GRANT=6546043;P01AG Prion diseases are a group of fatal and transmissible neurodegenerative disorders affecting both humans and animals. They include Creutzfeld-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia in humans, and scrapie, chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE) in animals. A wealth of data supports a causal role for an abnormal, protease-resistant isoform of the prion protein, PrP-Sc, in the pathogenesis of prion disease. The possibility of disease transmission between animals and humans has recently been highlighted due to the occurrence in young adults of a new variant form of CJD (vCJD) that is thought to originate rom BSE in cattle. The long-term objective of the present project is to understand the structural basis by which PrP-Sc modulates disease phenotypes in humans and animals, and in transmission of prion diseases from animals to humans. Toward this end, this project is focused on the following specific aims.
Specific Aim 1 will focus on a systemic comparison of all PrP-Sc subtypes in human and animal prion diseases. The sensitive and rapid PrP-Sc typing method developed in our laboratory will be used for the detection of all variants of PrP-Sc in humans and animals using Western blot analysis. Cases from human prion diseases include CJD of various phenotypes, FFI and GSS. The animal cases will be BSE of cattle, CWD in deer and elk, and experimental scrapie prion stains. Both conventional and two-dimensional gel electrophoresis will be used to separate different PrP-Sc subtypes and provide a comprehensive map of protein subtypes.
Specific Aim 2 will characterize the protease cleavage sites of PrP-Sc in human and animals to determine conformational variabilityof PrP-Sc. Purified PrP-Sc from diseased brains will be used to analyze the protease cleavage sites using multiple approaches such as N-terminal sequencing, enzymatic digestion, HPLC, and mass spectrometry.
Specific Aim 3 will examine the glycosylation patterns of PrP-Sc in human and animal brains since N-linked glycosylation contributes to the structural stability and heterogeneity of PrP-Sc. Purified PrP-Sc preparations will be denatured and trypsinized to allow isolation and purification by HPLC of glycopeptides containing N-linked glycosylation sites. The glycan structures will be determined by liquid chromatography and tandem mass spectrometry.
In Specific Aim 4 the secondary structure of purified animal and human PrP-Sc will be examined and compared using Fourier transform infrared spectroscopy. These proposed studies will lead to a better understanding of the role of the PrP-Sc structures in pathogenesis and transmission of prion disease.
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