Transmissible spongiform encephalopathies (TSE) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective anti-TSE therapeutics. The precise nature of the infectious agent of the TSE diseases is unknown. Susceptibility to infection can be influenced by amino acid homology between a normal host protein (PrP-sen) and the abnormal proteinase K-resistant form of this protein, PrP-res. Formation of PrP-res is closely associated with infectivity and PrP-res has been hypothesized to be the infectious agent in the TSE diseases. An understanding of how this protein is made is critical for our understanding of TSE pathogenesis and for devising therapeutic strategies to prevent its synthesis. <P> My studies address many different aspects of the TSE diseases at both the molecular and pathogenic level. In particular, my laboratory focuses on: 1) identifying the earliest events which occur during TSE infection, 2) precisely defining the different cellular compartments where PrP-res formation occurs, 3) determining the molecular basis of TSE strains, 4) understanding the pathways of PrP amyloid formation, 5) studying how mutations in PrP influence PrP-res formation in familial forms of TSE disease, and 6) development of effective therapeutic TSE agents.In the last year, we have continued our studies on the kinetics of PrP-res uptake for different scrapie strains and cell types. <P> We have developed a scrapie infected tissue culture cell line that may help us to study PrP-res formation in different species. We are also continuing our PrP fibrillization studies and are analyzing how different PrP-sen regions modulate the influence of the octapeptide repeat region on PrP amyloid formation.Using a tissue culture system developed last year that enables us to follow the process of TSE infection from the initial exposure of cells to TSE infectivity through to the establishment of a persistent infection, we have studied the uptake of PrP-res into the cell. We are currently investigating the cellular mechanisms involved in PrP-res uptake, where input PrP-res is localized in these cells, and whether or not that localization changes as the cells become persistently infected.PrP-res can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of TSE disease appear to be less transmissible than non-amyloid forms, suggesting a fundamental difference in their pathogenesis. Recent data suggest that the glycophosphatidyl-inositol (GPI) membrane anchor in PrP-sen may influence whether or not PrP-res forms amyloid. <P> Last year, in order to understand the basic molecular mechanisms underlying amyloid forms of TSE infection, we derived mouse fibroblast and neural cell lines expressing mouse PrP-sen without the GPI anchor and infected them with mouse scrapie. We are continuing our studies with these cells and are in the process of 1) determining the level of infectivity in these cells 2) determining how mouse PrP-sen without the GPI anchor is converted into PrP-res.Hereditary forms of TSE disease are associated with mutations within the PrP gene. One of these mutations is the insertion of extra copies of an eight amino acid motif (octapeptide repeat) into PrP. Last year we demonstrated that increasing the number of repeats increased the rate of abnormal PrP formation. Our data provided an explanation for the observation that, the greater the number of repeats, the earlier the onset of TSE disease. We now have evidence that other regions of the PrP molecule may modulate the effect of the octapeptide repeat region on amyloid forms of PrP-res. These preliminary data suggest that interactions between different regions of PrP-sen may help to determine whether or not PrP-res forms amyloid.