| In many experimental rodent scrapie models, as in natural sheep scrapie, the first documented prion disease which was found in sheep, the disease-specific isoform (PrPSc) of the host prion protein (PrPC) accumulates in the spleen and other lymphoid tissues prior to infecting the central nervous system. PrPSc builds up in germinal centres and is strongly associated with follicular dendritic cells (FDCs) and possibly circulating dendritic cells and macrophages. In addition, studies have shown that mature FDCs are critical for PrPSc replication in lymphoid tissues, and dedifferentiation of the cells increases the incubation period of the disease, presumably by lengthening the time for neuroinvasion to take place.;Previously, a small number of changes in expression during scrapie infection were observed when examining spleen tissue as a whole (unpublished observation by Booth lab). I believe that as the majority of cells that make up the spleen are not competent for prion replication, changes in expression in the small proportion of cells in which prions replicate are masked. Therefore, in this project I attempted to isolate a population of splenic cells enriched for FDCs, circulating dendritic cells and macrophages to identify an increased number of gene expression changes. The objective of this study is to use DNA microarrays to profile gene expression in spleen tissue with the aim of identifying changes that accompany prion replication. These may be host factors involved in prion propagation and/or may be useful as preclinical biomarkers for early diagnosis. To achieve these objectives I developed a cell isolation technique to target a more specific population of cells involved in prion disease, and then used microarray technology to identify differentially expressed genes between scrapie-infected and mock-infected tissues. The microarray platforms used in this project included BMAP 17K cDNA arrays manufactured in our laboratory, Operon mouse AROS v3 32K oligo arrays, and Agilent whole mouse genome 4x44K oligo arrays. Selected differentially expressed genes found upregulated from the microarray studies belonging to the small leucine-rich proteoglycan (SLRP) family were validated at the molecular level using qRT-PCR and at the protein level using both immunohistochemistry and western blot. These SLRPs could be involved in replication and propagation of the prion protein by assisting with the conversion process of PrPC to PrPSc and possibly as preclinical biomarkers for prion disease in the future. |
