Role of Lipid Rafts and GM1 in the Segregation and Processing of Prion Protein

<div><p>The prion protein (PrP^C) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrP^C is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrP^C raft association, together with raft lipid composition, appears essential for the conversion of PrP^C into the scrapie isoform PrP^Sc, and the development of prion disease. Controversial findings were reported on the nature of PrP^C-containing rafts, as well as on the distribution of PrP^C between rafts and non-raft membranes. We investigated PrP^C/ganglioside relationships and their influence on PrP^C localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrP^C conformations coexist: in lipid rafts PrP^C is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrP^Sc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrP^C conformation and interaction with lipid bilayers, without modifying PrP^C distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.</p></div

GM1-containing liposomes alter PrP^C structure.

<p><u>Panel A</u>- Circular dichroism spectra of recPrP (23–231) alone (black line) or mixed with POPC liposomes (blue line) or GM1-containing POPC liposomes (red line). <u>Panel B</u>- Results obtained replacing POPC with DPPC liposomes.</p

Influence of GM1 cells treatment in PrP^C processing.

<p>CGCs, after incubation with GM1/[³H]GM1 at a final concentration of 2×10⁻⁶ M at 37°C for 4 h, were treated with 1% Triton X-100-containing buffer for 30 min on ice. The cellular lysate was submitted to discontinuous sucrose density gradient centrifugation. 50 µg of proteins from gradient fractions were subjected to protein deglycosylation by PNGase F treatment and immunoblotted with 6H4Ab (<u>panel A</u>), SAF32Ab (<u>panel B</u>) and 8G8Ab (<u>panel C</u>). Bands were analyzed and quantified by Kodak Image Station 2000R interfaced with a Kodak Molecular Imaging Software. Representative blots from three independent experiments are shown. 5TQ =  fraction 5 not subjected to PNGase F treatment; f.l.  = full length-PrP^C; u  =  unglycosylated PrP^C; g =  glycosylated PrP^C.</p

Distribution of gangliosides radioactivity and proteins in the different fractions of the sucrose gradient.

<p>CGCs, after incubation with different gangliosides (GM3, GM1 or GD1a) and correspondent radiolabelled gangliosides ([³H]GM3, [³H]GM1 or [³H]GD1a), at a final concentration of 2×10⁻⁶ M at 37°C for 4 h, were treated with 1% Triton X-100-containing buffer for 30 min on ice. The cellular lysate was submitted to discontinuous sucrose density gradient centrifugation. One-milliliter fractions were withdrawn from the gradient and submitted to [³H]GM3, [³H]GM1 or [³H]GD1a radioactivity determination (p<u>anel A</u>) and evaluation of proteins distribution (p<u>anel B</u>). Data are means ± SD from at least three independent experiments performed in triplicate.</p

Cellular gangliosides treatment.

<p>CGCs were incubated with different gangliosides (GM3, GM1 or GD1a) and correspondent radiolabelled gangliosides ([³H]GM3, [³H]GM1 or [³H]GD1a), at a final concentration of 2×10⁻⁶ M at 37°C for 4 h. At the end of incubation, the ganglioside solution was removed and cells were washed 3 times with Locke's solution and maintained at 37°C for 20 min in 3 mL of FBS-BME. The lipids extract, from cell homogenates, were analysed to determine the ganglioside incorporation (p<u>anel A</u>) and metabolism by HPTLC following radioactivity imaging (p<u>anel B</u>). Lane 1: granule cell ganglioside pattern; lane 2: granule cells ganglioside extracted after incubation with GM3/[³H]GM3 2×10⁻⁶ M at 37°C for 4 h; lane 3: granule cells ganglioside extracted after incubation with GM1/[³H]GM1 2×10⁻⁶ M at 37°C for 4 h; lane 4: granule cells ganglioside extracted after incubation with GD1a/[³H]GD1a 2×10⁻⁶ M at 37°C for 4 h; lane 5: [³H]GM3 standard; lane 6. [³H]GM1 and [³H]GD1a standards. * Gangliosides endogenous content as reported by Palestini et al., 1991. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098344#pone.0098344-Palestini1" target="_blank">[40]</a></p

Immunofluorescence analysis of PrP^C distribution in CGCs.

<p><u>Panel A</u>: CGCs were immunolabelled with PrP^C 6H4Ab (green) and the Alexa Fluor 594 cholerae toxin B (red) to visualize lipid rafts. <u>Panel B</u>: CGCs were doubly immunolabelled with PrP^C SAF32 (green) and <i>cholerae toxin</i> B subunit (CTB). Insets show the different colocalization of the Abs with lipid rafts, indicating that 6H4Ab preferentially recognizes PrP^C resident in lipid rafts, while SAF32Ab show PrP^C that is widespread throughout the membrane. Arrows mark the position of CTB. <u>Panel C and D</u>: double immunofluorescence of PrP^C antibodies with Giantin (red) denoting a major presence of SAF32Ab-positive PrP^C in the Golgi apparatus with respect to 6H4Ab. Arrows mark the colocalization. <u>Panels E and F</u>: double immunofluorescence of PrP^C antibodies (green) showing a lack of colocalization with Na⁺-K⁺/ATPase, a non-lipid raft plasmamembrane marker (red). Scale bar: 10 µm.; insets: 20 µm.</p

Effect of gangliosides treatment on the localization of PKC, ADAM10 and Thy1 in gradient fractions from gangliosides treated-CGCs.

<p>Cells, after incubation with different gangliosides (GM3, GM1 or GD1a) and correspondent radiolabelled gangliosides ([³H]GM3, [³H]GM1 or [³H]GD1a), at a final concentration of 2×10⁻⁶ M at 37°C for 4 h, were treated with 1% Triton X-100-containing buffer for 30 min on ice. The cellular lysate was subjected to discontinuous sucrose density gradient centrifugation. One-milliliter fractions were analyzed by immunoblotting with anti-PKC (<u>panel A</u>), anti-ADAM10 (<u>panel B</u>) and anti-Thy1 (<u>panel C</u>) antibodies. Immunoblot bands were analyzed and quantified by Kodak Image Station 2000R interfaced with a Kodak Molecular Imaging Software. The enrichment of the proteins in DRM was calculated as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098344#pone.0098344-Botto1" target="_blank">[27]</a>. The data reported for each protein are the mean of 3 immunoblots ± S.D. obtained from 3 independent sucrose gradients. Ctrl =  CGCs control; GM3 = CGCs treated with GM3; GM1 =  CGCs treated with GM1; GD1a =  CGCs treated with GD1a. Ctrl <i>vs</i> GM1 *p<0.01 (one way ANOVA).</p

Characterization of PrP^C distribution in CGCs.

<p><u>Panel A</u>- Schematic diagram of the proteolysis of PrP^C and the epitope recognized by the antibodies used in this study. The native full-length PrP^C is shown with its C-terminal GPI-anchor, the two N-linked glycosylation sites, the three helical regions (HA, HB and HC), the octapeptide repeat region (black), and the “toxic” 106–126 domain (white). The epitopes for antibody SAF32 (residues 79–92, in the unstructured octapetidic stretch) and 6H4 (residues 114–152, localized in HA) are indicated. The two cleavage sites generating N1/C1 (α-cleavage) and N2/C2 (β-cleavage) are shown by arrows. C1 is recognized only by 6H4. <u>Panel B</u> Characterization of PrP^C localization in gradient fractions prepared from control CGCs. Cells were incubated with 1% Triton X-100-containing buffer for 30 min on ice. The suspension was subjected to discontinuous sucrose density gradient centrifugation. One-milliliter fractions were withdrawn from the gradient, submitted to 15% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with 6H4 or SAF32 antibodies against PrP^C (20 µg proteins/lane), followed by ECL detection. Representative blots from three independent experiments are shown. g: glycosylated PrP^C; u: unglycosylated PrP^C. <u>Panel C and D</u>: immunofluorescence images showing PrP^C distribution visualized by 6H4Ab (C) and SAF32Ab (D) in 8 DIV CGCs. Note the clusterized pattern visualized by 6H4Ab with respect to the diffuse staining of SAF32Ab. DAPI staining (blu) evidences nuclei. Scale bar: 10 µm.</p

Temperature and GM1 dose dependence of PrP^C distribution in GM1-treated CGCs.

<p>Cells after incubation with GM1/[³H]GM1 2×10⁻⁶ M at 4°C for 4 h or 1×10⁻⁶ M at 37°C for 4 h, were treated with 1% Triton X-100-containing buffer for 30 min on ice. A small amount of cells homogenates were analyzed to determine the gangliosides incorporation (<u>panel A</u>) and the residual was submitted to discontinuous sucrose density gradient centrifugation. One-milliliter fractions were withdrawn from the gradient and submitted to proteins and [³H]GM1 radioactivity determination (<u>panel B)</u>. 20 µg of proteins from different fractions were submitted to 15% SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with 6H4Ab or SAF32Ab followed by ECL detection (<u>panel C and D</u>). Immunoblot bands were analyzed and quantified by Kodak Image Station 2000R interfaced with a Kodak Molecular Imaging Software. Representative blots from three independent experiments are shown. Ctrl  = control CGCs; GM1  =  GM1-treated CGCs.</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