Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide
© Wilson et al; licensee BioMed Central Ltd. 2007
Received: 07 December 2006
Accepted: 22 November 2007
Published: 22 November 2007
Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases. The current study characterised the ingestion, internalization and intracellular trafficking of a neurotoxic peptide containing amino acids 105–132 of the murine prion protein (MoPrP105-132) in neuroblastoma cells and primary cortical neurons.
Fluorescence microscopy and cell fractionation techniques showed that MoPrP105-132 co-localised with lipid raft markers (cholera toxin and caveolin-1) and trafficked intracellularly within lipid rafts. This trafficking followed a non-classical endosomal pathway delivering peptide to the Golgi and ER, avoiding classical endosomal trafficking via early endosomes to lysosomes. Fluorescence resonance energy transfer analysis demonstrated close interactions of MoPrP105-132 with cytoplasmic phospholipase A2 (cPLA2) and cyclo-oxygenase-1 (COX-1), enzymes implicated in the neurotoxicity of prions. Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts. In squalestatin-treated cells, MoPrP105-132 was rerouted away from the Golgi/ER into degradative lysosomes. Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1.
As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways.
The Transmissible Spongiform Encephalopathies (TSE)s, otherwise known as prion diseases, are a family of neurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD) in man, Bovine Spongiform Encephalopathy (BSE) in cattle, and scrapie in sheep and goats. A notable feature of these diseases is the accumulation of (PrPSc) , a misfolded isoform of the host-encoded prion protein PrPc [2, 3]. Neuronal dysfunction and ultimately neuronal death are thought to arise following deposition of fibrils of PrPSc which accumulate in the brain of infected animals [4–6]. However, it remains unclear if PrPSc causes neuronal damage itself, or acts via other molecular forms of PrP that have been suggested as causative agents in prion disease [7–9].
The process of neuronal loss can be investigated in vitro using highly defined synthetic peptides derived from the protease-resistant core of PrPSc. The majority of neurotoxicity studies have employed a peptide consisting of amino acids 106–126 of the human prion protein (HuPrP106-126) which possesses many of the properties of the PrPSc isoform, notably a high β-pleated sheet content, fibril formation and toxicity for neurons in vitro . A corresponding peptide has been identified from the murine prion sequence (MoPrP105-132), which has also been shown to be neurotoxic . These peptides also encompass the major part of the putative transmembrane form of PrP (CtmPrP) that is thought to be important in prion disease pathogenesis as transgenic mice overexpressing such PrP molecules develop neurological disease, and the accumulation of PrPres is followed closely by an increase in CtmPrP [10, 11]. In the current study we employed labelled MoPrP105-132 to identify organelles involved in the trafficking pathways of neurotoxic peptides. We demonstrate that in neuroblastoma cells, MoPrP105-132 co-localises with cholera toxin subunit B (CTxB), which binds to the ganglioside GM1 [12, 13] and caveolin-1 [14, 15], markers of specialised microdomains called lipid rafts.
Lipid rafts are highly enriched in cholesterol, sphingolipids and a population of specific membrane proteins . In some cells, lipid rafts contain cholesterol-binding proteins called caveolins that define a subset of lipid raft called caveosomes . Lipid rafts also act as platforms for cell signalling processes [18, 19], suggesting that MoPrP105-132 might interact with signalling enzymes. As a close correlation exists between the production of prostaglandins (PG)s and neuronal death in prion disease [20, 21], the association between MoPrP105-132 and the enzymes PLA2 and COX responsible for the release of arachidonic acid (AA) and the metabolism of AA into PGs respectively, were studied.
There is increasing evidence that cholesterol levels within the brain may affect the progression of some neurodegenerative diseases. Cholesterol depletion in vitro has been shown to affect the integrity of lipid rafts [22, 23] and reducing cellular cholesterol levels reduces the sensitivity of neurons to prions . In the following studies we have demonstrated that pre-treatment of neurons with squalestatin, a drug that inhibits cholesterol production , alters the surface localisation and the intracellular trafficking of the MoPrP105-132 peptide. In addition, pre-treatment with squalestatin significantly reduced the association between MoPrP105-132 and cPLA2 or COX-1. Such observations raise the possibility that the neurotoxicity of PrP peptides is dependent on the specific intracellular trafficking pathways of such peptides and the association with signal transduction pathways.
MoPrP105-132 is located within lipid rafts
MoPrP105-132 traffics via a non-classical endocytic pathway
Squalestatin treatment alters MoPrP105-132 trafficking
Squalestatin alters the trafficking of MoPrP105-132 in neuroblastoma cells. The percentage of MoPrP105-132 that co-localised with cellular markers as shown in untreated neuroblastoma cells and cells treated for 24 hours with 1 μM squalestatin. Values shown are mean percent of peptide that co-localises with markers as shown, ± SD, which was calculated after counting a minimum of 3 cells.
% Co-localisation with MoPrP105-132
63% ± 8
5% ± 1
73% ± 3
9% ± 3
41% ± 5
7% ± 2
38% ± 4
3% ± 2
6% ± 3
68% ± 8
9% ± 2
40% ± 6
MoPrP105-132 associates with cPLA2and COX-1
Squalestatin treatment reduces the association between MoPrP105-132 and cPLA2/COX-1
Squalestatin reduces co-localisation between MoPrP105-132 and cPLA2/COX-1. The percentage of MoPrP105-132 that co-localised with the signalling enzymes cPLA2 or COX-1 in untreated neuroblastoma cells and cells treated for 24 hours with either 1 μM squalestatin or 2 Hexa-PAF, 5 minutes after the addition of peptide. Values shown are mean ± SD, calculated after counting a minimum of 3 cells.
Co-localisation with MoPrP105-132
38% ± 7
13% ± 4
22% ± 6
70% ± 5
5% ± 5
13% ± 3
Although neuronal dysfunction and ultimately neuronal loss are key features of prion diseases, the molecular mechanisms that that result in neuronal damage remain poorly understood. Synthetic peptides have been extensively used to model the process of neuronal degeneration in vitro and in the present study we analysed the trafficking of MoPrP105-132 in neuroblastoma cells and primary cortical neurones. Most of the MoPrP105-132 molecules co-localised with ganglioside GM1, a marker of lipid rafts identified by CTxB, and with caveolin-1, a marker of a lipid raft subset called caveolae. FRET analysis, a technique with spatial resolution beyond the limits of conventional fluorescence imaging, demonstrated that MoPrP105-132 was intimately associated with both GM1 and caveolin-1 (minimal spacing of 10–100 Å required to observe FRET signals). Separation of lipid rafts from cell membranes by detergent extraction confirmed the microscopy data (MoPrP105-132 was detected only in the GM1 and caveolin-1 positive fraction). It not clear why MoPrP105-132 localises to lipid rafts, one possibility is that lipid rafts contain a high percentage of saturated fatty acids, which provides a hydrophobic environment that stabilises the peptide within the membrane. Another possibility is that many of the putative receptors for prions  are found within lipid rafts, thus, peptide-receptor interactions may be responsible for targeting MoPrP105-132 to these domains.
The trafficking of molecules within cells is partly dependent on the mechanism of internalisation, thus, molecules that are ingested via clathrin-dependent endocytosis traffic via endosomes into degradative lysosomes . In contrast, agents targeted to lipid rafts or caveolae are internalised by clathrin-independent endocytosis and traffic into different cellular compartments. MoPrP105-132 trafficked into both the Golgi and ER (co-localised with the markers GM130 and Grp78); consistent with reports that the cargo of caveolae is directed into the Golgi [14, 15, 34]. These results are consistent with MoPrP105-132 trafficking via the non-classical endocytic pathway, recently defined through the trafficking of GM1 positive lipid rafts . Only a small percentage of MoPrP105-132 was associated with either TfR or LAMP-1, indicating that it was rarely found in early endosomes or lysosomes. Microscopy data was supported by organelle fractionation studies in which endosomes involved in the classical endocytic pathways were fractionated. MoPrP105-132 was not found in fractions containing either TfR or LAMP-1, but was found in an intermediate density fraction known to contain Golgi/ER . Taken together these results indicate that MoPrP105-132 does not traffic down the classical endocytic route and avoids the lysosomes. We speculate that the avoidance of proteolytic lysosomes may facilitate the build up of neurotoxic peptides during disease progression. The localisation of MoPrP105-132 to lipid rafts depends on the sequence of amino acids as a control peptide made up of the same amino acids synthesized in a scrambled sequence did not localise within lipid rafts. Subsequently, the scrambled MoPrP105-132 control peptide trafficked to endosomes and lysosomes, emphasising the sequence dependent nature of the intracellular trafficking of the peptide. Although most of the studies presented are on neuroblastoma cells we found that MoPrP105-132 trafficked in a similar manner in non-transformed primary cortical neurones. The sequence specificity of this effect would argue against MoPrP non-specifically associating with lipid rafts through for example, hydrophobicity, as the composition of the scrambled peptide was the same as MoPrP105-132.
Previous studies demonstrated that neurons treated with the cholesterol synthesis inhibitor squalestatin, a drug that inhibits squalene synthase and reduces cholesterol production without affecting the production of isoprenoid precursors , were protected against the otherwise toxic effects of prions . Since the formation of lipid rafts and lipid raft-dependent uptake is sensitive to cholesterol depletion , we examined the effects of squalestatin-treatment on the trafficking of MoPrP105-132. One possible explanation for the neuroprotective effects of squalestatin, that it prevents ingestion of MoPrP105-132, was excluded by studies that showed that there was no significant difference in the amounts of peptide taken up by untreated and treated cells. However, we did detect a difference in the trafficking of MoPrP105-132 between treated and untreated cells. Squalestatin-treatment of neuroblastoma cells greatly reduced the amounts of MoPrP105-132 that co-localised with either CTxB or with caveolin-1 indicating the most of the peptide was no longer present in lipid rafts. This observation was confirmed by Triton X-100 extractions which showed that most of the MoPrP105-132 peptide was now in the non-raft fraction. Fluorescence microscopy and cell fractionation studies both suggested that in squalestatin-treated cells MoPrP105-132 trafficked down a classical endocytic pathway into lysosomes, avoiding the Golgi/ER. These results are consistent with reports that cholesterol depletion results in the loss of lipid raft functions, including raft-dependent endocytosis and subsequent intracellular trafficking .
The neurotoxicity of prion-derived peptides depends on the activation of specific signalling pathways including PLA2 and the COX enzymes that convert AA into PGs [28–31]. Furthermore, prion-induced injury is associated with increased levels of PGE2 [20, 21]. Selective inhibitors indicated the importance of the COX-1 isoform in prion-mediated neurotoxicity . In untreated neuroblastoma cells a significant proportion of MoPrP105-132 co-localised with cPLA2 and COX-1, this association was short lived (seen at 5 minutes after the addition of peptide but not after 20 minutes). The brief association observed could be a result of peptide rapidly transiting the COX-1/cPLA2 containing compartment, or alternatively localisation of these enzymes is known to be transient and dependent on the activation state of the cell . These observations suggest that the association of MoPrP105-132 with cPLA2 and COX-1 activates these enzymes leading to the production of toxic second messengers. Pre-treatment of neuroblastoma cells with squalestatin reduced the co-localisation between MoPrP105-132 and cPLA2 or COX-1, and reduced prion-induced PGE2 production . Taken together these observations suggest that in normal neuroblastoma cells MoPrP105-132 accumulates in lipid rafts where they activate the PLA2/COX pathway resulting in the production of neurotoxins including PGE2 . Thus, the neuroprotective effect of squalestatin treatment may simply be by the dispersal of lipid rafts, which prevent the formation of MoPrP105-132/PLA2/COX-1 complexes and reduce the production of neurotoxins. Similar effects where found in neuroblastoma cells treated with the PAF antagonist (Hexa-PAF), which protects neurones against prion-mediated neuronal damage . Pre-treatment of neuroblastoma cells with the PAF antagonist reduced the co-localisation between MoPrP105-132 and cPLA2, and significantly between MoPrP105-132 and COX-1. This observation is consistent with reports that PAF antagonists reduce PGE2 production in prion peptide treated neuroblastoma cells .
In conclusion, our data demonstrate that in normal neuroblastoma cells the majority of MoPrP105-132 is found in lipid rafts and traffics via a non-classical endocytic, recycling pathway. This pathway involves internalisation via lipid rafts and trafficking into the Golgi and ER. During this process MoPrP105-132 co-localises with the signalling enzymes, cPLA2 and COX-1, enzymes that are involved in the production of prostaglandins, bioactive lipids closely associated with neuronal death in prion diseases. Treatment of neuroblastoma cells with squalestatin was shown to prevent peptide-induced neurotoxicity but did not prevent internalisation. However squalestatin treatment was associated with altered localisation and trafficking of MoPrP105-132 in neuroblastoma cells. In these cells MoPrP105-132 is not found in lipid rafts, did not associate with cPLA2 and COX-1, and trafficked into lysosomes. We speculate that the effect of squalestatin in negating prion peptide induced neurotoxicity may result from altered trafficking of MoPrP105-132 thus preventing interactions with specific signalling pathways. This hypothesis is strengthened by the observation that a second neuroprotective drug, Hexa-PAF, also prevented peptide association with cPLA2 and COX-1. As altering the trafficking of prion-derived peptides appears to be associated with protection against neurotoxicity, strategies to manipulate intracellular trafficking pathways could lead to novel therapeutic approaches in prion disease.
The murine neuroblastoma NB4-1A3 cell line (European Collection of Cell Cultures) was maintained in RPMI-1640 (Invitrogen, Paisley, UK) supplemented with 2 mM glutamine and 5% foetal calf serum (FCS). Cells were dispensed onto sterilised 13 mm glass coverslips pre-coated with poly-D-lysine (50 μg/ml) at 3 × 105 cells per well (24 well plates) and left to adhere overnight before further use.
Primary neuronal cultures
Primary cortical neurons were prepared from embryonic day 15.5 mice as previously described  and plated onto sterilised 13 mm glass coverslips pre-coated with poly-D-lysine (50 μg/ml) at 2 × 106 cells per well (24 well plates) . After 2 hours, media was changed to neurobasal medium (NBM) containing B27 components (Invitrogen), 2 mM glutamine and 5 μM cytosine arabinoside (Sigma, Dorset, UK) to prevent the proliferation of astroglia. Neuronal cultures were used 7 days after plating.
Synthetic PrP peptides were derived from the sequence of murine prion protein (MoPrP105-132; KTNLKHVAGAAAAGAVVGGLGGYMLGSA)  as was a control peptide containing the same amino acids in a scrambled order (scrambled MoPrP105-132; NGAGKAGMVGLYGAHGATAKVSLVGALA). Peptides were synthesized by solid-phase chemistry and purified by reverse-phase HPLC (gift from Dr. J. Langeveld, ID-Lelystad/Pepscan Systems, The Netherlands). To enable detection of the peptides, they were labelled with rhodamine, fluorescein or biotin.
NB4 1A3 neuroblastoma cells were incubated with 30 μM rhodamine or biotinylated MoPrP105-132 or scrambled peptide conjugated to rhodamine or biotin for 20 to 90 minutes at 37°C. Biotinylated MoPrP105-132 was detected using streptavidin Texas Red (Vector Laboratories, Peterborough, UK). Lipid rafts were identified using 1 μg/ml CTxB conjugated to Alexa Fluor 488 (Molecular Probes, The Netherlands) for 30 minutes at 37°C. FITC-dextran (70 kDa; Molecular Probes) was used as a marker for fluid-phase uptake. For intracellular staining, cells were fixed with 1% paraformaldehyde, Following incubation, cells attached to coverslips were washed once in PBS and fixed in 1% paraformaldehyde. Lipid rafts were identified using 1 μg/ml CTxB conjugated to Alexa Fluor 488 (Molecular Probes, The Netherlands) for 30 minutes at 37°C. For intracellular staining, cells were permeabilised (PBS containing 2% FCS, 2 mM EDTA, 0.1% w/v saponin) and incubated in blocking buffer (PBS containing 1% FCS) to prevent non-specific binding. Biotinylated MoPrP105-132 was detected using streptavidin Texas Red (Vector Laboratories, Peterborough, UK). Early endosomes and lysosomes were stained using antibodies specific for the transferrin receptor (TfR-FITC; BD Pharmingen, Oxford, UK) and lysosomal associated membrane protein-1 (LAMP-1-FITC; BD Pharmingen) respectively. The Golgi was stained using GM130 (Santa Cruz Biotechnology, CA, USA), a marker for the Golgi, and visualised using anti-rabbit FITC (Vector Laboratories). In addition, the ER was stained using anti-Grp78 (Stressgen Biotechnology, San Diego, USA) and visualised using anti-rabbit FITC (Vector Laboratories). Anti-COX-1-FITC (BD Biosciences) and anti-cPLA2-FITC (Santa Cruz Biotechnology, California, USA) were used for investigating signalling enzymes. Vectashieid containing DAPI (Vector Laboratories) was used to stain cell nuclei. Coverslips were mounted onto glass slides (Speci-Microsystems Ltd, Surrey, UK) and fluorescence microscopy was performed with an Axiovert S-100 Zeiss microscope using a 63× oil immersion lens. Serial images in z were captured using a Hamamatsu Orca LCD camera and unprocessed images (additional file 4) were deconvolved using OpenLab software (Improvision, Coventry, UK). Images shown are representative of at least 10 fields of view, with a cell density of typically 5 – 10 cells per field. The degree of co-localisation in fluorescent deconvolved images was evaluated from the number of co-localised pixels within a given cell for a minimum of 3 representative cells, as determined by OpenLab software. For drug treatment, cells were pre-treated under the following conditions prior to the addition of prion peptides: 1 μM squalestatin (GlaxoSmithKline, Herts, UK) or 2 μM 1-O-alkyl-2-acetyl-sn-glycerol-3-phospho-(N,N,N-trimethyl)-hexanolamine (Hexa-PAF) (Novabiochem, Nottingham, UK) overnight at 37°C before use.
NB4 1A3 cells were incubated with 30 μM MoPrP105-132 conjugated to rhodamine and either CTxB conjugated to Alexa Fluor 488, or FITC-labelled caveolin-1 for 30 minutes at 37°C. Fluorescence microscopy was performed with an Olympus BX50 fluorescence microscope equipped with appropriate excitation and emission filter sets. Images representing donor fluorescence, acceptor fluorescence (rhodamine) and FRET signal were taken. Donor and acceptor bleed through values were calculated and the FRET signal quantified and FRET images generated using OpenLab FRET module software .
Lipid raft isolation
Lipid raft isolation was performed as previously described . Briefly, NB4 1A3 cells were incubated with 30 μM MoPrP105-132-FITC for 30 minutes, before being lysed with distilled H2O and centrifuged (10 minutes at 1,000 × g). Pellets were suspended in 1% Triton-X 100 in PBS plus 5 mM phenylmethyl sulfonyl fluoride (PMSF) and incubated for 30 minutes on ice. After centrifugation (10 minutes at 14,000 × g), the supernatant was kept as the non-lipid raft fraction. Pellets were suspended in extraction buffer (10 mM Tris-HCL, 10 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40 and 0.5% Sodium Deoxycholate) and centrifuged (10 minutes at 14,000 × g). The supernatant obtained contained the lipid raft fraction. Using western blot analysis, fractions were transferred onto nitrocellulose and probed for MoPrP105-132 using anti-FITC horseradish peroxidase (HRP;Molecular Probes). Fractions were probed using anti-TfR-FITC and anti-Caveolin-1-FITC (Santa Cruz Biotechnology) and detected using anti-FITC HRP. Fractions were visualised using enhanced chemiluminescence (ECL). Using slot blot analysis, ganglioside-1 (GM1) was detected using CTxB-FITC (Sigma) followed by anti-FITC HRP. For drug treatment, NB4 1A3 cells were pre-treated with squalestatin as described above.
Cell homogenisation and fractionation
NBA 1A3 cells were fractionated using a combination of magnetic isolation of lysosomes  and gradient centrifugation [42, 43]. Cells were incubated with iron dextran, produced from 40 kDa dextran as previously described  for 1 hour at 37°C. Media containing iron dextran was removed and replaced with fresh medium and incubated overnight. After incubation with 30 μM MoPrP105-132-FITC for 1 hour at 37°C, NB4 1A3 cells were scraped into cold (4°C) homogenisation buffer (0.5 mM EGTA, 0.5 mM EDTA, 20 mM HEPES, 0.05% gelatin and 250 mM sucrose). Cells were centrifuged (250 × g for 8 minutes) and suspended in 1 ml homogenisation buffer and then lysed by repeat passage through a 25 G syringe. Nuclei and large fragments were removed by low speed centrifugation (110 × g for 6 minutes) and the resultant supernatant was then applied to a MACS column (Miltenyi Biotec, Surrey, UK) and allowed to run through the column by gravity to give a non-lysosomal fraction. The column was then washed with homogenisation buffer before being removed from the magnet and the remaining homogenate was eluted, giving a lysosomal fraction [F3]. Using a density gradient technique , the non-lysosomal fraction was layered on top of a two step 17% (w/w) Percoll (Amersham Biosciences, UK), 64% (w/w) sucrose cushion. After ultracentrifugation (1 hour at 80,000 × g) using a Beckman Type 70Ti fixed angle rotor, bands containing endosomal organelles [F1] and Golgi and ER [F2] were removed and concentrated by centrifugation (100,000 × g for 30 minutes). The protein content of these fractions were standardised following protein assay (Pierce MicroBCA kit; Pierce, Cheshire, UK). Gel electrophoresis of the endosomal fractions was performed using precast 4–12% Bis-Tris gradient gels with 2-(N-morpholino)ethanesulfonate (MES) running buffer (NuPAGE; Invitrogen) and proteins were transferred onto nitrocellulose, using western blot analysis and probed for MoPrP105-132 using anti-FITC HRP. Secondary antibody anti-FITC HRP was used to detect TfR-FITC and LAMP-1-FITC and fractions were visualised using enhanced chemiluminescence ECL. For drug treatment, NB4 1A3 cells were pre-treated with squalestatin as described above.
NB4 1A3 cells were incubated with FITC-conjugated MoPrP105-132 for 30 minutes at 37°C. Cells were then centrifuged and fixed with 1% paraformaldehyde for 10 minutes, washed and suspended in FACS flow (BD, Pharmingen). Cell associated fluorescence was determined by FACS analysis (FACSCalibur; Becton Dickinson), and expressed as arbitrary fluorescence units (mean fluorescence intensity) using Cell Quest software. Flow cytometry was also performed on MoPrP105-132-FITC treated, NB4 1A3 cells that had been pre-treated with either squalestatin as described above.
Treatment effects were compared using one and two way analysis of variance techniques as appropriate. For all statistical tests, significance was set at the 1% level. Percentage co-localisation in fluorescent deconvolved images was determined by counting the number of co-localised pixels against the number of pixels that did not co-localise, using OpenLab software. Percentage of co-localisation was presented as% ± standard deviation (SD), which was calculated after counting a minimum of 3 cells.
List of abbreviations
Scrapie isoform of PrP
Cellular isoform of prion protein
transmembrane form of PrP
murine prion protein
cholera toxin subunit B
platelet activating factor
cytoplasmic phospholipase A2
Fetal calf serum
lysosomal associated membrane protein-1
fluorescence resonance energy transfer
phenylmethyl sulfonyl fluoride
This work was supported by Scottish Hospitals Endowments Research Trust (SHERT; Grant number RG48/02), Tenovus, Scotland (Grant Number S04/6) and by the European Commission and FP6 "Neuroprion" – Network of Excellence.
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