Biology and Biomedical Sciences: Molecular Genetics and Genomics
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
ABSTRACT OF THE DISSERTATION Aggregation & Localization of a Disease-Associated Prion Protein: PrP) Mutant by Andrea Rhonda Zaragoza Medrano Doctor of Philosophy in Biology and Biomedical Sciences: Genetics) Washington University in St. Louis, 2009 Professor David A. Harris, Chairperson Prion protein: PrP) is a GPI-anchored sialoglycoprotein involved in the pathogenesis of infectious and inherited forms of transmissible spongiform encephalopathies: TSEs). A nine-octapeptide insertional mutation in the prion protein: PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular localization of this mutant PrP: designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo. Tg(PG14-EGFP) mice develop an ataxic neurological illness characterized by astrogliosis, PrP aggregation, and accumulation of a partially protease-resistant form of the mutant PrP. Strikingly, PG14-EGFP forms numerous fluorescent aggregates in the neuropil and white matter of multiple brain regions. These aggregates are particularly prominent along axonal tracts in both brain and peripheral nerve, and similar intracellular deposits are visible along the processes of cultured neurons. Our results reveal intra-axonal aggregates of a mutant PrP, which could contribute to the pathogenesis of familial prion disease by disrupting axonal transport. Wild-type PrP molecules lacking the GPI anchor: WT∆GPI) display aberrant glycosylation and are secreted into the extracellular space. When inoculated with scrapie, transgenic mice expressing WT∆GPI display dense intracerebral plaques that are larger and more dense than plaques found in their wild-type counterparts, suggesting that GPI anchor deletion promotes aggregation in infectious prion disorders. Thus far, the role of the GPI anchor has not been determined for disease-associated familial mutants of PrP. PrP mutant PG14 becomes glycosylated, forms spontaneous aggregates, and is partially retained within the ER and Golgi in cells. To determine whether the GPI anchor affects cellular behavior of the mutant protein, we generated a PG14ΔGPI construct and investigated its glycosylation, localization, and spontaneous aggregation in transfected mammalian cells. We demonstrate that deletion of the GPI anchor impairs PG14 glycosylation, but has no effect the mutant's ability to aggregate, as assayed by detergent insolubility and sucrose gradient assays in cells. Furthermore, PG14∆GPI shows similar intracellular retention and localization compared with full-length PG14. These studies show that the GPI anchor is crucial for proper glycosylation of PG14, but does not affect aggregation or localization of a genetic PrP mutant. Whether the GPI anchor affects clinical progression of familial prion disease remains to be determined. Our analysis provides foundational information for the continued study of the role of the GPI anchor in familial prion disease pathogenesis.
Medrano, Andrea Rhonda, "Aggregation & Localization of a Disease-Associated Prion Protein (PrP) Mutant" (2009). All Theses and Dissertations (ETDs). 238.
Permanent URL: http://dx.doi.org/10.7936/K7KH0KB6