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

Dynamin inhibitors abrogate the drug-hypersensitizing effect of ΔCR PrP.

<p><b>A.</b> The DBCA was employed to evaluate the anti-ΔCR PrP effects of the different dynamin inhibitors. The bar graph shows the quantification of the dose-dependent rescuing effect of each compound at the indicated concentrations. Mean values were obtained from a minimum of 4 independent experiments (n = 4), and expressed as percentage of cell viability rescue. Statistically-significant differences (*) between compound-treated and untreated cells were estimated by Student <i>t</i>-test: [Iminodyn-17, 10 μM], <i>p</i> = 0,00487; [Iminodyn-22, 10 μM], <i>p</i> = 0,00075.</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

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 does not affect prion amplification in vitro.

<p><b>A.</b> Brain homogenates from terminally ill voles infected with an Italian vole-adapted scrapie strain were diluted 1:10 (F, lanes 1 and 3) or 1:100 (A, lanes 2 and 4) in PMCA substrate in presence of vehicle (VHC, lanes 1–2) or 500 μM CPZ (lanes 3 and 4). Samples diluted 1:100 were subjected to a single PMCA round, while those diluted 1:10 were kept frozen and used to determine the amplification factor. Samples were PK-digested and analyzed by Western Blotting with antibody SAF84. The graph illustrates mean amplification factors (± standard error) obtained with CPZ or vehicle alone, from three independent experiments (n = 3). We detected no statistical differences between CPZ and vehicle control. <b>B.</b> A 50 μl aliquot of 1% Tg338 brain homogenate, seeded with different dilutions of scrapie (1:10–1:108) were mixed with 0.5 μl of DMSO, TMPyP (used as positive control) or CPZ diluted in DMSO (final concentrations as shown in the picture) and subjected to a unique 24 h round of standard PMCA. Amplified samples were digested with 200 μg/ml of proteinase K (PK) and analyzed by western blotting using monoclonal antibody SAF83 (1:400). While TMPyP exhibited a potent inhibitory activity toward prion amplification (estimated to be at least 10⁵ fold at 100 μM and 10−10² fold at 10 μM), CPZ did not show any inhibitory effect even at the highest concentration (500 μM). C: Normal, untreated brain homogenate. The results are representative of three independent replicates (n = 3).</p

CPZ inhibits prion replication in cells.

<p>N2a cells chronically infected with the 22L or RML prion strains were incubated with increasing concentrations (indicated) of CPZ, TP (10 μM), or vehicle (VHC) for 72h. The level of PrP^Sc in cell lysates was estimated by detecting the amount of PK-resistant PrP by Western blotting, using anti-PrP^C antibody D18. Signals from at least three independent experiments (n>3) were quantified by densitometric analysis of gel blots, normalized on the total amount of proteins (obtained by Ponceau staining) in PK-untreated replicates, and expressed in the bar graph as mean percentage (%) of the signal in vehicle-treated cells (± standard error). Statistical differences (*) between CPZ and vehicle control were estimated by Student <i>t</i>-test: for the 22L strain, [1 μM], <i>p</i> = 0.209; [3 μM], <i>p</i> = 0.074; [10 μM], <i>p</i> = 0.041; for RML strain, [1 μM] <i>p</i> = 0,274; [3 μM] <i>p</i> = 0,123; [10 μM] <i>p</i> = 0,0451. Estimated IC₅₀ values for CPZ were as follow: for the 22L strain, 4.28 ± 1.67 μM; for the RML strain, 7.32 ± 1.92.</p

CPZ suppresses the drug-hypersensitizing effect of ΔCR PrP.

<p><b>A.</b> The DBCA was employed to evaluate the anti-ΔCR PrP effects of CPZ. Stably transfected ΔCR HEK293 cells carrying the hygromycin B resistance cassette were plated in 24-well plates and incubated in medium containing 500 μg/mL of Zeocin, for 48h at 37°C. The picture shows an example of wells after MTT assay (CPZ 10 μM). <b>B.</b> The bar graph illustrates the quantification of the dose-dependent rescuing effect of CPZ. Mean values were obtained from a minimum of 6 independent experiments (n = 6), and expressed as percentage of cell viability rescue, using the following equation: R = (T-Z)/(U-Z) (R: rescuing effect; T: cell viability in CPZ-treated samples; Z: cell viability in zeocin-treated samples; U: cell viability in untreated samples). Statistically-significant differences (*) between CPZ-treated and untreated cells were estimated by Student <i>t</i>-test: [0.1 μM], <i>p</i> = 0.12381; [0.3 μM], <i>p</i> = 0.10209; [1 μM], <i>p</i> = 0.05764; [3 μM], <i>p</i> = 0.00109; [10 μM], <i>p</i> = 4.35 x 10⁻⁹.</p

Inhibitors of the GTPase domain of dynamin I and II induce the redistribution of PrP^C from the cell surface.

<p><b>A-B</b>. High-content analysis of HEK293 cells stably expressing EGFP-PrP^C after a 24h treatment with two dynamin inhibitors (at the indicated concentrations). The chemical structure of each compound, and representative images are shown. <b>C-D.</b> Graphs show the mean percentage of cells (± standard deviation) showing a ratio of surface vs intracellular EGFP-PrP^C signal higher than 1.5, after treatment with raising concentrations of each molecule. <b>E-F.</b> Toxicity profile of Iminodyn-17 and Iminodyn-22. The graphs show the mean percentage (± standard deviation) of the total number of cell nuclei in Iminodyn-17- or Iminodyn-22-treated cells, as detected by Hoechst staining.</p

Dynamin inhibitors block the replication of two different mouse prion strains.

<p><b>A.</b> N2a cells chronically infected with the 22L prion strain were incubated with increasing concentrations (indicated) of Iminodyn-17 or Iminodyn-22 for 72h, using untreated (Unt.) or vehicle (VHC)-treated cells as negative controls, and Fe(III)-TMPyP as positive control. PK-resistant PrP levels were quantified by Western blotting using anti-PrP^C antibody D18. <b>B-C.</b> The levels of PK-resistant PrP in RML-(B) or 22L-(C) infected N2a cells after treatment with dynamin inhibitors were quantified by densitometric analysis of gel blots, signals were normalized on the total amount of proteins (obtained by Ponceau staining) in PK-untreated replicates, and expressed as mean % of the signal in DMSO-treated cells (± standard error). * Statistical differences between dynamin inhibitors and untreated controls were estimated by Student <i>t</i>-test. For the 22L strain: [Iminodyn-17, 3 μM], <i>p</i> = 0.019671; [Iminodyn-17, 10 μM], p = 0,000685; [Iminodyn-22, 10 μM], <i>p</i> = 0,000317. For the RML strain: [Iminodyn-17, 1 μM], <i>p</i> = 0.034828; [Iminodyn-17, 3 μM], <i>p</i> = 0.026235; [Iminodyn-17, 10 μM], p = 0,009796; [Iminodyn-22, 3 μM], <i>p</i> = 0,021711; [Iminodyn-22, 10 μM], <i>p</i> = 0,009508.</p