An antipsychotic drug exerts anti-prion effects by altering the localization of the cellular prion protein

Abstract

Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrPC), an endogenous membrane glycoprotein of uncertain function, into PrPSc, a pathological isoform that replicates by imposing its abnormal folding onto PrPC molecules. A great deal of evidence supports the notion that PrPC plays at least two roles in prion diseases, by acting as a substrate for PrPSc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrPC may transduce neurotoxic signals elicited by other disease-associated protein aggregates. Thus, PrPC may represent a convenient pharmacological target for prion diseases, and possibly other neurodegenerative conditions. Here, we sought to characterize the activity of chlorpromazine (CPZ), an antipsychotic previously shown to inhibit prion replication by directly binding to PrPC. By employing biochemical and biophysical techniques, we provide direct experimental evidence indicating that CPZ does not bind PrPC at biologically relevant concentrations. Instead, the compound exerts anti-prion effects by inducing the relocalization of PrPC from the plasma membrane. Consistent with these findings, CPZ also inhibits the cytotoxic effects delivered by a PrP mutant. Interestingly, we found that the different pharmacological effects of CPZ could be mimicked by two inhibitors of the GTPase activity of dynamins, a class of proteins involved in the scission of newly formed membrane vesicles, and recently reported as potential pharmacological targets of CPZ. Collectively, our results redefine the mechanism by which CPZ exerts anti-prion effects, and support a primary role for dynamins in the membrane recycling of PrPC, as well as in the propagation of infectious prions.

Multimodal small-molecule screening for human prion protein binders

“…SYPRO Orange dye fluorescence in the presence of unfolded PrP was weak, which necessitated DSF screening at 30 µM protein concentration; compounds were accordingly screened at 100 µM, but solubility limitations may have prevented saturable binding with a maximum thermal shift. Our in silico screen utilized a homology model based on a crystal structure of promazine bound to mouse PrP, but promazine has not been shown to bind human PrP in solution, and promazine analogs that exert antiprion activity in cells appear to do so through an orthogonal mechanism 48 . In general, without a positive control available, it is difficult to guide the optimization of screening assays.…”

“…However, these binding events may not be monomeric 45 nor specific to PrP 35,46 . Still other compounds with demonstrated antiprion activity exhibit interaction with PrP only at concentrations orders of magnitude above their effective concentration in cell culture 47,48 .…”

Pharmacological Protein Inactivation by Targeting Folding Intermediates

“…In control conditions, EGFP-PrP localizes almost entirely in the Golgi apparatus and at the plasma membrane, with the latter giving rise to a typical "honeycomb-like" staining of the cell surface ( Figure 4E) 61 . Compounds altering PrP trafficking, as the phenothiazine derivative chlorpromazine, have previously been shown to alter such localization pattern 62 . Incubation with SM875 for 24 h induced a drastic reduction of cell surface EGFP-PrP at concentrations as low as 1 µM.…”

“…HEK293 and N2a cells were obtained from ATCC (ATCC CRL-1573 and CCL-131, respectively). We used a subclone (A23) of HEK293 stably expressing a mouse wild-type PrP or an EGFP-PrP construct, both already described and characterized previously 62 . L929 mouse fibroblasts and inducible RK13 cells were kindly provided by Ina Vorberg (DZNE, Bonn, Germany) 76 and Didier Villette (INRA, Toulouse, France) 72 , respectively.…”

The uptake of tau amyloid fibrils is facilitated by the cellular prion protein and hampers prion propagation in cultured cells

“…Surface staining of PrP C was performed as described in Stincardini et al (44). Cells were incubated with W226 antibody diluted 1:250 in Opti-MEM (Life Technologies) for 15 minutes at 4°C, followed by washing in PBS and fixation with 4% paraformaldehyde:PBS for 10 minutes.…”