David Peretz

Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Strain-specified relative conformational stability of the scrapie prion protein

Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease‐causing prion protein (PrPSc). To explore this postulate, we examined the properties of PrPSc from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrPSc molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrPSc as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half‐maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT‐C5 isolates, all of which have ∼75‐d incubation periods; a concentration of 1.08 M was found for the DY strain with a ∼170‐d incubation period and ∼1.25 M for the SHa(RML) and 139H isolates with ∼180‐d incubation periods. A mean value of 1.39 M GdnHCl for the Me7‐H strain with a ∼320‐d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrPSc conformers encipher the biological properties of prion strains.

Prion Protein of 106 Residues Creates an Artificial Transmission Barrier for Prion Replication in Transgenic Mice

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.

A change in the conformation of prions accompanies the emergence of a new prion strain.

To investigate the role of the pathogenic prion protein (PrP(Sc)) in controlling susceptibility to foreign prions, two Syrian hamster (SHa) prion strains, Sc237 and DY, were transmitted to transgenic mice expressing chimeric SHa/mouse PrP genes, Tg(MH2M). First passage of SHa(Sc237) prions exhibited prolonged incubation times, diagnostic of a species barrier. PrP(Sc) of the new MH2M(Sc237) strain possessed different structural properties from those of SHa(Sc237), as demonstrated by relative conformational stability measurements. This change was accompanied by a disease phenotype different from the SHa(Sc237) strain. Conversely, transmission of SHa(DY) prions to Tg(MH2M) mice showed no species barrier, and the MH2M(DY) strain retained the conformational and disease-specific properties of SHa(DY). These results suggest a causal relationship between species barriers, changes in PrP(Sc) conformation, and the emergence of new prion strains.

Trafficking of prion proteins through a caveolae-mediated endosomal pathway

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A–gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes

On passaging synthetic prions, two isolates emerged with incubation times differing by nearly 100 days. Using conformational-stability assays, we determined the guanidine hydrochloride (Gdn·HCl) concentration required to denature 50% of disease-causing prion protein (PrPSc) molecules, denoted as the [Gdn·HCl]1/2 value. For the two prion isolates enciphering shorter and longer incubation times, [Gdn·HCl]1/2 values of 2.9 and 3.7 M, respectively, were found. Intrigued by this result, we measured the conformational stabilities of 30 prion isolates from synthetic and naturally occurring sources that had been passaged in mice. When the incubation times were plotted as a function of the [Gdn·HCl]1/2 values, a linear relationship was found with a correlation coefficient of 0.93. These findings demonstrate that (i) less stable prions replicate more rapidly than do stable prions, and (ii) a continuum of PrPSc structural states enciphers a multitude of incubation-time phenotypes. Our data argue that cellular machinery must exist for propagating a large number of different PrPSc conformers, each of which enciphers a distinct biological phenotype as reflected by a specific incubation time. The biophysical explanation for the unprecedented plasticity of PrPSc remains to be determined.

Prions in skeletal muscle

Considerable evidence argues that consumption of beef products from cattle infected with bovine spongiform encephalopathy (BSE) prions causes new variant Creutzfeldt–Jakob disease. In an effort to prevent new variant Creutzfeldt–Jakob disease, certain “specified offals,” including neural and lymphatic tissues, thought to contain high titers of prions have been excluded from foods destined for human consumption [Phillips, N. A., Bridgeman, J. & Ferguson-Smith, M. (2000) in The BSE Inquiry (Stationery Office, London), Vol. 6, pp. 413–451]. Here we report that mouse skeletal muscle can propagate prions and accumulate substantial titers of these pathogens. We found both high prion titers and the disease-causing isoform of the prion protein (PrPSc) in the skeletal muscle of wild-type mice inoculated with either the Me7 or Rocky Mountain Laboratory strain of murine prions. Particular muscles accumulated distinct levels of PrPSc, with the highest levels observed in muscle from the hind limb. To determine whether prions are produced or merely accumulate intramuscularly, we established transgenic mice expressing either mouse or Syrian hamster PrP exclusively in muscle. Inoculating these mice intramuscularly with prions resulted in the formation of high titers of nascent prions in muscle. In contrast, inoculating mice in which PrP expression was targeted to hepatocytes resulted in low prion titers. Our data demonstrate that factors in addition to the amount of PrP expressed determine the tropism of prions for certain tissues. That some muscles are intrinsically capable of accumulating substantial titers of prions is of particular concern. Because significant dietary exposure to prions might occur through the consumption of meat, even if it is largely free of neural and lymphatic tissue, a comprehensive effort to map the distribution of prions in the muscle of infected livestock is needed. Furthermore, muscle may provide a readily biopsied tissue from which to diagnose prion disease in asymptomatic animals and even humans.

Inactivation of Prions by Acidic Sodium Dodecyl Sulfate

ABSTRACT Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of ≤4.5 or ≥10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 107; however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.

Aβ40 Oligomers Identified as a Potential Biomarker for the Diagnosis of Alzheimer's Disease

Alzheimers Disease (AD) is the most prevalent form of dementia worldwide, yet the development of therapeutics has been hampered by the absence of suitable biomarkers to diagnose the disease in its early stages prior to the formation of amyloid plaques and the occurrence of irreversible neuronal damage. Since oligomeric Aβ species have been implicated in the pathophysiology of AD, we reasoned that they may correlate with the onset of disease. As such, we have developed a novel misfolded protein assay for the detection of soluble oligomers composed of Aβ x-40 and x-42 peptide (hereafter Aβ40 and Aβ42) from cerebrospinal fluid (CSF). Preliminary validation of this assay with 36 clinical samples demonstrated the presence of aggregated Aβ40 in the CSF of AD patients. Together with measurements of total Aβ42, diagnostic sensitivity and specificity greater than 95% and 90%, respectively, were achieved. Although larger sample populations will be needed to confirm this diagnostic sensitivity, our studies demonstrate a sensitive method of detecting circulating Aβ40 oligomers from AD CSF and suggest that these oligomers could be a powerful new biomarker for the early detection of AD.

Transmission Barriers for Bovine, Ovine, and Human Prions in Transgenic Mice

ABSTRACT Transgenic (Tg) mice expressing full-length bovine prion protein (BoPrP) serially propagate bovine spongiform encephalopathy (BSE) prions without posing a transmission barrier. These mice also posed no transmission barrier for Suffolk sheep scrapie prions, suggesting that cattle may be highly susceptible to some sheep scrapie strains. Tg(BoPrP) mice were also found to be susceptible to prions from humans with variant Creutzfeldt-Jakob disease (CJD); on second passage in Tg(BoPrP) mice, the incubation times shortened by 30 to 40 days. In contrast, Tg(BoPrP) mice were not susceptible to sporadic, familial, or iatrogenic CJD prions. While the conformational stabilities of bovine-derived and Tg(BoPrP)-passaged BSE prions were similar, the stability of sheep scrapie prions was higher than that found for the BSE prions but lower if the scrapie prions were passaged in Tg(BoPrP) mice. Our findings suggest that BSE prions did not arise from a sheep scrapie strain like the one described here; rather, BSE prions may have arisen spontaneously in a cow or by passage of a scrapie strain that maintains its stability upon passage in cattle. It may be possible to distinguish BSE prions from scrapie strains in sheep by combining conformational stability studies with studies using novel Tg mice expressing a chimeric mouse-BoPrP gene. Single-amino-acid substitutions in chimeric PrP transgenes produced profound changes in incubation times that allowed us to distinguish prions causing BSE from those causing scrapie.