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 bloodbrain 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.protein misfolding | high-throughput screening | prion therapeutics

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD(+)) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD(+) followed by decreased ATP production, and are completely rescued by treatment with NAD(+) or its precursor nicotinamide because of restoration of physiological NAD(+) levels. Toxic prion protein-induced NAD(+) depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD(+). Intranasal NAD(+) treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD(+) starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD(+) replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases.

Prion diseases are infectious and belong to the group of protein misfolding neurodegenerative diseases. In these diseases, neuronal dysfunction and death are caused by the neuronal toxicity of a particular misfolded form of their cognate protein. The ability to specifically target the toxic protein conformer or the neuronal death pathway would provide powerful therapeutic approaches to these diseases. The neurotoxic forms of the prion protein (PrP) have yet to be defined but there is evidence suggesting that at least some of them differ from infectious PrP (PrP Sc ). Herein, without making an assumption about size or conformation, we searched for toxic forms of recombinant PrP after dilution refolding, size fractionation, and systematic biological testing of all fractions. We found that the PrP species most neurotoxic in vitro and in vivo (toxic PrP, TPrP) is a monomeric, highly α-helical form of PrP. TPrP caused autophagy, apoptosis, and a molecular signature remarkably similar to that observed in the brains of prion-infected animals. Interestingly, highly α-helical intermediates have been described for other amyloidogenic proteins but their biological significance remains to be established. We provide unique experimental evidence that a monomeric α-helical form of an amyloidogenic protein represents a cytotoxic species. Although toxic PrP has yet to be purified from prion-infected brains, TPrP might be the equivalent of one highly neurotoxic PrP species generated during prion replication. Because TPrP is a misfolded, highly neurotoxic form of PrP reproducing several features of prion-induced neuronal death, it constitutes a useful model to study PrP-induced neurodegenerative mechanisms.

Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.

Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrP C into PrP Sc in vitro . In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrP Sc -converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA.