Models for ΔCR PrP cis-toxicity and WT PrP trans-rescue.

<p>(<b>A</b>) Data from previous reports suggest that ΔCR PrP forms a channel or pore in the plasma membrane, which allows cationic molecules (illustrated as yellow balls) such as the antibiotics used in the DBCA, to enter the cell. (<b>B</b>) WT PrP on the surface of one cell exerts its protective effect <i>in trans</i> by silencing the channel on neighboring cells. A similar effect could be produced by PrP molecules released into the medium or physically transferred from neighboring cells (not shown). The N-terminal polybasic domain (residues 23–31, indicated in green) plays an important role in both the cis-toxicity of ΔCR PrP and the trans-rescuing effect of WT PrP.</p

PrP is expressed on the plasma membrane in differentiated NSCs.

<p>Surface staining of differentiated NSCs shows PrP (green) expression on the surface of both neuronal (MAP-2 positive cells [red]) and non-neuronal (MAP-2 negative) cells from Tg<i>a</i> (A), WT (B), and ΔCR(C), but not KO (D) cells. PrP was detected with 6D11 antibody.</p

Differentiated NSCs express markers for astrocytes, oligodendrocytes, and neurons.

<p>(<b>A–B</b>) Differentiation of NSCs gives rise to cells expressing the astrocytic marker GFAP (red) and the oligodendrocyte marker MBP (green). (<b>C–D</b>) Differentiated NSC cultures also include neurons expressing dendritic (MAP-2, red) and axonal (SMI-31, green) markers. The absence of co-localization of the two neuronal markers indicates polarization of neurons after differentiation. For both (A) and (B), DAPI staining is shown in blue. Representative pictures from WT and ΔCR NSCs are shown. Scale bars = 20 µm.</p

WT PrP rescuing activity can be exerted <i>in trans</i>.

<p>Mixed cultures of GFP-negative or GFP-positive NSCs from of Tg<i>a</i> or ΔCR mice were treated and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033472#pone-0033472-g008" target="_blank">Figure 8</a>. The bar graph shows the number of TUNEL-positive cells, expressed as a percentage of the number of DAPI-stained cells, as determined in 5 fields for each sample group. Bars show means ± SEM (n = 4 independent experiments). The number of TUNEL-positive ΔCR cells, either GFP-positive or GFP-negative, was significantly reduced when these cells were co-cultured with WT PrP-expressing Tg<i>a</i> cells (** p<0.01).</p

Nestin expression is decreased in differentiated NSCs.

<p>(A) NSCs grown in complete media have high levels of nestin expression (green), which decreased after 10 days in differentiation media. DAPI staining is shown in blue. Representative pictures from ΔCR NSCs are shown. Scale bar = 50 µm. (B) NSCs harvested after differentiation for the indicated number of days were immunoblotted with anti-nestin and anti-MAP-2 antibodies. After 10 days of differentiation, nestin is almost undetectable, coincident with a slight increase in MAP-2 expression. Molecular size markers are given in kDa.</p

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

<div><p>Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrP^C), an endogenous membrane glycoprotein of uncertain function, into PrP^Sc, a pathological isoform that replicates by imposing its abnormal folding onto PrP^C molecules. A great deal of evidence supports the notion that PrP^C plays at least two roles in prion diseases, by acting as a substrate for PrP^Sc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrP^C may transduce neurotoxic signals elicited by other disease-associated protein aggregates. Thus, PrP^C 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 PrP^C. By employing biochemical and biophysical techniques, we provide direct experimental evidence indicating that CPZ does not bind PrP^C at biologically relevant concentrations. Instead, the compound exerts anti-prion effects by inducing the relocalization of PrP^C 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 PrP^C, as well as in the propagation of infectious prions.</p></div

CPZ changes the cell surface distribution of EGFP-PrP^C.

<p>HEK293 cells stably expressing EGFP-PrP^C were grown to ~60% confluence on glass coverslips, and then treated with the indicated concentrations of CPZ or Fe(III)-TMPyP for 24h. After fixation and washing, the intrinsic green signal of EGFP-PrP^C was acquired with an inverted microscope coupled with a high-resolution camera equipped with a 488 nm excitation filter.</p

CPZ alters the cell surface localization of PrP^C.

<p><b>A.</b> Cells were seeded on glass coverslips and grown for 24 h to ~60% confluence. For surface staining of PrP, cells were first incubated at 4°C with antibody D18 diluted, then fixed with paraformaldehyde and incubated with fluorescently-labelled secondary antibody. For total PrP staining, cells were permeabilized with Triton X-100, fixed with paraformaldehyde, and then incubated with primary and secondary antibodies. Coverslips were mounted with Fluor-save Reagent (Calbiochem), and analyzed with a Zeiss Imager M2 microscope. <b>B.</b> N2a cells stably expressing mouse WT PrP^C were grown to confluence on glass coverslips, and treated with the indicated concentrations of Fe(III)-TMPyP or CPZ for 24h. For detection of surface PrP^C (SC#1, shown in the picture), coverslips were incubated in ice with antibody 6D11 (this step was omitted for detection of total PrP^C, not shown). Coverslips were blotted on a nitrocellulose membrane soaked in lysis buffer, and incubated with horseradish peroxidase-conjugated secondary antibody. For detection of total PrP^C, cell blots were incubated with the primary and secondary antibodies. The PrP^C signal was revealed by enhanced chemiluminescence. <b>C.</b> PrP^C signal was quantitated by densitometry. The bar graph shows the % ratio of surface to total PrP^C. Each bar represents the mean (± standard error) of three independent experiments (n = 3). Statistically-significant differences (*), estimated by Student <i>t</i>-test, between CPZ-treated and untreated cells were as follow: [3 μM], <i>p</i> = 0.0058; [10 μM], <i>p</i> = 0.00034.</p

CPZ is a weak ligand of PrP^C.

<p><b>A.</b> The interaction of CPZ with recombinant PrP^C was evaluated by SPR. Starting at time 0, the indicated concentrations of CPZ were injected for 130 sec over sensor chip surfaces (GL-H chip, Bio-Rad) on which 16.000 resonance units (RU) of full-length, mouse recombinant PrP^C had previously been captured by amine coupling. The chip was then washed with PBST buffer alone to monitor ligand dissociation. Sensorgrams show CPZ binding in RU. The data were obtained by subtracting the reference channels. No reliable fitting was obtained for any of the curves, a fact that undermined the calculation of the kinetic constants for the interaction. <b>B.</b> CPZ-PrP^C interaction by DMR. Different concentrations of CPZ were added to label-free microplate well surfaces (EnSpire-LFB HS microplate, Perkin Elmer) on which full-length human recombinant PrP^C or BSA had previously been immobilized. Measurements were performed before (baseline) and after (final) adding the compound. The response (pm) was obtained subtracting the baseline output to the final output signals. The output signal for each well was obtained by subtracting the signal of the protein-coated reference area to the signal of uncoated area. The CPZ signals (red dots) were fitted (black line) to a sigmoidal function using a 4 parameter logistic (4PL) non-linear regression model; <i>R</i>^<i>2</i> = 0.99; <i>p</i> = 0.00061.</p