Structural characterization of infectious bovine spongiform encephalopathy prion strains

  • Author / Creator
    Kamali Jamil, Razieh
  • Prion diseases, also termed transmissible spongiform encephalopathies (TSEs), are progressive and fatal neurodegenerative disorders in humans and animals. The condition is associated with spongiform changes in the brain tissue. Bovine spongiform encephalopathy (BSE), commonly known as “mad cow disease,” belongs to a group of prion diseases primarily affecting cattle. BSE was first reported in the 1980s in the UK and later spread to other parts of the world, including Canada. The first case of BSE in Canada was detected in Alberta in May 2003. Since then, a total of 19 BSE cases have been detected in Canada. Transmission studies suggest a causal link between BSE and variant Creutzfeldt-Jakob disease (vCJD) in humans. Three BSE strains have been known to cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. These BSE strains exhibit distinct biochemical and histopathological characteristics.The main pathological hallmark in prion disease is a structural transition from a normal host-encoded prion protein (PrPC) to an infectious and disease-causing isoform (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, with a high propensity to self-assemble into amyloid fibrils. Although the high-resolution structure of PrPC has been resolved, despite extensive efforts, the high-resolution structure of infectious PrPSc molecules has yet remained to be determined, mainly due to its insolubility and tendency to aggregate. The previous structural studies on different prion strains have provided some insights into the overall architecture of these transmissible agents. However, no structural characterization has been reported for infectious BSE prions. To fill this gap, in this research, I performed structural characterization of infectious, brain-derived C-, H-, and L-type BSE prions employing transmission electron microscopy (TEM) and image processing approaches. First, I purified BSE prions from transgenic mice brains expressing bovine PrP sequence using phosphotungstate anion (PTA) precipitation and sucrose density gradient purification techniques. I performed morphological characterization of the isolated BSE prion amyloid fibrils on negatively stained electron micrographs and examined whether there are conformational differences between the three BSE strains. In addition, to gain insights into the three-dimensional structure of these fibrils, I employed helical reconstruction and 2D average techniques on the EM micrographs of the purified BSE samples. Afterwards, I developed immunogold labeling methods using various gold-conjugated anti-PrP monoclonal antibodies and Fab fragments to confirm the presence of BSE prions at the ultrastructural level. Bioassay experiments were conducted by intracerebral inoculation of Tg4092 transgenic mice, expressing bovine prion protein, to assess the infectivity of the purified prion samples. In this study, I presented morphological information and 3D reconstructions of C-, L-, and H-type BSE fibrils. EM analysis of the purified samples of the BSE strains revealed amyloid fibrils exhibiting helical twist, which were morphologically distinguishable between the three strains. Helical reconstruction of the amyloid fibrils provided further insights into the structure of these agents. Our results could establish that the conformational features of different BSE strains dictate their distinct biochemical and phenotypic properties. Our data are compatible with the proposed model of four-rung beta-solenoid for the infectious prion protein structure. The findings of this research may have implications for understanding the relationship between pathological properties and conformations in different prion strains and could provide a basis for elucidating the 3D structure of other infectious prion strains. Moreover, our findings may facilitate therapeutic interventions against prion disease.

  • Subjects / Keywords
  • Graduation date
    Spring 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.