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Characterization of Infectious Human Prions

  • Author / Creator
    Acevedo Morantes, Claudia Y
  • Human prion diseases present as sporadic, familial, infectious, or iatrogenic forms. They include diseases such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and Fatal Familial Insomnia (FFI). The wide range of phenotypic variation in human prion diseases is caused by aberrantly folded versions of the prion protein, termed PrPSc. Evidence indicates that distinct aggregated forms with heterogeneous morphologies regarding length, width, shape, longitudinal twist, and the number of protofilaments exist, which originate during prion replication. However, the insolubility of PrPSc and its propensity to aggregate make it inaccessible to high-resolution techniques. Another piece of the puzzle is that prions have different proteinase K (PK)-cleavage sites and distinct pathogenic mechanisms, characterized by diverse symptoms, despite an identical primary structure.
    Thus, the proposed hypothesis of this study is that human prions have different PK-cleavage sites despites an identical primary structure, suggesting that prion types exist due to different pathological protein conformations. The central objective of this study is to evaluate the intrinsic heterogeneity between sporadic and familial forms of human prions by characterizing their structural conformations, via electron microscopy (EM) approaches. It is expected that these results will provide new knowledge regarding the conformational structure of these particles and serves as a platform to evaluate the current proposed structural models. Previous studies have used in vitro- and rodent- derived prions to elucidate the structure of prions and the heterogeneity of fibrils in sample populations. Hence, this study is based on the rationale that, regardless that these models have contributed and will continue to contribute in our understanding of the molecular basis of the structural organization of diverse prion species, these conformers are imperfect substitutes to prions derived from human brain.
    To accomplish this objective, I used tilted-beam–transmission electron microscopy (TB-TEM), to determine the mass-per-length (MPL) of PrPSc fibrils. This data can be used to determine the number of protofilaments per fibril, establish the intrinsic heterogeneity of individual samples, and provide a platform to evaluate the current proposed models for prion fibrils.
    The main findings of this work were to delineate a biochemical profile of each human PrP species based on their limited proteolysis, electrophoretic mobility, TEM imaging and MPL measurements. Thus, MPL measurements determined from PrPSc fibrils of sCJD and fCJD, showed a value of ~60 kDa/nm, suggesting that PrPSc fibrils are composed of more than one protofilament. Bioassays in transgenic humanized PrP mice (TgHu(PrP)) also confirmed the infectious nature of these PrPSc species. These results also suggested that no significant differences are present in the structural conformation of PrPSc fibrils derived from sCJD and fCJD.
    PrP species derived from brain tissue of a patient diagnosed with GSS and with the alanine to valine mutation at codon 117 (A117V), and the common methionine to valine polymorphism at codon 129 (M129V), were examined by a series of enzymatic digestions, electrophoretic mobility and TEM imaging. The presence of PK- and thermolysin (TL)- resistant fragments of ~7 kDa/nm and ~16 kDa/nm, respectively was revealed. Although no significant differences were found regarding the amount of PrP species derived from cortex and cerebellum, enzymatic digestions exhibited that PrP species from cortex were more sensitive to PK-digestion when compared with those derived from cerebellum. These findings suggest that brain tropism might have an effect in the structural conformation of PrP species, regardless that they present the same neuropathological features. Evaluation of the infectivity of these particles in Tg(HuPrP) mice, also revealed that these particles maintained their infectious nature, a hallmark of prion particles. Elucidating the structure of prion proteins may aid the design of effective structure-based therapies against different proteinopathies.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-g93k-v635
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.