Prion diseases are fatal neurodegenerative disorders of humans and animals that are characterized by dementia, motor dysfunction, and cerebral amyloidosis. These disorders are unique because they can be either infectious, genetic or sporadic in origin. All three forms result from a posttranslational alteration in the conformation of a host-encoded membrane glycoprotein called PrPc. Infectious cases are caused by prion particles composed of a protease-resistant isoform of PrPc called PrPSc, and inherited cases are linked to mutations in the host gene that encodes PrPc. Although cell culture models for infectious forms of prion disease are available, there has been no analogous system for familial forms. Applicant and his colleagues have recently developed such a model, in which mutant prion proteins associated with each of the known inherited prion disorders undergo conversion to PrPSc in cultured cells. The purpose of this application is to use this system to carry out a detailed analysis of the cellular and molecular mechanisms responsible for familial prion diseases, and to elucidate general features that are shared by familial, infectious and sporadic forms of these disorders. First, we propose to define the molecular mechanisms responsible for attachment of mutant and infectious forms of PrPsc to cellular membranes, using differential extraction, lipid-soluble labeling reagents, mapping of antibody and protease accessibility, and characterization of the aggregation state of protein on the cell surface. Second, we plan to analyze the kinetics of PrPsc biosynthesis in cells expressing mutant PrPs using pulse-chase metabolic labeling, in conjunction with assays of membrane attachment, aggregation state, and association with molecular chaperons. Third, we will investigate the localization and cellular trafficking of mutant PrPs by light and electron microscopy, subcellular fractionation, pharmacological treatments, temperature block, and surface labeling to monitor endocytosis. Fourth, we propose to characterize """"""""strain-specific"""""""" differences between mutant PrPs in their glycosylation patterns and proteinase K cleavage sites, and in the efficiency with which they are converted to PrPsc in cultured neurons from several brain regions. Fifth, we will develop in vitro systems for generation of PrPsc from mutant PrPs, using permeabilized cells, isolated fractions of microsomes and Golgi, and purified proteins.
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