Recent successful investigations into CNS degeneration caused by prions demand that some striking observations be exploited. To take advantage of these discoveries, we propose to develop and extend several new and innovative approaches to transmissible neurodegenerative diseases utilizing molecular genetics, transgenic animals and cultured cells. Molecular genetic studies in mice and Gerstmann-Straussler syndrome (GSS) in humans - is genetically linked to polymorphisms in the open reading frame of the prion protein (PrP) gene. These polymorphisms result in nonconservative amino acid substitution.s Linkage of a PrP missense variant to the development of GSS established that GSS is a genetic disease which is also infectious and raises the possibility that the leucine variant at PrP codon 102 causes GSS. Recently, we have found that transgenic (Tg) mice inoculated with Ha scrapie prions exhibit scrapie infectivity, incubation times and PrP amyloid plaques characteristic of hamsters. These studies are the first demonstration of the synthesis of infectious scrapie prions programmed by a recombinant DNA molecule; furthermore, they have created a shift in prion research from an area restricted to descriptive and correlative observations to one now amenable to experimental manipulation. We propose to determine whether a GSS mutant PrP transgene will spontaneously produce scrapie in mice. The GSS mutant PrP will also be expressed in human cells to determine whether prions are produced spontaneously. We shall use Tg mice with chimeric Ha/Mo transgenes to identify specific domains of HaPrP which are required for Ha prion infectivity synthesis. Additional studies will focus on cultured cells for the synthesis of PrPSc from expression vectors and use of these cells to define the steps of PrPSc synthesis. Considerable effort will be devoted to attempts to develop a new bioassay for prions in cultured cells. We shall also try to develop a system for cell-free synthesis of PrPSc; such a system should lead to the purification of macromolecules involved in the conversion of PrPC or a precursor into PrPSc. The unprecedented discoveries in studies on prion diseases signal new directions in CNS degenerative disease research and may have profound implications for understanding a variety of neurodegenerative processes, most notably some disorders of unknown etiology such as Alzheimer's disease.
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