Hana Serban

Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice

There is increasing concern over the extent to which bovine spongiform encephalopathy (BSE) prions have been transmitted to humans, as a result of the rising number of variant Creutzfeldt–Jakob disease (vCJD) cases. Toward preventing new transmissions, diagnostic tests for prions in livestock have been developed using the conformation-dependent immunoassay (CDI), which simultaneously measures specific antibody binding to denatured and native forms of the prion protein (PrP). We employed high-affinity recombinant antibody fragments (recFab) reacting with residues 95–105 of bovine (Bo) PrP for detection and another recFab that recognizes residues 132–156 for capture in the CDI. We report that the CDI is capable of measuring the disease-causing PrP isoform (PrPSc) in bovine brainstems with a sensitivity similar to that of end-point titrations in transgenic (Tg) mice expressing BoPrP. Prion titers were ∼107 ID50 units per gram of bovine brainstem when measured in Tg(BoPrP) mice, a figure ∼10 times greater than that determined by bioassay in cattle and ∼10,000× greater than in wild-type mice. We also report substantial differences in BoPrPSc levels in different areas of the obex region, where neuropathology has been consistently observed in cattle with BSE. The CDI was able to discriminate between PrPSc from BSE-infected cattle and Tg(BoPrP) mice as well as from chronic wasting disease (CWD)-infected deer and elk. Our findings argue that applying the CDI to livestock should considerably reduce human exposure to animal prions.

Paradoxical shortening of scrapie incubation times by expression of prion protein transgenes derived from long incubation period mice.

Prolonged incubation times for experimental scrapie in I/LnJ mice are dictated by a dominant gene linked to the prion protein gene (Prn-p). Transgenic mice were analyzed to discriminate between an effect of the I/LnJ Prn-pb allele and a distinct incubation time locus designated Prn-i. Paradoxically, 4 independent Prn-pb transgenic mouse lines had scrapie incubation times shorter than nontransgenic controls, instead of the anticipated prolonged incubation periods. Aberrant or overexpression of the Prn-pb transgenes may dictate abbreviated incubation times, masking genuine Prn-p/Prn-i congruence; alternatively, a discrete Prn-i gene lies adjacent to Prn-p.

Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation.

The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.

Structural intermediates in the putative pathway from the cellular prion protein to the pathogenic form.

Abstract The conversion of the αhelical, protease sensitive and noninfectious form of the prion protein (PrP) into an insoluble, protease resistant, predominantly βsheeted and infectious form (PrP) is the fundamental event in prion formation. In the present work, two soluble and stable intermediate structural states are newly identified for recombinant Syrian hamster PrP(90 231) (recPrP), a dimeric αhelical state and a tetra or oligomeric, βsheet rich state. In 0.2% SDS at room temperature, recPrP is soluble and exhibits αhelical and random coil secondary structure as determined by circular dichroism. Reduction of the SDS concentration to 0.06% leads first to a small increase in αhelical content, whereas further dilution to 0.02% results in the aquisition of βsheet structure. The reversible transition curve is sigmoidal within a narrow range of SDS concentrations (0.04 to 0.02%). Size exclusion chromatography and chemical crosslinking revealed that the αhelical form is dimeric, while the βsheet rich form is tetra or oligomeric. Both the αhelical and βsheet rich intermediates are soluble and stable. Thus, they should be accessible to further structural and mechanistic studies. At 0.01% SDS, the oligomeric intermediates aggregated into large, insoluble structures as observed by fluorescence correlation spectroscopy. Our results are discussed with respect to the mechanism of PrP formation and the propagation of prions.

Spontaneous neurodegeneration in transgenic mice with prion protein codon 101 proline----leucine substitution.

Gerstmann-Sträussler-Scheinker syndrome (GSS) is an autosomal, dominantly inherited, human neurodegenerative disease that can sometimes be transmitted to non-human primates and rodents through intracerebral inoculation of brain homogenates from patients. Recent studies of GSS demonstrated significant genetic linkage between GSS and a leucine substitution at codon 102 of the human prion protein (PrP) gene. Transgenic mice were created to test the biologic activity of this mutation. Spontaneous neurologic disease with spongiform degeneration developed in one of three lines of transgenic mice containing murine PrP genes with a leucine substitution at codon 101 (homologous to codon 102 in humans). Transmission studies of brain homogenates from affected mice are in progress. These results indicate that some of the clinical and pathologic features of GSS can be reproduced in a transgenic mouse paradigm; this represents the first time a dominantly inherited, neurodegenerative process similar to a human disease has been genetically modeled in an experimental animal (Hsiao and Prusiner 1990).

15 Some Strategies and Methods for the Study of Prions

The discovery of the prion protein (PrP) by enriching fractions prepared from scrapie-infected hamster brains transformed research on the prion diseases. Prior to identification of PrP 27–30, the protease-resistant core of the scrapie prion protein (PrP Sc ), almost all studies of prions required bioassays. With the isolation of PrP 27–30 came the ability to apply the tools of molecular cloning, genetics, immunology, cell biology, and structural biology. Many different areas of the prion diseases can now be investigated using a wide variety of approaches. Recombinant PrP expressed in bacteria or mammalian cells can be isolated in large quantities and used for structural studies (Mehlhorn et al. 1996; Blochberger et al. 1997). The biology of PrP Sc formation as well as the molecular pathogenesis of prion diseases can be studied in cultured cells and transgenic (Tg) mice. Similarly, potential therapeutics might be evaluated by measuring the inhibition of PrP Sc formation in scrapie-infected cultured cells or Tg mice. The biology of prions is sufficiently advanced that there are now three reasonably rational approaches available for the development of effective therapies and preventive measures in humans. First, drugs that alter the conformation of PrP Sc and allow the cell to degrade it might prove to be efficacious. Second, drugs that block the formation of nascent PrP Sc by interfering with the binding of PrP C to PrP Sc might also prove effective. Third, pharmacotherapeutics that disrupt the binding of PrP C to protein X may prove the most effective of all the approaches currently available (Kaneko et al. 1997c). In...

Biophysical Studies on Structure Structural Transitions and Infectivity of the Prion Protein

Prions are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrP) designated PrPSc. A protease resistant polypeptide, PrP 27–30, can be derived from PrPSc by limited proteolysis with retention of infectivity. Both PrPSc and the cellular isoform PrPC are encoded by a chromosomal gene; PrPSc is produced from the cellular isoform by a posttranslational process (for review see Prusiner, 1991).