Prion Strain Conformational Stability and the Role of PrP(c) in Prion Strain Interference.

摘要

Prions are the causative agent of transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases characterized by the accumulation of PrPSc, an abnormal conformation of the normal host protein PrPC. Different conformations of PrPSc are thought to be the cause of different phenotypes of TSE disease which are called prion strains. These different prion strains can interact with one another. Prion strain interference is the ability of a long incubation period strain (blocking strain) to extend the incubation period or block a shorter incubation period strain (superinfecting strain) from causing disease. The mechanism of prion strain interference is of interest because this is one of the few known mechanisms where the ability of a prion strain to cause disease is attenuated.;To determine if a single blocking strain can interfere with multiple superinfecting strains, we inoculated hamsters in the sciatic nerve with uninfected or DY TME-infected brain homogenate and superinfected 120 days later with brain homogenate from one of three superinfecting strains. The superinfecting strain caused disease in all hamsters initially inoculated with uninfected brain homogenate. DY TME caused disease in all hamsters initially inoculated with DY TME. This demonstrates that a single blocking strain has the ability to interfere with several superinfecting strains, suggesting that prion strain interference may be a property common to many different prion strains.;To determine if prion strain interference can occur through a natural route of inoculation, hamsters were fed DY TME-infected food pellets and then superinfected with HY TME-infected food pellets. HY TME caused disease in all hamsters. However, with 120 days between inoculations, DY TME extended the incubation period of HY TME, showing that prion strain interference can occur following per os inoculation, suggesting that prion strain interference may be part of the natural biology of TSEs.;To determine if prion strain interference was due to depletion of PrP C by the blocking strain, I developed an assay that isolated PrP C from PrPSc. We inoculated DY TME or uninfected brain homogenate into the sciatic nerve of hamsters, and different time points post inoculation, the spinal cord between vertebrae T10-T13 was removed, dissected into ipsilateral and contralateral halves. PrPC was isolated from each half of the spinal cord and the concentration of PrPC was standardized to the concentration of total protein. I could not detect any change in PrPC level over time, between each half of the spinal cord or between DY TME-infected and uninfected spinal cords. While there was no change in PrPC level, I confirmed DY PrP Sc accumulation in the spinal cord over time. This data shows that PrPC depletion is not part of the mechanism of prion strain interference.;I also developed an assay to differentiate different hamster prions strains based on their conformational stability. Conformational stability is the ability of PrPSc to resist being denatured by a chaotropic agent. I incubated brain homogenate in 0-2% (w/v) sodium dodecyl sulfate (SDS) or 0-2 M guanidine hydrochloride (Gdn HCl) at 70ºC prior to proteinase K digestion and dot blot analysis. Based on this assay, shorter incubation period strains were the most stable and the longer incubation period strains were less stable. This data suggests that conformational stability is a strain property that can be used to differentiate prion strains. Combined with immunohistochemical and in vitro conversion of PrPC data, this data suggests that relatively stable hamster prion strains convert PrP C more efficiently and better resist host cell clearance compared to less stable hamster prion strains.