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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
3R01NS035496-05S2
Application #
6345618
Study Section
Special Emphasis Panel (ZRG1 (01))
Program Officer
Kerza-Kwiatecki, a P
Project Start
1996-08-01
Project End
2003-04-30
Budget Start
2000-05-01
Budget End
2001-04-30
Support Year
5
Fiscal Year
2000
Total Cost
$50,000
Indirect Cost
Name
Washington University
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Stewart, Richard S; Harris, David A (2005) A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons. J Biol Chem 280:15855-64
Stewart, Richard S; Harris, David A (2003) Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection. J Biol Chem 278:45960-8
Drisaldi, Bettina; Stewart, Richard S; Adles, Cheryl et al. (2003) Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation. J Biol Chem 278:21732-43
Stewart, R S; Harris, D A (2001) Most pathogenic mutations do not alter the membrane topology of the prion protein. J Biol Chem 276:2212-20
Ivanova, L; Barmada, S; Kummer, T et al. (2001) Mutant prion proteins are partially retained in the endoplasmic reticulum. J Biol Chem 276:42409-21
Stewart, R S; Drisaldi, B; Harris, D A (2001) A transmembrane form of the prion protein contains an uncleaved signal peptide and is retained in the endoplasmic Reticulum. Mol Biol Cell 12:881-9
Soto, C; Kascsak, R J; Saborio, G P et al. (2000) Reversion of prion protein conformational changes by synthetic beta-sheet breaker peptides. Lancet 355:192-7
Chiesa, R; Harris, D A (2000) Nerve growth factor-induced differentiation does not alter the biochemical properties of a mutant prion protein expressed in PC12 cells. J Neurochem 75:72-80
Narwa, R; Harris, D A (1999) Prion proteins carrying pathogenic mutations are resistant to phospholipase cleavage of their glycolipid anchors. Biochemistry 38:8770-7
Daude, N; Lehmann, S; Harris, D A (1997) Identification of intermediate steps in the conversion of a mutant prion protein to a scrapie-like form in cultured cells. J Biol Chem 272:11604-12

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