Novel in vitro approaches to delineate prion strain conformational variation
Date
2019
Authors
Selwyn, Vanessa Villegas, author
Telling, Glenn, advisor
Zabel, Mark, committee member
Ross, Eric, committee member
Dobos, Karen, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Prions cause in invariably lethal, transmissible neurodegenerative diseases. There are no effective treatments or cures for prion diseases. Unlike other known pathogens, prions replicate in the absence of nucleic acids. Prion diseases stem from the conformational corruption of the cellular prion protein (PrPC) by the pathogenic form (PrPSc) (Prusiner, 1982). The prion phenomenon, protein-templated misfolding, is no longer limited to the prion protein (PrP). Other neurodegenerative disorders, including but not limited to Alzheimer's, Parkinson's, Huntington's are now being recognized as prion-like disorders (Soto, 2012). By exploring the intricacies of prion protein- misfolding, therapeutic approaches might emerge that will be useful in treating other neurodegenerative protein-misfolding disorders. Although the structure of PrPC has been solved (Riek et al 1997, Zahn et al 2000, Garcia et al 2000, Donne et al 2007, Antonyuk et al 2009), the three-dimensional structure of PrPSc has yet to be resolved. A confounding issue to identifying PrPSc structure is the existence of prion strains (Bett et al 2012). In the absence of nucleic acids, prion strain properties are propagated though variations in the conformational structure of PrPSc (Telling et al 1996). As such, prion strains can be defined as an infectious prion protein particle with a specific tertiary conformation that produces a specific neurodegenerative phenotype (Colby et al., 2009). Specifically, a prion strain can be considered to have a strain-specific (Peretz et al 2001) disease phenotype (Collinge et al 1996) based on the prion's ability to be stably propagated, fidelity to neuropathology, disease length, glycosylation profile, molecular weight of PK-resistant PrPSc, resistance to denaturation, amyloid seeding potential and other molecular characteristics. Ultimately, revealing PrPSc structure will provide better understanding of the basis of strains, species adaption and ultimately the species barrier. The traditional methodologies to examine prion strains are costly, time consuming, and do not provide adequate resolution of the PrPSc structure. The overarching aim of my research is to better understand how prions encrypt strain information. In Chapter 1, I outline essential background regarding prions and prion strains. In Chapter 2 and 3, I address the creation of the expanded Cell-Based Conformational Stability Assay, Epitope Stability Assay, and use of a new 7-5 ELISA Conformational Stability Assay. These represent novel tools that use chaotropic agents to probe epitope-mapped regions to identify subtle differences in prion strain structure. The prion strains evaluated were cervid (deer and elk) chronic wasting disease, murine- adapted scrapie (RML, 22L, 139A), murine-adapted chronic wasting disease (mD10) and cervid-adapted (deer and elk) RML. These techniques revealed subtle but significant prion strain structural variations within and between these strains. In Chapter 4, the techniques were used to better understand drug-induced prion evolution and strain evolution in cell culture. Drug-induced prion evolution of PrPSc structure was subtle but detectable within 24 hours of treatment. Additionally, the structural changes were not stable, but in flux. Prion strains evolve in cell culture through serial passaging, they do not recapitulate molecular characteristics of a biological prion infection. Moreover, the prion structure is not stably passaged into naïve cells, or transgenic mice. This makes reliance on chronically infected cells as a basis for anti- prion therapeutic testing inadvisable. In conclusion, the subtle variations encoded in prion strain structure can be detected with the three new techniques in this dissertation: C-CSA, ESA, and 7-5 ELISA-CSA.
Description
Rights Access
Subject
denaturant
RML
CWD
strain
prion