Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

Autophagy flux induced by ginsenoside-Rg3 attenuates human prion protein-mediated neurotoxicity and mitochondrial dysfunction.

Mitochondrial quality control is a process by which mitochondria undergo successive rounds of fusion and fission with dynamic exchange of components to segregate functional and damaged elements. Removal of mitochondrion that contains damaged components is accomplished via autophagy. In this study, we investigated whether ginsenoside Rg3, an active ingredient of the herbal medicine ginseng that is used as a tonic and restorative agent, could attenuate prion peptide, PrP (106-126)-induced neurotoxicity and mitochondrial damage. To this end, western blot and GFP-LC3B puncta assay were performed to monitor autophagy flux in neuronal cells; LC3B-II protein level was found to increase after Rg3 treatment. In addition, electron microscopy analysis showed that Rg3 enhanced autophagic vacuoles in neuronal cells. By using autophagy inhibitors wortmannin and 3-methyladenine (3MA) or autophagy protein 5 (Atg5) small interfering RNA (siRNA), we demonstrated that Rg3 could protect neurons against PrP (106-126)-induced cytotoxicity via autophagy flux. We found that Rg3 could also attenuate PrP (106-126)-induced mitochondrial damage via autophagy flux. Taken together, our results suggest that Rg3 is a possible therapeutic agent in neurodegenerative disorders, including prion diseases.

Bcl-2 overexpression protects against amyloid-beta and prion toxicity in GT1-7 neural cells.

In this study we analysed the effect of Bcl-2 on the cytotoxicity induced by the amyloid-beta (Abeta(25-35)) and prion (PrP(106-126)) peptides by using GT1-7puro and GT1-7bcl-2 (overexpressing the anti-apoptotic protein Bcl-2) neural cells. Exposure to Abeta(25-35) (1-5 microM) and PrP(106-126) (25 microM) caused a decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These data were correlated with Abeta(25-35) and PrP(106-126)-induced activation of caspase-9, which is linked to the mitochondrial death pathway, and the activation of the effector caspase-3, suggesting cell death by apoptosis. Furthermore, Bcl-2 overexpression protected from loss of cell viability and caspase-9 and -3 activation induced by Abeta(25-35) and PrP(106-126), showing that Bcl-2 is neuroprotective against apoptotic cell death caused by amyloidogenic peptides.

In vivo toxicity of prion protein in murine scrapie: ultrastructural and immunogold studies.

Prion protein (PrP) is a cell surface, host coded, sialoglycoprotein which accumulates in excess in scrapie, Creutzfeldt-Jakob disease, bovine spongiform encephalopathy and other transmissible spongiform encephalopathies. Infection of mice with the 87 V or ME7 scrapie strains results in distinctive and very different light microscopical patterns of vacuolation and disease specific PrP accumulation. In both of these scrapie strains immunogold electron microscopy was used to locate PrP to the plasmalemma of neurons from where it was released into the neuropil. Initial PrP accumulation around neurons and in early plaques lacking amyloid fibrils was generally not associated with morphological changes either of the neuron or dendrite releasing the PrP or in the adjacent neuropil in which excess PrP accumulated. However, accumulation of pre-amyloid PrP in some brain areas was associated with specific degeneration of dendritic spines and axon terminals. Initial PrP aggregation into fibrils was also associated with tissue damage with both ME7 and 87 V plaques and diffuse accumulations. Tissue damage associated with fibrillogenesis was localized and would not be expected to have clinical significance. We conclude that pre-amyloid PrP release and accumulation is not invariably toxic, either to the neuron releasing PrP or to the neuropil into which it is released. However, axon terminal degeneration and dendritic spine loss in some neuroanatomical areas may be indicative of specific PrP toxicity and may be the main cause of neurological dysfunction in murine scrapie.