Glypican-1 mediates both prion protein lipid raft association and disease isoform formation

Abstract

In prion diseases, the cellular form of the prion protein, PrP(C), undergoes a conformational conversion to the infectious isoform, PrP(Sc). PrP(C) associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrP(C) from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP(C). We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP(C) to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP(C) from rafts, promoting its endocytosis. Glypican-1 and PrP(C) colocalised on the cell surface and both PrP(C) and PrP(Sc) co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP(Sc) formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP(C) on the beta-secretase cleavage of the Alzheimer's amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP(C) and PrP(Sc) in lipid rafts.

Figure 1. Heparin stimulates the endocytosis of PrP^C in a dose-dependent manner and displaces it from detergent-resistant lipid rafts.

(A) SH-SY5Y cells expressing PrP^C were surface biotinylated and then incubated for 1 h at 37°C in the absence or presence of various concentrations of heparin diluted in OptiMEM. Prior to lysis cells were, where indicated, incubated with trypsin to digest cell surface PrP^C. Cells were then lysed and PrP^C immunoprecipitated from the sample using antibody 3F4. Samples were subjected to SDS PAGE and western blot analysis and the biotin-labelled PrP^C detected with peroxidase-conjugated streptavidin. (B) Densitometric analysis of multiple blots from four separate experiments as described in (A) is shown. (C) SH-SY5Y cells expressing PrP^C were surface biotinylated and then incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP^C was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to SDS-PAGE and western blotting. The gradient fractions from both the untreated and heparin treated cells were analysed on the same SDS gel and immunoblotted under identical conditions. The biotin-labelled PrP^C was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. (D) Densitometric analysis of the proportion of total PrP^C in the detergent soluble fractions of the plasma membrane. (E) Untransfected SH-SY5Y cells and SH-SY5Y cells expressing either PrP^C or PrP-TM were grown to confluence and then incubated for 1 h in the presence or absence of 50 µM heparin prepared in OptiMEM. Media samples were collected and concentrated and cells harvested and lysed. Cell lysate samples were immunoblotted for PrP^C using antibody 3F4, with β-actin used as a loading control. (F) Quantification of PrP^C and PrP-TM levels after treatment of cells with heparin as in (E). Experiments were performed in triplicate and repeated on three occasions. * P<0.05.

Figure 2. The association of PrP-TM with DRMs is disrupted by treatment of cells with either heparin or bacterial PI-PLC.

SH-SY5Y cells expressing PrP-TM were surface biotinylated and then (A) incubated in the absence or presence of 50 µM heparin prepared in OptiMEM for 1 h at 37°C or (B) incubated in the absence or presence of 1 U/ml bacterial PI-PLC for 1 h at 4°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrP-TM fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions respectively. (C) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after heparin and PI-PLC treatment. Experiments were performed in triplicate and repeated on three occasions. * P<0.05.

Figure 3. Depletion of glypican-1 inhibits the association of PrP-TM with DRMs.

SH-SY5Y cells expressing PrP-TM were treated with either control siRNA or siRNA targeted to glypican-1 and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C in the presence of Tyrphostin A23 to block endocytosis. The media was removed and the cells washed in phosphate-buffered saline prior to homogenisation in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. (A) Quantification of glypican-1 and PrP-TM expression in cell lysates. To detect glypican-1, cell lysate samples were treated with heparinase I and heparinase III prior to electrophoresis as described in the materials and methods section. (B) PrP-TM was immunoprecipitated from equal volumes of each gradient fraction using 3F4 and then subjected to western blotting with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected by immunoblotting as markers for DRM and detergent-soluble fractions, respectively. (C) Densitometric analysis of the proportion of total PrP-TM present in the detergent soluble fractions of the plasma membrane after siRNA treatment from multiple blots from three independent experiments. * P<0.05.

Figure 4. Depletion of glypican-1 stimulates the endocytosis of PrP^C.

SH-SY5Y cells expressing wild type PrP^C were treated with either control or glypican-1 siRNA and then incubated for 60 h. Cells were surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Where indicated, cells were treated with trypsin to remove remaining cell surface PrP^C. Cells were then lysed and total PrP^C immunoprecipitated from the sample using antibody 3F4. (A) Samples were subjected to western blot analysis and the biotin-labelled PrP^C fraction was detected with peroxidase-conjugated streptavidin. (B) Densitometric analysis (mean ± s.e.m.) of multiple blots from three separate experiments in (A) is shown. (C) Expression of glypican-1 (in lysate samples treated with heparinase I and heparinase III) and PrP^C in the cell lysates from (A). β-actin was used as a loading control. (D) SH-SY5Y cells expressing PrP^C were treated with either control siRNA or glypican-1 siRNA and then allowed to reach confluence for 48 h. Cells were subsequently surface biotinylated and incubated in OptiMEM for 1 h at 37°C. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. (E) Densitometric analysis of the proportion of total PrP^C present in the detergent soluble fractions of the plasma membrane after siRNA treatment from three independent experiments. (F) SH-SY5Y cells expressing PrP^C were seeded onto glass coverslips and grown to 50% confluency. Cells were fixed, and then incubated with anti-PrP antibody 3F4 and a glypican-1 polyclonal antibody. Finally, cells were incubated with Alexa488-conjugated rabbit anti-mouse and Alexa594-conjugated goat anti-rabbit antibodies and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Scale bars equal 10 µm. * P<0.05.

Figure 5. PrP^C and PrP^Sc immunoprecipitate with glypican-1.

(A) SH-SY5Y cells expressing PrP^C, (B) N2a cells or (C and D) ScN2a cells were lysed in the indicated detergents and then immunoprecipitated with a polyclonal glypican-1 antibody and where indicated, co-incubated with 50 µM heparin. Those samples pretreated with heparinase I and heparinase III were lysed with Triton X-100 followed by immunoprecipitation with glypican-1 antibody. In (D), immunopreciptiates from ScN2a cells were digested with PK. All immunoprecipitates were subsequently blotted for PrP. TX, Triton X-100; OG, octylglucoside; SK, sarkosyl.

Figure 6. Depletion of glypican-1 by siRNA reduces PrP^Sc formation.

ScN2a cells were either untreated or incubated with either control siRNA or one of four siRNAs targeted to glypican-1. After 48 h incubation the treatments were repeated. After a total incubation period of 96 h cells were harvested, lysed and protein concentration determined. (A) For detection of PK-resistant PrP^Sc, samples containing 200 µg protein were digested with 4 µg PK for 30 min at 37°C. Protein was then recovered by methanol precipitation and immunoblotted for PrP using antibody 6D11. (B) Densitometric analysis (mean ± s.e.m.) of PK-resistant PrP^Sc levels for each treatment, relative to those of mock-treated cells, from multiple blots from four independent experiments. (C) To confirm that glypican-1 depletion had been achieved in the ScN2a cells, cell lysate samples were immunoblotted for glypican-1, as well as PrP and β-actin. (D) To confirm the specificity of glypican-1 in modulating PrP^Sc formation, ScN2a cells were treated with control, syndecan-1 or glypican-1 siRNA reagents. Samples were processed as described in (A). (E) Densitometric analysis (mean ± s.e.m.) of PK-resistant PrP^Sc levels for each treatment in (D), from multiple blots from three independent experiments. (F) Confirmation of syndecan-1 and glypican-1 knockdown by immunblotting.

Figure 7. Depletion of glypican-1 does not affect cell division or surface levels of PrP^C.

(A) ScN2a cells were seeded into 96 well plates and treated with transfection reagent only or incubated with either control siRNA or one of the four siRNAs targeted to glypican-1. Those experiments exceeding 48 h were dosed with a second treatment of the indicated siRNAs. Cells were then rinsed with PBS and fixed with 70% (v/v) ethanol. Plates were allowed to dry, stained with Hoescht 33342 and the fluorescence measured. (B) ScN2a cells were treated with control or glypican-1 siRNA. After 96 h, cell monolayers were labelled with a membrane impermeable biotin reagent. Biotin-labelled cell surface PrP was detected by immunoprecipitation using 6D11 and subsequent immunoblotting using HRP-conjugated streptavidin. Total PrP and PK-resistant PrP (PrP^Sc) were detected by immunoblotting using antibody 6D11. (C) Densitometric analysis of the proportion of the relative amount of biotinylated cell surface PrP in the absence or presence of glypican-1 siRNA from three independent experiments.

Figure 8. Depletion of glypican-1 does not affect the inhibition of BACE1 by PrP^C.

SH-SY5Y cells co-expressing APP695 and PrP or cells expressing APP695 and an empty pIRESneo vector were treated with control siRNA or siRNA targeted to glypican-1. After 30 h the medium was replaced and cells incubated with reduced-serum medium for a further 24 h. Conditioned medium was harvested and cell lysates prepared. (A) Expression of glypican-1, full-length APP and PrP^C in cell lysates, with β-actin as a loading control. Immunodetection of sAPPβ in conditioned medium. (B) Densitometric analysis of sAPPβ levels in glypican-1 depleted cells relative to control siRNA cells, calculated from multiple blots from three independent experiments.

Figure 9. Proposed model for glypican-1 in prion conversion.

Under normal circumstances, glypican-1 (light grey) with its heparan sulfate side chains (black lines) constitutes a lipid raft targeting determinant for PrP^C. (A) For PrP^C (dark grey) to exit lipid rafts and undergo endocytosis through its interaction with low density lipoprotein receptor-related protein 1 (LRP1, thick black line [59]), its interaction with glypican-1 must be disrupted (arrow 1). In prion disease, we hypothesise that glypican-1 provides a scaffold to facilitate interaction between PrP^C and PrP^Sc (chequered) and allow misfolding of PrP^C to proceed (arrow 2). (B) Prion therapeutic strategies may act, in part, by disrupting the interaction of PrP^C and PrP^Sc that is facilitated through the heparan sulfate sidechains of glypican-1.