intervention. These diseases are also transmissible and possible routes of transmission include in utero mother-to-child, meat or milk consumption, blood transfusion and organ grafts. Currently, the definitive method of diagnosis is at post-mortem and robust, sensitive ante-mortem diagnostics are much needed for both cattle and people.

Initial experiments infected sheep of defined Prnp genotypes with scrapie agent and investigated the progression of disease in different tissues in the different genotypes. We found that peripheral lymph glands (lymph nodes, spleen, tonsils) were the first organs to show PrPSc accumulation. Infection eventually reached the brain; the hind brain is affected first and then the thalamus and eventually the frontal cortex. PrP genotype affects the actual time of progression but not the pattern; although the resistant genotype sheep never show signs of disease. We also measured the levels of PrP and the related protein Sho, but unexpectedly found that the pattern of disease progression was not related to quantitative levels of expression.

This project used functional genomic technologies, developed for the human genome project, to try and identify changes to brain and spleen that occur as a result of scrapie infection. Of particular interest are events that occur soon after infection to try and identify changes to cells at an early stage of disease before the onset of clinical symptoms. This required the cloning and identification of more than 20,000 genes, with more than 4,000 genes being identified that were novel for the sheep. New high-throughput sequencing has recently been used to compare the 'gene profiles' of infected and normal brain and spleen at different times after infection to try and (1) understand the molecular basis of TSE pathogenesis; (2) identify novel risk genes; (3) identify potential biomarkers of infection..

In terms of diagnostics, we have identified EGR-1 as a possible diagnostic marker of pre-clinical infection that has potential for post-mortem diagnostic test that could be used for disease surveillance of cattle and sheep in relation to food safety.

Scrapie in sheep results in the accumulation of the misfolded protein PrPSc in both the central nervous system (CNS) and the peripheral lymphoid system (PLS). This accumulation of PrPSc is thought to play a major role is the disease process.

Functional genomic analysis of the CNS during the progression of scrapie highlight that regions of the brain are affected at different times after infection and respond differently to that infection, as assessed by differential expression of specific gene transcripts. However, the major physiological pathways associated with scrapie progression are 'inflammation', and 'cell death and apoptosis'.

In the PLS the 'cell death and apoptosis' pathway is also up-regulated but in contrast to the CNS,the 'inflammation' and 'oxidative stress' pathways are repressed.

The research also highlights the suitability of EGR1 as a potential surrogate diagnostic marker for scrapie infection, suggested by its differential expresion in the PLS, the low level of intrinsic variation of expression and by the fact that EGR1 expression is unaffected by PRNP genotype.