Diagram of the 15B3-immunopurification procedure.

<p>The scheme summarizes the main steps of the purification protocol (A), and illustrates the putative mechanism by which sonication elutes aggregated PrP from 15B3-coated beads (B).</p

Mutant PrP aggregates from Tg(PG14) and Tg(CJD) mice are toxic to primary neurons.

<p>Cortical neurons from C57BL/6 mice were exposed to ∼15 nM of PrP aggregates purified from Tg(PG14) and Tg(CJD) mice, to the vehicle alone (PBS), or to 15B3-immunoprecipitated P3 fractions from <i>Prnp</i>^0/0 mice. Cell survival was quantified by MTT assay after seven days, and expressed as a percentage of untreated cells. Data are the mean±SEM of 8–24 replicates from three independent experiments. **<i>p</i><0.005 and ***<i>p</i><0.0001 <i>vs.</i> untreated (Mann-Whitney test).</p

Immunopurified aggregates of PrP^Sc support <i>in vitro</i> misfolding of PrP^C by PMCA.

<p>Aggregates of PrP^Sc immunopurified from P3 fractions of RML-infected C57BL/6 mice (lanes 1–8), or immunoprecipitates of P3 fractions of <i>Prnp</i>^0/0 mice (lanes 9–12) were resuspended in PBS (lanes 1–4 and 9–12) or immobilized on glass coverslips by ultracentrifugation (lanes 5–8), and mixed with brain homogenates of Tg(WT) mice overexpressing 3F4-tagged PrP^C. Mixtures were either rapidly frozen (lanes 1 and 2, 5 and 6, 9 and 10) or subjected to 90 cycles of sonication/incubation (lanes 3 and 4, 7 and 8, 11 and 12). Samples were then either subjected to proteinase K (PK) (lanes 2, 4, 6, 8, 10 and 12), or left undigested (lanes 1, 3, 5, 7, 9 and 11). All samples were analyzed by Western blotting with 3F4 antibody.</p

15B3 immunoprecipitation yields pure preparations of infectious and non-infectious mouse PrP aggregates.

<p>(A) PrP aggregates were immunoprecipitated from P3 fractions of Tg(PG14) brains with antibody 15B3. PG14 PrP eluted (E lanes) by incubation in 0.1 M Tris-Glycine buffer at different pH (lanes 1–7) or by sonication in PBS (lanes 8–9), and the residual protein eluted by boiling the Dynabeads in SDS (B lanes), were detected by Western blotting with anti-PrP antibody 3F4. (B) PrP aggregates, immunopurified and eluted by sonication, from un-inoculated Tg(PG14) (lane 1) and Tg(CJD) (lane 2) mice, or RML-infected C57BL/6 (lane 3) and Tg(PG14) (lane 4) mice were visualized by silver staining (top panel) or by Western blotting (bottom panel) using anti-PrP antibody 8H4. No protein bands were detected when the purification was done using <i>Prnp</i>^0/0 mouse brains (lane 5). (C) Two samples of PrP^Sc immunopurified from RML-infected C57BL/6 mice were run on SDS-PAGE, and the gels stained to saturation with silver (lanes 1–2) or immunoblotted with antibody 8H4 (lanes 3–4).</p

Surface plasmon resonance shows that monoclonal antibody 15B3 selectively captures PG14 PrP aggregates from P3 fractions.

<p>Fractions P3 prepared from whole brains of Tg(PG14) or <i>Prnp</i>^0/0 mice were perfused for 5 min (bars) over the sensor surface of SPR chips on which antibody 15B3 (A) or 3F4 (B) had been immobilized. The sensorgrams (time course of the SPR signal in Resonance Units, RU) refer to the specific binding to the antibody (binding was negligible on sensor surfaces without antibody). Significant binding was detected when P3 fractions containing PG14 PrP (black line) were flowed over the 15B3-coated chip (A), but not on the 3F4-coated chip (B). No binding was observed when a P3 fraction from <i>Prnp</i>^0/0 mice was analyzed (gray line).</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 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

Image_1_A cytokine/PTX3 prognostic index as a predictor of mortality in sepsis.tiff

BackgroundEarly prognostic stratification of patients with sepsis is a difficult clinical challenge. Aim of this study was to evaluate novel molecules in association with clinical parameters as predictors of 90-days mortality in patients admitted with sepsis at Humanitas Research Hospital.MethodsPlasma samples were collected from 178 patients, diagnosed based on Sepsis-3 criteria, at admission to the Emergency Department and after 5 days of hospitalization. Levels of pentraxin 3 (PTX3), soluble IL-1 type 2 receptor (sIL-1R2), and of a panel of pro- and anti-inflammatory cytokines were measured by ELISA. Cox proportional-hazard models were used to evaluate predictors of 90-days mortality.ResultsCirculating levels of PTX3, sIL-1R2, IL-1β, IL-6, IL-8, IL-10, IL-18, IL-1ra, TNF-α increased significantly in sepsis patients on admission, with the highest levels measured in shock patients, and correlated with SOFA score (PTX3: r=0.44, pConclusionThese data suggest that a prognostic index based on selected cytokines, PTX3 and clinical parameters, and hence easily adoptable in clinical practice, performs in predicting 90-days mortality better than SOFA. An independent validation is required.</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

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