The transmissible spongiform encephalopathies (TSEs) are a class of neurodegenerative diseases that are characterized by proteinaceous deposits in the brain. The deposits consist largely of an abnormal form of prion protein which is highly aggregated and resistant to degradation by proteases. The function of prion protein (PrP) is unknown and its normal form (PrPC) is sensitive to protease digestion. Some of the TSEs include scrapie in sheep, mice, and hamsters, bovine spongiform encephalopahty, and Creutzfeldt-Jakob disease in humans. Animals with scrapie accumulate a disease-specific form of PrP designated PrPSC. The identity of the infectious agent of the TSEs is unclear. No conventional agent or disease-specific nucleic acid has been found and treatments that destroy most normal viruses have no effect. Based on that information the infectious particle has been surmized to consist solely of protein.The "protein only" theory has been strengthened by the discovery of PrP and that preparations of PrPSc are greatly enriched for infectivity. Previously, the simplest system producing protease-resistant PrP was cell culture. Here is described a system that produces protease-resistant PrP in vitro. This system was used to study the mechanism of the conversion of PrPC to the protease-resistant form. A threshold concentration of aggregates of PrP S c was required for conversion. Guanidine-HCI solubilized PrPSC had no converting activity. The need for aggregates and a threshold concentration favors a seeded polymerization mechanism of conversion. The system was also used to study species barriers and different strains of scrapie. Different combinations of mouse and hamster PrPs were used in conversion reactions. The in vitro conversion results correlated with infectivity data on transmission. Scrapie strains with different PrPSc N-terminal protease cleavage sites were used with this system. These different cleavage sites were passed to newly-resistant PrPC depending on which strain was in the reaction. This system provided evidence that a "protein only" theory is compatible with the properties of TSEs.Thesis Supervisor: Dr. Peter T. Lansbury, Jr. Title:Associate Professor of Chemistry Chapter 1 CJD is characterized by dementia, ataxia (a loss of muscle coordination), and the formation of small holes in the brain known as spongiform degeneration.This spongiform degeneration is the result of a loss of neurons. CJD is often accompanied by abnormal proteinaceous deposits known as amyloid plaques.The onset of CJD can be months to years after infection, but once the disease presents itself a gradual deterioration of mental capabilities ensues. The deterioration ends in death usually within a year of onset. Heparin sulfates have been shown to offer some protection to mice inoculated with scrapie,7 but as of now there is no way to stop the deterioration and the disease is always fatal.GSS is a familial version of CJD with different pathology. All GSS cases are accompanied by the formation of amyloid plaques 2 and it i...

The infectious agents causing scrapie and other transmissible spongiform encephalopathies have been postulated to consist solely of the protease-resistant form of prion protein (PrPSc). One unprecedented requirement of the protein-only model is that the 'inheritance' of pathogen strain differences must be mediated by stable variations in PrPSc structure, rather than mutations in an agent-specific nucleic acid. Strain differences in PrPSc structure have been described for the hyper (HY) and drowsy (DY) strains of hamster transmissible mink encephalopathy (TME), a scrapie-like disease originating in mink. Although HY and DY PrPSc are both post-translationally derived from the precursor prion protein (PrPC) they are cleaved at different amino-terminal sites by proteinase K (ref. 8). Here we investigate whether this strain-specific property of PrPSc is transmitted to PrPC during formation of new PrPSc. PrPSc from the HY and DY TME strains converted the protease-sensitive PrPC into two distinct sets of protease-resistant PrP products in a cell-free system. These data provide evidence that self-propagation of PrPSc polymers with distinct three-dimensional structures could be the molecular basis of scrapie strains.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrP9) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to proteaseresistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction.The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPC and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

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More than a million cattle infected with bovine spongiform encephalopathy (BSE) may have entered the human food chain. Fears that BSE might transmit to man were raised when atypical cases of Creutzfeldt-Jakob disease (CJD), a human transmissible spongiform encephalopathy (TSE), emerged in the UK. In BSE and other TSE diseases, the conversion of the protease-sensitive host prion protein (PrP-sen) to a protease-resistant isoform (PrP-res) is an important event in pathogenesis. Biological aspects of TSE diseases are reflected in the specificities of in vitro PrP conversion reactions. Here we show that there is a correlation between in vitro conversion efficiencies and known transmissibilities of BSE, sheep scrapie and CJD. On this basis, we used an in vitro system to gauge the potential transmissibility of scrapie and BSE to humans. We found limited conversion of human PrP-sen to PrP-res driven by PrP-res associated with both scrapie (PrP[Sc]) and BSE (PrP[BSE]). The efficiencies of these heterologous conversion reactions were similar but much lower than those of relevant homologous conversions. Thus the inherent ability of these infectious agents of BSE and scrapie to affect humans following equivalent exposure may be finite but similarly low.

The entity responsible for the converting activity was many times larger than a soluble PrP monomer and required a threshold concentration of PrPSc. These results are consistent with a nucleated polymerization mechanism of PrPSc formation and inconsistent with a heterodimer mechanism.

The dipolar coupling in rotating solids, averaged to zero when the rotation is about the magic angle, can be reintroduced in the spin-locking interaction frame by rotor-synchronized 90° phase shifts of an applied spin-locking field. This new technique is an interaction frame analogue of previously proposed experiments. Because the spin-locking fields both truncate and play the role of the chemical shift terms in the Hamiltonian, this technique is insensitive to the chemical shifts as well as chemical shift anisotropies of the coupled spins. Through numerical simulations of the magnetization exchange trajectory (e.g., the mixing time dependence of the cross-peak intensity connecting two coupled spins), the through-space distance between two spin nuclei can be estimated. This technique can also be applied to obtain two-dimensional correlation spectra, or alternatively it can be used as a double-quantum filter. Preliminary experiments demonstrate that the cross peaks in the two-dimensional correlation spectra obtained with this technique have a ‘‘negative–positive–negative’’ pattern to the first, second, etc., coupled neighbor. This spectral feature is useful in the assignment of complex magic angle spinning spectra.

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