Prion diseases such as Creutzfeldt–Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP–FRET-enabled high throughput assay (PrP–FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood–brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.

Prions consist mainly of PrPSc, a pathogenic conformer of host-encoded PrPC. Prion populations with distinct phenotypes but associated with PrPSc, having the same amino acid sequence, constitute distinct strains. Strain identity is thought to be encoded by the conformation of PrPSc and to be maintained by seeded conversion. Prion strains can be distinguished by the cell panel assay, which measures their ability to infect distinct cell lines. Brain-derived 22L prions characteristically are able to infect R33 cells (i.e., are “R33 competent”), as well as PK1 cells in the presence of the inhibitor swainsonine (i.e. are “swa resistant”). Here we report that 22L prions retained their characteristic cell tropism and swa resistance when transferred from brain to R33 cells. However, when transferred from the R33 cells to PK1 cells, they gradually became R33 incompetent and swa sensitive, unless the transfer was in the presence of swa, in which case swa resistance and R33 competence were retained. PrPSc associated with swa-resistant/R33-competent and swa-sensitive/R33-incompetent prions had different conformational stabilities. When cloned R33-incompetent/swa-sensitive prions were again propagated in brain, their properties gradually reverted to those of the original brain-derived 22L prions. Our results support the view that 22L prion populations are heterogeneous and that distinct prion variants are selected in different cellular environments.

Prions are transmissible agents that cause lethal neurodegeneration in humans and other mammals. Prions bind avidly to metal surfaces such as steel wires and, when surface-bound, can initiate infection of brain or cultured cells with remarkable efficiency. While investigating the properties of metal-bound prions by using the scrapie cell assay to measure infectivity, we observed, at low frequency, positive assay results in control groups in which metal wires had been coated with uninfected mouse brain homogenate. This phenomenon proved to be reproducible in rigorous and exhaustive control experiments designed to exclude prion contamination. The infectivity generated in cell culture could be readily transferred to mice and had strain characteristics distinct from the mouse-adapted prion strains used in the laboratory. The apparent ”spontaneous generation” of prions from normal brain tissue could result if the metal surface, possibly with bound cofactors, catalyzed de novo formation of prions from normal cellular prion protein. Alternatively, if prions were naturally present in the brain at levels not detectable by conventional methods, metal surfaces might concentrate them to the extent that they become quantifiable by the scrapie cell assay.

Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental advance was the cell culture-based scrapie cell assay, allowing prion titres to be estimated more precisely and an order of magnitude faster than by animal bioassays. Here we describe a bioassay method that exploits the marked binding affinity of prions to steel surfaces. Using steel wires as a concentrating and sensitization tool and combining with an adapted scrapie cell endpoint assay we can achieve, for mouse prions, a sensitivity 100× higher than that achieved in standard mouse bioassays. The rapidity and sensitivity of this assay offers a major advance over small animal bioassay in many aspects of prion research. In addition, its specific application in assay of metal-bound prions allows evaluation of novel prion decontamination methods.

Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA).