A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrPC) into a disease related, alternatively folded isoform (PrPSc). The accumulation of PrPSc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.
Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrPC from the surface of prion-infected neuronal cells. The cell surface is a site where PrPC molecules may be converted to PrPSc and glimepiride treatment reduced PrPSc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A2 (cPLA2) and the production of prostaglandin E2 that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrPC expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.
Citation: Bate C, Tayebi M, Diomede L, Salmona M, Williams A (2009) Glimepiride Reduces the Expression of PrPC, Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity. PLoS ONE 4(12): e8221. https://doi.org/10.1371/journal.pone.0008221
Editor: Colin Combs, University of North Dakota, United States of America
Received: September 21, 2009; Accepted: November 11, 2009; Published: December 9, 2009
Copyright: © 2009 Bate et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a grant from the European Commission FP6 NeuroPrion Network of Excellence (http://www.neuroprion.org/en/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The transmissible spongiform encephalopathies, otherwise known as prion diseases include Creutzfeldt-Jakob disease and kuru in humans, as well as important livestock diseases such as scrapie in sheep and goats and bovine spongiform encephalopathy in cattle. The central event in these diseases is the conversion of a host encoded cellular prion protein (PrPC) into abnormally folded, disease-associated isoforms (PrPSc) in the brains of infected animals . Although the primary amino acid sequence remains the same, during the conversion process a portion of the α-helix and random coil structure in PrPC is refolded into a β-pleated sheet in PrPSc . This change in secondary structure is accompanied by changes in the biological and biochemical properties of the PrPSc protein, including reduced solubility and an increased resistance to proteases . Consequently, aggregates of PrPSc accumulate in association with neurones in affected brain areas , a process which is thought to lead to synapse degeneration and ultimately neuronal death. PrPSc is believed to constitute the major and perhaps only component of the infectious particle . While the correlation between PrPSc and infectivity is not completely clear , cell based studies routinely measure the amount of PrPSc as an indicator of infectivity.
The production of PrPSc and the progression of prion diseases are dependent upon the presence of PrPC , , . PrPC is linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor  and can be released from the surface of cells by treatment with phosphatidylinositol-phospholipase C (PI-PLC) . Treatment of prion-infected neuronal cells with PI-PLC reduced PrPSc formation ,  indicating that PrPSc is formed from PrPC expressed at the cell surface, or from PrPC that had been expressed at the cell surface. This conclusion is supported by observations that PrPC reactive antibodies reduced PrPSc formation in prion-infected neuronal cells , . Thus, any treatment that affected the amount of PrPC expressed at the cell surface may also be expected to affect PrPSc formation.
Glimepiride is a sulphonylurea used to treat non insulin-dependent diabetes mellitus. It some cells it activates an endogenous GPI-PLC , . Thus, in adipocytes glimepiride treatment released some GPI-anchored proteins from surface membranes ,  and caused the redistribution of other GPI-anchored proteins . We therefore investigated the effect of glimepiride treatment on the amount of PrPC at the surface of primary cortical neurones and prion-infected neuronal cell lines. Glimepiride treatment reduced the amount of PrPC at the surface of neuronal cell lines and primary cortical neurones. The effects of glimepiride were similar to the effects of PI-PLC; both caused the release of a soluble, deacylated PrPC. Treatment with glimepiride also reduced PrPSc formation in 3 prion-infected neuronal cell lines (ScGT1, SMB and ScN2a cells). In addition, the effects of glimepiride treatment on prion neurotoxicity were examined. PrPC is required for the neurotoxicity of PrPSc  and the process of prion-induced neurodegeneration is commonly examined by incubating neurones with either recombinant PrP or specific PrP-derived peptides. A synthetic peptide containing amino acids 82 to 146 of the human PrP protein (PrP82–146) corresponding to a major PrP fragment isolated from the brains of patients with Gerstmann-Sträussler-Scheinker disease (GSS) , was toxic to cultured cortical neurones , . Here we report the effects of glimepiride on PrP82–146 induced activation of cytoplasmic phospholipase A2 (cPLA2) and neuronal survival.
Prion-infected neuronal cell lines (ScGT1, ScN2a and SMB cells) , ,  were grown in Ham's F12 medium supplemented with 2 mM glutamine, 2% foetal calf serum (FCS) and standard antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were plated in 6 well plates (105 cells/well) and allowed to adhere overnight before the addition of test compounds. The medium was changed twice daily and the amount of cell-associated PrPSc measured after 7 days. Cells were washed twice in phosphate buffered saline (PBS) and homogenised at 106 cells/ml in an extraction buffer containing 10 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40 and 0.5% sodium deoxycholate. Nuclei and large fragments were removed by centrifugation (300×g for 5 minutes) and the supernatant digested with 5 µg/ml proteinase K for 1 hour at 37°C, digestion was stopped using mixed protease inhibitors (AEBSF, Aprotinin, Leupeptin, Bestain, Pepstatin A and E-46) (Sigma, Poole, UK). Culture supernatants were also collected to see if PrPSc was released from cells. They were digested with 5 µg/ml proteinase K for 1 hour at 37°C and stopped with mixed protease inhibitors (as above). In some studies cell extracts/supernatants were digested with 50 µg/ml thermolysin, which is reported to digest PrPC without affecting protease sensitive PrPSc , . The digested supernatant was concentrated by centrifugation with a 10 kDa filter (Sartorius vivaspin) and adjusted to an equivalent of 106 cells/ml. Samples were heated to 95°C for 5 minutes and tested in a PrP specific enzyme-linked immunosorbent assay (ELISA). Uninfected N2a, GT1 or SMB-PS cells were used as controls.
Primary neuronal cultures
Primary cortical neurones were prepared from the brains of mouse embryos (day 15.5) after mechanical dissociation . Neuronal precursors were plated (2×105 cells/well in 48 well plates pre-coated with 5 µg/ml poly-L-lysine) in Ham's F12 medium containing 5% FCS for 2 hours. Cultures were shaken (600 r.p.m for 5 minutes) and non-adherent cells removed by 2 washes in PBS. Neurones were subsequently grown in neurobasal medium (NBM) containing B27 components (Invitrogen, Paisley, UK) for 7 days. Immunolabelling studies showed that after 7 days cultures contained less than 5% glial cells (∼3% GFAP positive and less than 1% MAC-1 positive cells). Hippocampal neurones were prepared from the brains of adult mice as described . Briefly, hippocampi were dissected from the adult brain tissue and triturated in Ham's F12 containing 5% FCS, 0.35% glucose, 0.025% trypsin, and 0.1% type IV collagenase (Invitrogen). After 30 minutes at 37°C, the cells were triturated with a 1 ml pipette and passed through a 100 µM cell strainer. Cells were washed twice in Ham's F12 medium containing 5% FCS and plated in 48 well plates pre-coated with 5 µg/ml poly-L-lysine (2×105 cells/well) for 24 hours. Cultures were shaken (600 r.p.m for 5 minutes) to remove non-adherent cells, washed twice with PBS and cultured in NBM containing B27 components and 10 ng/ml glial-derived neurotrophic factor (Sigma) for 7 days. Neurones were subsequently pre-treated with test compounds (glimepiride, glipizide, glibenclamide, p-chloromercuriphenylsulphonate (p-CMPS) or PI-PLC derived from Bacillus cereus, obtained from Sigma) and washed before the addition of PrP peptides or further analysis. Stock solutions of drugs were prepared in di-methyl sulphoxide (DMSO) and diluted on the day of use, vehicle controls were equivalent dilutions of DMSO. The survival of neurones was determined 5 days later using 25 µM thiazlyl blue tetrazolium (MTT); neuronal survival was reported as a percentage of control, vehicle treated neurones.
After treatment, cells were washed twice in PBS and homogenised in an extraction buffer containing 10 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate and 0.2% sodium dodecyl sulphate (SDS) at 106 cells/ml and nuclei and large fragments were removed by centrifugation (300×g for 5 minutes). Mixed protease inhibitors were added to cell extracts where appropriate.
Measurement of cell surface PrPC
The amount of PrPC expressed at the cell surface was determined by two methods. In the first, 106 treated neurones were subsequently pulsed with PBS containing 50 µg/ml of membrane-impermeable sulfo-biotin-X-NHS (Pierce, Cramlington, UK) for 10 minutes. Cells were then washed 4 times with ice cold PBS containing 10% FCS to remove unbound biotin and the amount of biotinylated PrPC measured in a modified ELISA. Maxisorb Immunoplates (Nunc, Roskilde, Denmark) were pre-coated with 10 µg/ml streptavidin (Sigma) and blocked with 10% milk powder. Samples were added for 1 hour and the amount of bound biotinylated PrPC was determined by incubation with the PrP-specific mAb ICSM18, anti-mouse IgG-alkaline phosphate and 1 mg/ml 4-nitrophenyl phosphate. Absorbance was measured at 450 nm and the amount of biotinylated PrPC was calculated by reference to a standard curve of biotinylated recombinant PrP (Prionics, Zurich, Switzerland). The second method involved incubating treated cells with 0.2 units of PI-PLC/106 cells for 1 hour at 37°C. PI-PLC acts on the GPI anchored proteins including PrPC at the cell surface. The amount of PrPC released into the supernatant following PI-PLC digestion was measured by PrP ELISA.
Reverse phase chromatography of PrPC
Supernatants from glimepiride treated primary cortical neurones were applied to C18 columns (Waters, Elstree, UK). For comparison, PrPC from cell extracts and PrPC that had been digested with PI-PLC (0.2 units/ml for 1 hour at 37°C) were also added to C18 columns. Proteins were eluted by reverse phase chromatography under a gradient of acetonitrile in water and 0.1% trifluoroacetic acid (TFA). Fractions were collected, lyophilised, solubilised in extraction buffer and tested in a PrP ELISA.
The amount of PrP in cell extracts/supernatants was measured by ELISA as described , . Maxisorb Immunoplates were coated with a PrP-specific mAb (ICSM18 which recognises amino acids 146 to 159 of murine PrP). Samples were applied and detected with biotinylated mAb ICSM35 (which recognises a region between amino acids 91 and 110). This was detected using extravidin-alkaline phosphatase and 1 mg/ml 4-nitrophenyl phosphate. Absorbance at 450 nm was measured on a microplate reader and the amount of PrP calculated by reference to a standard curve of recombinant murine PrP (Prionics); its limit of detection was 0.05 ng/ml.
The amount of PrP82–146 in cell extracts was also determined by ELISA. Maxisorb Immunoplates were coated with 0.5 µg/ml of mouse mAb 3F4 (reactive with residues 109–112 of human PrP (Abcam, Chandler's Ford, UK), this mAb does not bind to murine PrP . Samples were applied and detected with biotinylated ICSM35 (D-gen), followed by extravidin-alkaline phosphatase and 1 mg/ml 4-nitrophenyl phosphate. Absorbance was measured on a microplate reader at 450 nm and the amount of PrP82–146 was calculated by reference to a standard curve of PrP82–146.
Activated cPLA2 ELISA
The activation of cPLA2 is accompanied by phosphorylation of the 505 serine residue, which can be measured by phospho-specific antibodies. The amount of activated cPLA2 in cell extracts was measured by ELISA as described . Nunc Maxisorb Immunoplates were coated with 0.5 µg/ml of mouse mAb anti-cPLA2, clone CH-7 (Upstate, Milton Keynes, UK) for 1 hour and blocked with 10% FCS. Samples were incubated for 1 hour and the amount of activated cPLA2 was detected using a rabbit polyclonal anti-phospho-cPLA2 (Cell Signalling Technology). Bound antibodies were then detected using biotinylated anti-rabbit IgG (Dako), extravidin-alkaline phosphatase and 1 mg/ml 4-nitrophenyl phosphate. Absorbance was measured at 450 nm and the amount of activated cPLA2 present calculated by reference to a standard curve, using nonlinear regression. Samples were expressed as “units cPLA2”, 100 units being defined as the amount of cPLA2 in 106 untreated cortical neurones. A standard curve ranging from 100 to 1.56 units/well was prepared from this sample using doubling dilutions.
The amount of PGE2 produced by cells was determined by using an enzyme-immunoassay kit (Amersham Biotech, Amersham, UK). The detection limit of this assay is 20 pg/ml.
Peptides containing amino acids 82 to 146 of the human PrP protein (PrP82–146) corresponding to a PrP fragment found in certain prion-infected human brains , and a control peptide (PrP82–146scrambled) were synthesised by solid-phase chemistry and purified by reverse-phase HPLC. Stock solutions were thawed on the day of the experiment and sonicated for 10 minutes before addition to cells.
Glimepiride reduced PrPC at the surface of neurones
Since glimepiride has been reported to stimulate the release or redistribution of GPI-anchored proteins in adipocytes , , its effect on PrPC in cortical neurones was examined. Treatment of neurones for 1 hour with glimepiride reduced the amount of PrPC in cell extracts in a dose-dependent manner (Figure 1). This effect of glimepiride was not shared by glibenclamide or glipizide, two other sulphonylureas used to treat diabetes mellitus, which did not alter the PrPC content of neurones. The reduction in the PrPC content of neurones achieved following treatment with 5 µM glimepiride (19.6 ng/106 cells±1.7 compared with 28.5±3.4, n = 12, P<0.01) was similar in magnitude to that observed in neurones treated with 0.2 units PI-PLC/ml (19.2 ng/106 cells±2.8 compared with 28.5±3.4, n = 12, P<0.01), results consistent with the hypothesis that glimepiride activates an endogenous GPI-PLC. Higher concentrations of glimepiride or PI-PLC, or prolongation of treatment to 4 hours, did not cause any further reduction in the neuronal PrPC content. Neither glimepiride nor PI-PLC affected the survival of neurones as measured by thiazyl blue tetrazolium.
The amount of PrPC in whole cell extracts from primary cortical neurones treated for 1 hour with different concentrations of glimepiride (•), glibenclamide (○) or glipizide (▪) as shown. Values shown are the mean average amount of PrPC in neuronal extracts (ng/106 cells) ± SD, n = 12.
Cortical neurones treated with glimepiride for 1 hour were subsequently pulsed with a membrane-impermeable biotin ester to determine whether glimepiride affects the amount of PrPC expressed at the cell surface. Treatment with glimepiride, but not glipizide or glibenclamide, reduced the amount of biotinylated PrPC in cell membranes (Figure 2A). Treatment with 0.2 units PI-PLC/ml also reduced the amount of biotinylated PrPC in cell membranes (0.6 ng/106 cells±0.2 compared to 7.4±1.1, n = 9, P<0.01). Time course studies showed that the effect of glimepiride on the expression of PrPC on the surface of cortical neurones was transient. Thus the amount of PrPC labelled with cell-impermeable biotin in neurones pulsed with 5 µM glimepiride for 1 hour remained low for 2 hours after the cessation of glimepiride treatment and only returned to the levels seen in untreated cells after 12 hours (Figure 2B). When these experiments were conducted in the presence of 20 µg/ml cycloheximide, which inhibits the synthesis of proteins, the return of PrPC to the cell surface was delayed, indicating that the PrPC that appeared at the surface of glimepiride treated cells had been newly synthesised rather than rerouted from an existing intracellular pool.
(A) The amount of cell surface PrPC on neurones treated for 1 hour with different concentrations of glimepiride (•), glibenclamide (○) or glipizide (▪) as shown. Values shown are the mean average amount of biotinylated PrPC in cell extracts (ng/106 cells) ± SD, n = 9. (B) The amount of cell surface PrPC in cell extracts from cortical neurones taken at different time points after the addition of 5 µM glimepiride alone (□) or a mixture containing 5 µM glimepiride and 20 µg/ml cycloheximide (▪) as shown. Values shown are the mean average amount of biotinylated PrPC in cell extracts (ng/106 cells) ± SD, n = 9.
Glimepiride stimulated the release of PrPC from neurones
Next we sought to determine if glimepiride stimulated the release of PrPC from the cell by measuring the amount of PrPC in the supernatants of cultured neurones. The addition of glimepiride for 1 hour, but not glibenclamide or glipizide, increased the amount of PrPC released into cell supernatant (Figure 3A). Similarly, the addition of 0.2 units PI-PLC/ml for 1 hour increased the amount of PrPC in the supernatants of neurones (9.9 ng/106 cells±1.1 compared to 0.6±0.3 in untreated supernatants, n = 9, P<0.01). We noted that glimepiride treatment caused the release of specific GPI-anchored proteins. Thus, while digestion with 0.2 units PI-PLC/ml released both PrPC and Thy-1 from neurones, treatment with 5 µM glimepiride released PrPC but did not release Thy-1 (data not shown).
(A) The amount of PrPC in the supernatant of cortical neurones treated for 1 hour with different concentrations of glimepiride (•), glibenclamide (○) or glipizide (▪). Values shown are the mean average amount of soluble PrPC (ng/106 cells) ± SD, n = 9. (B) The amount of PrPC molecules eluted from C18 columns following elution with a gradient of acetonitrile in water containing 0.1% TFA. Fractions eluted from C18 columns loaded with whole cell extracts (○) or with supernatants from cortical neurones treated with 5 µM glimepiride (•). Values shown are the mean average amount of PrPC (ng/ml) ± SD, n = 6.
PrPC can be released from cells by different mechanisms , , , , . Therefore we sought evidence that the PrPC in the supernatant of glimepiride treated cells was released following its digestion by a GPI-PLC. When cell-associated PrPC containing an intact GPI anchor was bound to a C18 column and exposed to acetonitrile:water gradients, it was found in those fractions containing 70–76% acetonitrile. In contrast, cell-associated PrPC molecules that had been digested by 0.2 units PI-PLC/ml did not bind to C18 columns. The PrPC in supernatants from glimepiride treated neurones did not bind to C18 columns indicating that these PrPC molecules had lost their hydrophobic acyl chains, consistent with the view that they had been digested by GPI-PLC (Figure 3B). Next we sought to determine if the effect of glimepiride could be reversed by the addition of p-CMPS, which inhibited GPI-PLC . Whereas treatment with as much as 500 µM p-CMPS alone had no detectable effect on cell surface PrPC (not shown), addition of p-CMPS to cortical neurones treated with 5 µM glimepiride increased the amount of PrPC at their surface to control levels (Figure 4), confirming that glimepiride activates an endogenous GPI-PLC.
The amount of PrPC at the surface of cortical neurones treated for 1 hour with 5 µM glimepiride alone (□) or with combinations containing 5 µM glimepiride and different concentrations of p-CMPS as shown (▪). Values shown are the mean average amount of biotinylated-PrPC (ng/106 cells) ± SD, n = 8.
Glimepiride reduced the PrPSc content of prion infected neuronal cells
Since the cell surface is a possible site of the conversion of PrPC to PrPSc ,  the effect of glimepiride on the PrPSc content of ScGT1 cells was determined. Treatment with 5 µM glimepiride for 1 hour released PrP molecules from ScGT1 cells into the supernatant (8.4 ng/106 cells). These PrP molecules were sensitive to digestion with 5 µg/ml proteinase K which reduced the PrP content to less than 0.05 ng/106 cells, indicating that the PrP released was PrPC or protease-sensitive PrPSc. The PrP molecules were also sensitive to digestion with 50 µg/ml thermolysin (following digestion the PrP content of supernatants was reduced to less than 0.05 ng/106 cells) indicating that the PrP released was PrPC rather than protease-sensitive PrPSc . Next, the effect of longer term treatment was examined. Twice daily treatment for 7 days with glimepiride, but not glibenclamide, caused a dose-dependent reduction in the amount of PrPSc in ScGT1 cells (Figure 5A). We were unable to detect PrPSc in ScGT1 cells treated with 5 µM glimepiride for 7 days. Moreover, these cells remained free of PrPSc when grown for a further month after the cessation of treatment. The amount of PrPSc in ScGT1 cells treated for 7 days with increasing concentrations of glimepiride showed a significant correlation with the amount of PrPC released from the surface of glimepiride treated ScGT1 cells 1 hour after treatment, Pearson's coefficient = 0.868, P<0.01 (Figure 5B). This effect of glimepiride was not cell line specific as similar dose-dependent reductions in PrPSc content were observed in both ScN2a and SMB cells (Figure 6A and B).
(A) The amount of PrPSc in ScGT1 cells treated for 7 days with different concentrations of glimepiride (▪) or with glibenclamide (□). Values shown are the mean average amount of PrPSc (ng/106 cells) ± SD, n = 12. (B) Correlation between the amount of PrPC at the surface of ScGT1 cells after 1 hour incubation with different concentrations of glimepiride and the amount of PrPSc in ScGT1 cells following treatment for 7 days.
(A) The amount of PrPSc in ScN2a cells following treatment for 7 days with control medium (□) or with different concentrations of glimepiride (▪). Values shown are the mean average amount of PrPSc (ng/106 cells) ± SD, n = 12. (B) The amount of PrPSc in SMB cells treated for 7 days with control medium (□) or with different concentrations of glimepiride (▪). Values shown are the mean average amount of PrPSc (ng/106 cells) ± SD, n = 12.
Recent studies showed that glimepiride treatment of adipocytes released GPI anchored proteins in exosomes , . Since PrPSc can also be released from prion-infected rabbit kidney cells in exosomes , the possibility that glimepiride might also stimulate exosome formation and the release of PrPSc from prion-infected neuronal cells was examined. The amount of PrPSc present in supernatants from ScGT1, ScN2a or SMB cells treated with 5 µM glimepiride for 7 days, was measured by ELISA. Treatment with glimepiride significantly reduced the amount of PrPSc in supernatants collected from ScGT1 cells (0.4 ng PrPSc/106 cells±0.3 compared with 1.97 ng±0.58, n = 9, P<0.01), SMB cells (0.3 ng PrPSc/106 cells±0.4 compared with 1.21 ng±0.28, n = 9, P<0.01) and ScN2a cells (0 ng PrPSc/106 cells compared with 0.45 ng±0.01, n = 9, P<0.01). These data indicated that the reduction of cell associated PrPSc observed after glimepiride treatment was not related to a stimulation of PrPSc release.
Glimepiride treated neurones are resistant to PrP82-146 toxicity
The addition of PrP82–146 reduced the survival of cortical neurones , . Pre-treatment with 5 µM glimepiride protected cortical neurones against the toxic effect of PrP82–146 (Figure 7A). The concentration of PrP82–146 required to kill 50% of neurones (LD50) was at least 20 times greater, from 10 µM in mock-treated neurones to more than 200 µM PrP82–146 in glimepiride treated cells. The protective effect of glimepiride was dose-dependent (Figure 7B) and was not observed with glipizide. This effect of glimepiride was not specific for cortical neurones, the survival of hippocampal neurones incubated with 5 µM PrP82–146 was also significantly increased by pre-treatment with 5 µM glimepiride (24% cell survival±9 compared to 94%±8, n = 10, P<0.01). Glimepiride treated neurones were not resistant to all neurotoxins and did not protect against staurosporine (data not shown). The protective effect of glimepiride was dependent on pre-treatment and was not observed when 5 µM glimepiride was added 1 hour after addition of 20 µM PrP82–146 (41% cell survival±7 compared with 38%±5, n = 10, P = 0.4). It was also transient and was lost after 12 hours (Figure 8). The return of sensitivity to the toxic action of PrP82–146 coincided with the return of PrPC to the surface of the neurones as shown in Figure 2.
(A) The survival of cortical neurones pre-treated for 1 hour with control medium (•) or with 5 µM glimepiride (○) and incubated with varying concentrations of PrP82–146 for 5 days. Values shown are the mean average neuronal survival ± SD, n = 12. (B) The survival of cortical neurones pre-treated for 1 hour with varying concentrations of glimepiride (○) or glipizide (•) and incubated with 20 µM PrP82–146 for 5 days. Values shown are the mean average neuronal survival ± SD, n = 9.
The survival of vehicle treated cortical neurones (□) or cortical neurones pre-treated with 5 µM glimepiride for the time periods as shown (▪) and incubated with 20 µM PrP82–146 for a further 5 days. Values shown are the mean average neuronal survival ± SD, n = 12.
Since glimepiride activates an endogenous GPI-PLC ,  the effects of PI-PLC on neuronal responses to PrP82–146 were also examined. Pre-treatment with 0.2 units PI-PLC/ml was found to increase the survival of cortical neurones subsequently incubated with PrP82–146 (Figure 9A). Next we tested the hypothesis that the protective effect of glimepiride was due to activation of an endogenous GPI-PLC. First we showed that pre-treatment with a GPI-PLC inhibitor (500 µM p-CMPS) did not affect the survival of neurones subsequently incubated with PrP82–146. Next, we showed that the protective effect of 5 µM glimepiride was reversed by the inclusion of 500 µM p-CMPS indicating that protection was dependent upon activation of an endogenous GPI-PLC (Figure 9B).
(A) The survival of cortical neurones pre-treated for 1 hour with 0.2 units PI-PLC/ml (○) or with control medium (•) and incubated with varying concentrations of PrP82–146 as shown. Values shown are the mean average neuronal survival ± SD, n = 9. (B) The survival of cortical neurones pre-treated for 1 hour with a vehicle control (•), with 5 µM glimepiride (○), with 500 µM p-CMPS (□) or with a combination of 5 µM glimepiride and 500 µM p-CMPS (▪) and incubated with varying concentrations of PrP82–146. Values shown are the mean average neuronal survival ± SD, n = 9.
Glimepiride reduced the binding of PrP82–146 by neurones
Since PrPC acts as a receptor for PrP peptides , the effect of glimepiride on the binding of 10 µM PrP82–146 to cortical neurones was examined. The amount of PrP82–146 bound was time-dependent over 60 minutes. Neurones pre-treated with 5 µM glimepiride bound significantly less PrP82–146 than mock-treated neurones (Figure 10). It was noted that glimepiride treatment did not completely block the binding of PrP82–146 to neurones. Thus, 60 minutes after the addition of 10 µM PrP82–146, glimepiride treated neurones bound 6.2 nM PrP82–146±0.8 compared to 9.9 nM PrP82–146±0.8 in controls (n = 8). Similar results were obtained when neurones were pre-treated with 0.2 units PI-PLC/ml, which after 60 minutes had bound 5.3 nM PrP82–146±0.7, n = 8.
The amount of PrP82–146 in cell extracts from cortical neurones pre-treated for 1 hour with a vehicle control (□) or with 5 µM glimepiride (▪) and exposed to 10 µM PrP82–146 for different times periods as shown. Values shown are the mean average amount of PrP82–146 (µM) ± SD, n = 9.
Glimepiride reduced activation of cPLA2 by PrP82–146
Unregulated activation of PLA2 is recognized as a key event in some neurodegenerative diseases , , . The activation of PLA2 is the first step in the production of eicosanoids, docosanoids and platelet activating factors, high concentrations of which cause glial cell activation, synapse degeneration and neuronal death. The addition of PrP82–146 increased the amount of activated cPLA2 in neurones. Treatment with 5 µM glimepiride alone did not alter the amount of activated cPLA2 in cortical neurones (100 units activated cPLA2±8 compared to 97±10, n = 9, P = 0.4), but greatly reduced the activation of cPLA2 induced by PrP82–146 (Figure 11A); treatment with 5 µM glibenclamide or 5 µM glipizide had no effect. Digestion of neurones with PI-PLC also reduced activation of cPLA2 induced by PrP82–146. To confirm the effect of glimepiride on PrP82–146 induced PLA2 activation, neuronal PGE2 production was also measured. As would be predicted, PGE2 production from neurones pre-treated with 5 µM glimepiride, or with PI-PLC, was significantly lower than that of untreated neurones after incubation with 10 µM PrP82–146 for 24 hours (Figure 11B).
The amount of activated cPLA2 in cell extracts from cortical neurones pre-treated for 1 hour with a vehicle control (•) 5 µM glimepiride (○) or PI-PLC (▪) and incubated with varying concentrations of PrP82–146 for 24 hours. Values shown are the mean average amount of activated cPLA2 (units) ± SD, n = 12. (B) The amount of PGE2 in cell extracts from cortical neurones pre-treated for 1 hour with control medium (□), 5 µM glimepiride (▪) or PI-PLC (striped bars) and incubated with control medium or 10 µM PrP82–146. Values shown are the mean average amount of PGE2 (pg/ml) ± SD, n = 9.
In this study we report that treatment with glimepiride reduced the amount of PrPC expressed at the surface of primary cortical neurones, neuronal cells and prion-infected neuronal cells. Subsequently, glimepiride treatment significantly reduced the amount of PrPSc within 3 prion-infected neuronal cell lines (ScGT1, SMB and ScN2a cells) and glimepiride treated cortical neurones showed increased resistance to the toxic effects of PrP82–146. The protective effect of glimepiride was accompanied by reduced binding of PrP82–146 to neurones, reduced activation of cPLA2 and PGE2 production. These effects of glimepiride were observed at physiological concentrations , however they were not shared by 2 other sulphonylureas used to treat diabetes, glibenclamide or glipizide.
The cellular location of PrPC is controversial and although PrPC is expressed at the cell surface, it is also found within cells , . Our findings support the view that a proportion of the PrPC in cortical neurones is in an intracellular pool, since it resisted both digestion with PI-PLC and biotinylation with a cell impermeable biotin-conjugate. Treatment of cortical neurones, neuronal cells and prion-infected neuronal cells with glimepiride reduced the amount of PrPC expressed at the cell surface and caused its release into the supernatant whereas the amount present in cell extracts was reduced by about a third. The release of PrPC into supernatants may occur as a consequence of exosome formation, or following digestion by phospholipases and proteases , , , , . Glimepiride activates an endogenous GPI-PLC in adipocytes  and many of its effects on neurones were replicated by the addition of PI-PLC. In addition, glimepiride induced release of PrPC was reversed by the inclusion of p-CMPS, an inhibitor of GPI-PLC. Whereas PrPC bound to C18 columns, PrPC released from glimepiride treated cells did not bind, consistent with the loss of hydrophobic acyl chains. Why glimepiride affects PrPC at the surface of neurones, but not intracellular PrPC, is unclear. It is possible that glimepiride does not penetrate the cell, an alternative explanation may be that GPI-PLC is associated with cell surface PrPC, but is absent from intracellular stores of PrPC.
Glimepiride treatment did not affect all GPI-anchored proteins. Thus, while digestion of neurones with PI-PLC released several GPI-anchored proteins into the supernatant, including PrPC and Thy-1, glimepiride treatment released PrPC but not Thy-1 (unpublished data). This specific effect of glimepiride may be due to the activation of an endogenous GPI-PLC that is closely associated with specific GPI-anchored proteins such as PrPC, but not with others including Thy-1 or CD55 which occupy separate domains upon the cell surface . An alternative explanation is that CD55 and Thy-1 may contain GPI anchors that are resistant to endogenous GPI-PLC.
The precise cellular location in which PrPC is converted to PrPSc remains controversial with advocates for the endosomal recycling compartment , . However, anti-PrP antibodies reduced PrPSc formation, suggesting that conversion occurs either at the cell surface, or after PrPC has been internalised from the cell surface , , . Such observations indicate that surface expression of PrPC is a prerequisite for PrPSc formation and that glimepiride reduced PrPSc formation by shedding PrPC from the cell surface. Our finding that glimepiride treatment released PrP molecules from ScGT1 cells raised concerns that glimepiride might cause the release of PrPSc and facilitate its spread throughout the brain. However, all PrP released from cells within 1 hour of treatment was sensitive to digestion with proteinase K. Although the presence of proteinase K sensitive PrPSc is well documented , the released PrP was also sensitive to digestion with thermolysin which has been reported to digest PrPC but not PrPSc . Collectively these results indicate that the PrP released from ScGT1 cells was PrPC. The longer term effects of glimepiride treatment showed that twice daily treatment for 7 days caused a dose-dependent reduction in the PrPSc content of ScGT1, ScN2a and SMB cells. It also reduced the amount of PrPSc released into supernatants over this period, excluding the possibility that the reduction in cell-associated PrPSc was due to glimepiride induced the release of PrPSc from cells. Our findings are consistent with the hypothesis that glimepiride acts by limiting the supply of PrPC to cellular sites that are essential for PrPSc formation.
Glimepiride treatment of cortical neurones affected cell surface PrPC but not intracellular PrPC. Similar results were obtained with GT1 and ScGT1 cells in which approximately 70% of PrPC molecules remained after treatment with glimepiride or PI-PLC and about the same amount resisted labelling by membrane impermeable biotin. These results suggest that PrPSc is formed from a subset of PrPC molecules that recycle to and from the cell surface. Perhaps more significantly they indicate that the intracellular PrPC molecules were poor substrates for conversion to PrPSc. In addition, the repopulation of surface PrPC in glimepiride treated cells was from newly synthesised PrPC rather than from the intracellular pool, as it was delayed by the inclusion of the protein synthesis inhibitor cycloheximide. We conclude that there are at least 2 pools of PrPC: one that consists of PrPC molecules that recycle to and from the cell surface and are susceptible to conversion to PrPSc and another pool of PrPC molecules that are mostly intracellular and are not readily converted to PrPSc suggesting that they follow different trafficking pathways. Such results are consistent with reports that altering the trafficking of PrPC alters PrPSc formation , .
Pre-treatment with glimepiride protected cortical and hippocampal neurones against the toxic action of PrP82–146; it increased the LD50 of PrP82–146 by more than 20 fold, from 10 µM to over 200 µM. However, glimepiride treated neurones remained sensitive to staurosporine (data not shown). The protective effect of glimepiride required pre-treatment; glimepiride did not rescue neurones that had been incubated with PrP82–146 for 30 minutes indicating that it affected an early event in PrP82–146 induced toxicity. Moreover, the protective effect of glimepiride was transient. Restoration of PrPC to the surface of neurones following glimepiride treatment was associated with their increased sensitivity of neurones to PrP82–146. Neurones treated with PI-PLC were also protected against PrP82–146 suggesting that glimepiride-mediated neuroprotection was due to activation of GPI-PLC. This hypothesis was strengthened by the finding that the protection induced by glimepiride was reversed following the addition of the GPI-PLC inhibitor p-CMPS.
PrPC has been proposed as a receptor for PrP peptides  and glimepiride reduced the binding of PrP82–146 to neurones. It is worth noting that although treatment with glimepiride or PI-PLC significantly reduced the binding of PrP82–146 to neurones, these cells still bound significant amounts of PrP82–146 (about 50% of the binding of untreated cells) indicating that PrP82–146 can bind to neurones via a PrPC independent mechanism. Many proteins have been proposed to be prion receptors  and it seems likely that glimepiride treated cells express some of them, or that PrP82–146 binds to cells independently of specific protein interactions. Since the reduction in PrP82–146 binding alone did not fully explain the protective effects of glimepiride, the effects of glimepiride on PrP82–146 induced cell signalling were examined. The unregulated activation of PLA2 is recognized as a key event in some neurodegenerative diseases ,  and is the first step in the production of eicosanoids, docosanoids and platelet activating factors, high concentrations of which can cause glial activation, synapse damage and neuronal death. Furthermore, PLA2 plays a critical role in neurotoxicity caused by PrP peptides . Our experiments showed that PrP82–146 activated cPLA2 in neurones and increased PGE2 production, a marker of PLA2 activation that is increased in scrapie infected mice ,  and in the cerebrospinal fluid of patients with CJD , . Pre-treatment with glimepiride significantly reduced PrP82–146 induced activation of cPLA2 and the production of PGE2. Although the precise mechanism is not clear, the activation of cPLA2 occurs in cholesterol-sensitive lipid rafts , . Since glimepiride induced digestion of GPI anchored proteins affects membrane cholesterol in adipocytes  our results are consistent with the hypothesis that glimepiride modifies lipid rafts required for cPLA2 activation. Other studies suggest that the neurotoxicity of PrP peptides is through the amplication of PrPC associated signalling pathways  which may be downregulated following glimepiride treatment.
Prion infection increased cholesterol in cell membranes . Since the insulin receptor is found within lipid rafts  and insulin signalling is cholesterol dependent , , prion infection induced changes in cell cholesterol may modify insulin signalling. This is consistent with observations that prion infection affects insulin and insulin-like growth factor receptors in cell lines ,  and that scrapie infection induced diabetes mellitus in hamsters is directly damaging the central nervous system, without affecting the pancreas . Thus, glimepiride treatment may also reverse prion-induced effects on insulin signalling.
One important consequence of the effect of glimepiride on PrPC may be of relevance to the treatment of Alzheimer's disease. PrPC was identified as a receptor that mediated the impairment of synaptic plasticity induced by amyloid-β oligomers . Thus, the pharmacological regulation of PrPC by glimepiride could provide a new approach to the inhibition of amyloid-β mediated synapse damage in Alzheimer's disease patients.
New approaches to the treatment of neurodegenerative conditions including prion diseases are urgently required. We have demonstrated that treatment of neurones with glimepiride caused the shedding of PrPC from the cell surface through activation of an endogenous GPI-PLC. Treatment reduced the formation of PrPSc in prion-infected neuronal cell lines and increased the survival of neurones incubated with PrP82–146. Glimepiride treatment reduced both the amount of PrP82–146 ingested by neurones and PrP82–146 induced activation of cPLA2. Our results suggest that glimepiride could prove beneficial in the treatment of prion diseases.
Conceived and designed the experiments: CB MS AW. Performed the experiments: CB. Analyzed the data: CB MT LD MS AW. Contributed reagents/materials/analysis tools: MT MS. Wrote the paper: CB MT LD AW.
- 1. Prusiner SB (1998) Prions. ProcNatlAcadSciUSA 95: 13363–13383.
- 2. Pan KM, Baldwin M, Nguyen J, Gasset M, Serban A, et al. (1993) Conversion of alpha-helices into beta-sheets features in the formation of the scrapie prion proteins. ProcNatlAcadSciUSA 90: 10962–10966.
- 3. Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, et al. (1983) Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35: 349–358.
- 4. Jeffrey M, Halliday WG, Bell J, Johnston AR, MacLeod NK, et al. (2000) Synapse loss associated with abnormal PrP precedes neuronal degeneration in the scrapie-infected murine hippocampus. NeuropatholApplNeurobiol 26: 41–54.
- 5. Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216: 136–144.
- 6. Barron RM, Campbell SL, King D, Bellon A, Chapman KE, et al. (2007) High Titers of Transmissible Spongiform Encephalopathy Infectivity Associated with Extremely Low Levels of PrPSc in Vivo. J Biol Chem 282: 35878–35886.
- 7. Bueler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, et al. (1993) Mice devoid of PrP are resistant to scrapie. Cell 73: 1339–1347.
- 8. Mallucci G, Dickinson A, Linehan J, Klohn PC, Brandner S, et al. (2003) Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science 302: 871–874.
- 9. Manson JC, Clarke AR, McBride PA, McConnell I, Hope J (1994) PrP gene dosage determines the timing but not the final intensity or distribution of lesions in scrapie pathology. Neurodegeneration 3: 331–340.
- 10. Stahl N, Borchelt DR, Hsiao K, Prusiner SB (1987) Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51: 229–240.
- 11. Stahl N, Borchelt DR, Prusiner SB (1990) Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. Biochemistry 29: 5405–5412.
- 12. Caughey B, Raymond GJ (1991) The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J Biol Chem 266: 18217–18223.
- 13. Enari M, Flechsig E, Weissmann C (2001) Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. ProcNatlAcadSciUSA 98: 9295–9299.
- 14. Beringue V, Vilette D, Mallinson G, Archer F, Kaisar M, et al. (2004) PrPSc Binding Antibodies Are Potent Inhibitors of Prion Replication in Cell Lines. J Biol Chem 279: 39671–39676.
- 15. Peretz D, Williamson RA, Kaneko K, Vergara J, Leclerc E, et al. (2001) Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 412: 739–743.
- 16. Movahedi S, Hooper NM (1997) Insulin stimulates the release of the glycosyl phosphatidylinositol-anchored membrane dipeptidase from 3T3-L1 adipocytes through the action of a phospholipase C. Biochem J 326 (Pt 2): 531–537.
- 17. Müller G, Dearey EA, Punter J (1993) The sulphonylurea drug, glimepiride, stimulates release of glycosylphosphatidylinositol-anchored plasma-membrane proteins from 3T3 adipocytes. Biochem J 289 (Pt 2): 509–521.
- 18. Müller G, Dearey EA, Korndorfer A, Bandlow W (1994) Stimulation of a glycosyl-phosphatidylinositol-specific phospholipase by insulin and the sulfonylurea, glimepiride, in rat adipocytes depends on increased glucose transport. J Cell Biol 126: 1267–1276.
- 19. Müller G, Jung C, Straub J, Wied S, Kramer W (2009) Induced release of membrane vesicles from rat adipocytes containing glycosylphosphatidylinositol-anchored microdomain and lipid droplet signalling proteins. Cellular Signalling 21: 324–338.
- 20. Müller G, Wied S, Walz N, Jung C (2008) Translocation of Glycosylphosphatidylinositol-Anchored Proteins from Plasma Membrane Microdomains to Lipid Droplets in Rat Adipocytes Is Induced by Palmitate, H2O2, and the Sulfonylurea Drug Glimepiride. Mol Pharmacol 73: 1513–1529.
- 21. Brandner S, Isenmann S, Raeber A, Fischer M, Sailer A, et al. (1996) Normal host prion protein necessary for scrapie-induced neurotoxicity. Nature 379: 339–343.
- 22. Salmona M, Morbin M, Massignan T, Colombo L, Mazzoleni G, et al. (2003) Structural properties of Gerstmann-Straussler-Scheinker disease amyloid protein. JBiolChem 278: 48146–48153.
- 23. Bate C, Salmona M, Diomede L, Williams A (2004) Squalestatin cures prion-infected neurones and protects against prion neurotoxicity. JBiolChem 279: 14983–14990.
- 24. Fioriti L, Angeretti N, Colombo L, De Luigi A, Colombo A, et al. (2007) Neurotoxic and Gliotrophic Activity of a Synthetic Peptide Homologous to Gerstmann-Straussler-Scheinker Disease Amyloid Protein. J Biol Chem 27: 1576–1583.
- 25. Taraboulos A, Raeber A, Borchelt D, Serban D, Prusiner S (1992) Synthesis and trafficking of prion proteins in cultured cells. Mol Biol Cell 3: 851–863.
- 26. Schatzl HM, Laszlo L, Holtzman DM, Tatzelt J, DeArmond SJ, et al. (1997) A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. JVirol 71: 8821–8831.
- 27. Birkett CR, Hennion RM, Bembridge DA, Clarke MC, Chree A, et al. (2001) Scrapie strains maintain biological phenotypes on propagation in a cell line in culture. EMBO J 20: 3351–3358.
- 28. Owen JP, Rees HC, Maddison BC, Terry LA, Thorne L, et al. (2007) Molecular profiling of ovine prion diseases by using thermolysin-resistant PrPSc and endogenous C2 PrP fragments. J Virol 81: 10532–10539.
- 29. Cronier S, Gros N, Tattum MH, Jackson GS, Clarke AR, et al. (2008) Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin. Biochem J 416: 297–305.
- 30. Brewer GJ (1997) Isolation and culture of adult rat hippocampal neurons. J Neurosci Meth 71: 143–155.
- 31. Wadsworth JDF, Joiner S, Linehan JM, Cooper S, Powell C, et al. (2006) Phenotypic heterogeneity in inherited prion disease (P102L) is associated with differential propagation of protease-resistant wild-type and mutant prion protein. Brain 129: 1557–1569.
- 32. Bate C, Tayebi M, Williams A (2008) Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A2 activation. BMC Biol 6: 8.
- 33. Kascsak RJ, Rubenstein R, Merz PA, Tonna-DeMasi M, Fersko R, et al. (1987) Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J Virol 61: 3688–3693.
- 34. Fevrier B, Vilette D, Archer F, Loew D, Faigle W, et al. (2004) Cells release prions in association with exosomes. ProcNatlAcadSciUSA 101: 9683–9688.
- 35. Vella LJ, Sharples RA, Lawson VA, Masters CL, Cappai R, et al. (2007) Packaging of prions into exosomes is associated with a novel pathway of PrP processing. J Pathol 211: 582–590.
- 36. Parkin ET, Watt NT, Turner AJ, Hooper NM (2004) Dual mechanisms for shedding of the cellular prion protein. JBiolChem.
- 37. Borchelt DR, Rogers M, Stahl N, Telling G, Prusiner SB (1993) Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor. Glycobiology 3: 319–329.
- 38. Stanton JD, Rashid MB, Mensa-Wilmot K (2002) Cysteine-less glycosylphosphatidylinositol-specific phospholipase C is inhibited competitively by a thiol reagent: evidence for glyco-mimicry by p-chloromercuriphenylsulphonate. Biochem J 366: 281–288.
- 39. Müller G, Over S, Wied S, Frick W (2008) Association of (c)AMP-Degrading Glycosylphosphatidylinositol-Anchored Proteins with Lipid Droplets Is Induced by Palmitate, H2O2 and the Sulfonylurea Drug, Glimepiride, in Rat Adipocytes. Biochemistry 47: 1274–1287.
- 40. Brown DR, Herms J, Kretzschmar HA (1994) Mouse cortical cells lacking cellular PrP survive in culture with a neurotoxic PrP fragment. Neuroreport 5: 2057–2060.
- 41. Farooqui AA, Ong W-Y, Horrocks LA (2006) Inhibitors of Brain Phospholipase A2 Activity: Their Neuropharmacological Effects and Therapeutic Importance for the Treatment of Neurologic Disorders. Phamacol Rev 58: 591–620.
- 42. Sun GY, Xu J, Jensen MD, Simonyi A (2004) Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases. Journal of Lipid Research 45: 205–213.
- 43. Sanchez-Mejia RO, Newman JW, Toh S, Yu G-Q, Zhou Y, et al. (2008) Phospholipase A2 reduction ameliorates cognitive deficits in a mouse model of Alzheimer's disease. Nat Neurosci 11: 1311–1318.
- 44. Becker ML, Visser LE, Trienekens PH, Hofman A, van Schaik RHN, et al. (2007) Cytochrome P450 2C9 *2 and *3 Polymorphisms and the Dose and Effect of Sulfonylurea in Type II Diabetes Mellitus. Clin Pharmacol Ther 83: 288–292.
- 45. Magalhaes AC, Silva JA, Lee KS, Martins VR, Prado VF, et al. (2002) Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein. J Biol Chem 277: 33311–33318.
- 46. Prado MA, Alves-Silva J, Magalhaes AC, Prado VF, Linden R, et al. (2004) PrPc on the road: trafficking of the cellular prion protein. JNeurochem 88: 769–781.
- 47. Madore N, Smith KL, Graham CH, Jen A, Brady K, et al. (1999) Functionally different GPI proteins are organized in different domains on the neuronal surface. EMBO J 18: 6917–6926.
- 48. Marijanovic Z, Caputo A, Campana V, Zurzolo C (2009) Identification of an intracellular site of prion conversion. PLoS Pathog 5: e1000426.
- 49. Godsave SF, Wille H, Kujala P, Latawiec D, DeArmond SJ, et al. (2008) Cryo-Immunogold Electron Microscopy for Prions: Toward Identification of a Conversion Site. J Neurosci 28: 12489–12499.
- 50. White AR, Enever P, Tayebi M, Mushens R, Linehan J, et al. (2003) Monoclonal antibodies inhibit prion replication and delay the development of prion disease. Nature 422: 80–83.
- 51. Tzaban S, Friedlander G, Schonberger O, Horonchik L, Yedidia Y, et al. (2002) Protease-Sensitive Scrapie Prion Protein in Aggregates of Heterogeneous Sizes. Biochemistry 41: 12868–12875.
- 52. Beranger F, Mange A, Goud B, Lehmann S (2002) Stimulation of PrP(C) retrograde transport toward the endoplasmic reticulum increases accumulation of PrP(Sc) in prion-infected cells. J Biol Chem 277: 38972–38977.
- 53. Gilch S, Winklhofer KF, Groschup MH, Nunziante M, Lucassen R, et al. (2001) Intracellular re-routing of prion protein prevents propagation of PrP(Sc) and delays onset of prion disease. EMBO J 20: 3957–3966.
- 54. Lee KS, Linden R, Prado MAM, Brentani RR, Martins VR (2003) Towards cellular receptors for prions. Reviews in Medical Virology 13: 399–408.
- 55. Bate C, Salmona M, Williams A (2004) The role of platelet activating factor in prion and amyloid-β neurotoxicity. Neuroreport 15: 509–513.
- 56. Williams A, Van Dam AM, Ritchie D, Eikelenboom P, Fraser H (1997) Immunocytochemical appearance of cytokines, prostaglandin E2 and lipocortin-1 in the CNS during the incubation period of murine scrapie correlates with progressive PrP accumulations. Brain Res 754: 171–180.
- 57. Williams AE, Van Dam AM, Man AHW, Berkenbosch F, Eikelenboom P, et al. (1994) Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice. Brain Res 654: 200–206.
- 58. Minghetti L, Cardone F, Greco A, Puopolo M, Levi G, et al. (2002) Increased CSF levels of prostaglandin E(2) in variant Creutzfeldt-Jakob disease. Neurology 58: 127–129.
- 59. Minghetti L, Greco A, Cardone F, Puopolo M, Ladogana A, et al. (2000) Increased brain synthesis of prostaglandin E2 and F2-isoprostane in human and experimental transmissible spongiform encephalopathies. JNeuropatholExpNeurol 59: 866–871.
- 60. Gaudreault SB, Chabot C, Gratton JP, Poirier J (2004) The Caveolin Scaffolding Domain Modifies 2-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate Receptor Binding Properties by Inhibiting Phospholipase A2 Activity. J Biol Chem 279: 356–362.
- 61. Bate C, Williams A (2007) Squalestatin protects neurons and reduces the activation of cytoplasmic phospholipase A2 by Aβ1–42. Neuropharmacology 53: 222–231.
- 62. Müller G, Schulz A, Wied S, Frick W (2005) Regulation of lipid raft proteins by glimepiride- and insulin-induced glycosylphosphatidylinositol-specific phospholipase C in rat adipocytes. Biochemical Pharmacology 69: 761–780.
- 63. Pietri M, Caprini A, Mouillet-Richard S, Pradines E, Ermonval M, et al. (2006) Overstimulation of PrPC Signaling Pathways by Prion Peptide 106–126 Causes Oxidative Injury of Bioaminergic Neuronal Cells. J Biol Chem 281: 28470–28479.
- 64. Vainio S, Heino S, Mansson JE, Fredman P, Kuismanen E, et al. (2002) Dynamic association of human insulin receptor with lipid rafts in cells lacking caveolae. EMBO Rep 3: 95–100.
- 65. Parpal S, Karlsson M, Thorn H, Stralfors P (2001) Cholesterol Depletion Disrupts Caveolae and Insulin Receptor Signaling for Metabolic Control via Insulin Receptor Substrate-1, but Not for Mitogen-activated Protein Kinase Control. J Biol Chem 276: 9670–9678.
- 66. Müller G, Hanekop N, Wied S, Frick W (2002) Cholesterol depletion blocks redistribution of lipid raft components and insulin-mimetic signaling by glimepiride and phosphoinositolglycans in rat adipocytes. MolMed 8: 120–136.
- 67. Nielsen D, Gyllberg H, Ostlund P, Bergman T, Bedecs K (2004) Increased levels of insulin and insulin-like growth factor-1 hybrid receptors and decreased glycosylation of the insulin receptor alpha- and beta-subunits in scrapie-infected neuroblastoma N2a cells. Biochem J 380: 571–579.
- 68. Ostlund P, Lindegren H, Pettersson C, Bedecs K (2001) Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie-infected neuroblastoma cells. J Biol Chem 276: 36110–36115.
- 69. Srinivasappa J, Asher DM, Pomeroy KL, Murphy LJ, Wolff AV, et al. (1989) Scrapie-induced diabetes mellitus in hamsters. Microbial Pathogenesis 7: 189–194.
- 70. Lauren J, Gimbel DA, Nygaard HB, Gilbert JW, Strittmatter SM (2009) Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers. Nature 457: 1128–1132.