Molecular Genetics Of Scrapie Pathogenesis

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

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. 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.[unreadable] [unreadable] In FY 08, we have studied how PrP-res initially interacts with the cell during acute TSE infection (i.e. the first 96 hours). We approached this issue using a tissue culture system previously developed in my laboratory which utilizes PrP-res tagged with a unique antibody epitope. This epitope tag allows us to distinguish PrP-res associated with exogenous TSE infectivity from PrP-res newly made by the host cell. Our data showed that, for all TSE strains and cell lines tested, PrP-res uptake was a rapid process. However, the size of the PrP-res particle influenced its cellular localization and rate of uptake. Furthermore, the rate of uptake was also influenced by the type of PrP-res preparation with PrP-res in crude brain homogenate being taken up most efficiently. Our data suggest that the mechanism of PrP-res uptake is similar for all TSE strains and cell types. Thus, acute infection of a cell is likely scrapie strain and cell-type independent. We have also completed preliminary experiments which show that we can track our epitope tagged PrP-res in mice, allowing us to follow the course of acute TSE infection in vivo.[unreadable] [unreadable] PrP-res can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of TSE appear to be less transmissible than non-amyloid forms, suggesting that there is a fundamental difference in how they trigger disease. We are interested in understanding the molecular mechanisms underlying this fundamental difference and have begun to approach this issue using both in vitro and in vivo model systems. One in vitro system uses cells previously developed in my laboratory that express a mouse PrP-sen molecule without the glycophosphatidyl-inositol (GPI) membrane anchor. This PrP mutant was chosen because of previous work demonstrating that the PrP-sen GPI anchor may influence whether or not PrP forms amyloid. These cells have now been infected with mouse scrapie and the course of infection monitored. Our current results suggest that, in scrapie-infected cells in vitro, GPI-anchorless PrP-sen does not make amyloid but is essential for the establishment of a persistent TSE infection.[unreadable] [unreadable] A second in vitro system utilizes PrP peptides in a cell-free system of fibrillization to examine how different regions of the PrP molecule may influence PrP amyloid formation. Our most recent data suggest that complex interactions between different regions of PrP determine whether or not PrP forms amyloid. We are in the process of determining whether these PrP interactions are inter- or intra-molecular as well as the precise regions of PrP involved.[unreadable] [unreadable] Finally, for our in vivo amyloid studies, we have produced several transgenic mouse lines that express human PrP-sen without the GPI anchor. These mice have been infected with different forms of human TSE and are currently being monitored for disease. Our expectation is that, if these mice develop PrP amyloid, we can utilize them as an in vivo model system for human amyloid disease. [unreadable] [unreadable] 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. We have used an in vitro fibrillization model of this hereditary mutation to study how the repeat region influences the formation of both PrP-res and PrP amyloid. Last year, we demonstrated that environmental conditions could change the mechanism of fibril formation which in turn altered the ultrastructural properties of the amyloid. In FY08, we have gathered data suggesting that the octapeptide repeat region can significantly influence the final structure of both PrP-res and PrP amyloid. The structural influence of the octapeptide repeat region appears to be strain specific and thus may help to explain the different pathologies associated with different strains of TSE infectivity.