The human prion protein peptide fragment HuPrP106-126 was used to investigate the effect of PrP upon PLA₂ in primary cortical neurons. It is known that cPLA₂ is promptly activated within 1 hour by agonists including phorbol 12-myristate 13-acetate (PMA) A23187 and ionomycin 24 25 26 , this was confirmed in murine primary cortical neurons via preliminary experiments (Additional file 1: Figure S1), and therefore neurons were initially treated for 30 minutes. p-cPLA₂ was visualised by confocal microscopy using an anti-phospho cPLA₂ antibody against the serine-505 residue.

In untreated neurons a low basal level of p-cPLA₂ labelling in the nuclear region could be seen, however exposure to 40 μM HuPrP106-126 resulted in a significant increase in the intensity of p-cPLA₂ labelling (P < 0.001), indicating amplified levels of the enzyme (Figure 1A). In addition, p-cPLA₂ appeared to relocate from the cell body to the neurites (Figure 1A), an occurrence not previously noted in cPLA₂ activation. The effect of HuPrP106-126 on PLA₂ activation was amino acid sequence specific and dependent on the presence of PrP, as the intensity and localisation of p-cPLA₂ labelling was not altered in cells exposed to 40 μM scrambled HuPrP106-126 peptide or in PrP null neurons exposed to HuPrP106-126 (Figure 1A and B). In contrast known activators of cPLA₂, PMA and A23187, induced p-cPLA₂ activation and localisation to the nuclear region in both cell types (P < 0.05). This is consistent with previous studies which reported that cPLA₂ translocates to the ER and nuclear membrane upon activation 23 .

The effect of HuPrP106-126 on the cPLA₂ cascade was further analysed by measuring the release of arachidonic acid (AA). Cleavage and subsequent release of AA by cPLA₂ can be used as a marker for PLA₂ activity. Primary cortical neurons were exposed to 40 μM HuPrP106-126 or scrambled HuPrP106-126. After 15 and 30 minutes PMA and A23187 induced significant [³ H]-AA release (P < 0.01 and P < 0.001 respectively) (Figure 2A). In contrast, HuPrP106-126 did not induce [³ H]-AA release above untreated or scrambled control levels. When incubation was prolonged to 24 hours, PMA and A23187 again induced a significantly higher [³ H]-AA release compared to controls (P < 0.001), and at this time HuPrP106-126 also induced a significant [³ H]-AA release (P < 0.001) (Figure 2B). Levels of AA following exposure to scrambled peptide controls remained comparable to untreated controls.

To examine the specific involvement of cPLA₂ in HuPrP106-126-induced cellular toxicity cells were pre-treated with the PLA₂ inhibitor PACOCF₃, a known cPLA₂ inhibitor 27 . Primary cortical neuron samples were analysed by Western immunoblotting and levels of activated and total cPLA₂ were measured. After exposure to 40 μM HuPrP106-126 for a period of 1 hour and 24 hours, the relative density of p-cPLA₂/cPLA₂ was significantly higher than in untreated neurons (P < 0.05 and P < 0.01 respectively) (Figure 3A). In contrast, relative expression levels of p-cPLA₂ in neurons treated with 1 μM PACOCF₃ and samples pre-treated with PACOCF₃ prior to the addition of peptide for 1 hour were not significantly different from control samples. However after 24 hours, cPLA₂ activation was reduced markedly in samples pre-treated with PACOCF₃ in comparison to neurons treated with peptide alone (P < 0.01).

Localisation of p-cPLA₂ in the presence of PACOCF₃ was subsequently determined (Figure 3B). Incubation with HuPrP106-126 for 30 minutes resulted in increased p-cPLA₂ labelling in the neurites (P < 0.01) (left panel middle row) as seen previously. In contrast, neurons pre-treated with PACOCF₃ showed a distinct decrease in levels of p-cPLA₂ to levels and location comparable to that of untreated controls (left panel, bottom row). The increase in p-cPLA₂ labelling in neurons exposed to HuPrP106-126 compared to untreated neurons was still apparent after 1 hour (P < 0.01) and 24 hours (P < 0.05) (middle and right panel). In agreement with Western immunoblot data, cells pre-treated with PACOCF₃ showed reduced levels of cPLA₂ phosphorylation in comparison to those induced by HuPrP106-126 at all time points measured (P < 0.001, P < 0.01).

The effect of PACOCF₃ on [³ H]-AA release was examined to determine if it would prevent cPLA₂ from accessing its substrate and inhibit HuPrP106-126-induced activation of cPLA₂. Cells exposed to PACOCF₃ alone released similar amounts of AA to untreated controls (Figure 4A). Again primary cortical neurons exposed to 40 μM HuPrP106-126 for 24 hours released a significant (P < 0.001) amount of [³ H]-AA as seen previously in Figure 2B. However, when neurons were pre-treated with PACOCF₃ and then exposed to PrP peptide for 24 hours, only 35% of total [³ H]-AA was released (Figure 4A). Interestingly, PACOCF₃ pre-treatment did not affect [³ H]-AA release induced by PMA or A23187.

To assess the extent of the role of cPLA₂ in PrP peptide action the effect of PACOCF₃ on HuPrP106-126-induced cell toxicity was also analysed. The toxicity of HuPrP106-126 was measured via MTT assay and confirmed that HuPrP106-126 was lethal to primary cortical neurons, but only after a period of around 5 days (Figure 4B). This was comparable to other studies using cortical neurons 28 . Concentrations of 10 μM and 20 μM HuPrP106-126 did not significantly affect cell survival but 40 μM HuPrP106-126 caused a 70% decrease in neuronal survival (P < 0.001). HuPrP106-126 scrambled peptide had no effect on cell viability. Pre-treatment with PACOCF₃, was protective cells pre-treated with 1 μM or 10 μM PACOCF₃ prior to HuPrP106-126 treatment decreased the amount of cell death to a 16% and 15% loss respectively (Figure 4C).

Further investigation of the neurodegenerative effects of HuPrP106-126 was investigated by staining neurones for the synapse-related protein synaptophysin. Synapse loss is one of the signs of pathology seen in prion infection 29 30 and Alzheimer’s disease 31 . Synaptophysin can therefore be used as a neurodegenerative marker for these disorders. Cortical neurons exposed to HuPrP106-126 for 30 minutes and then examined by confocal microscopy showed no significant changes in levels of synaptophysin (Figure 5A left panel). However, longer incubations of neurones with HuPrP106-126 peptide resulted in a significant loss of synaptophysin labelling at 1 and 24 hours (P < 0.05) (Figure 5A middle and right panel). PACOCF_3, in addition to inhibiting HuPrP106-126-induced cPLA₂ activation and AA-release, also protected against synapse damage with synaptophysin levels in neurons pre-treated with PACOCF₃ prior to peptide treatment being equivalent to untreated controls (Figure 5 bottom row).

As the translocation of p-cPLA₂ within neuronal neurites in the presence of HuPrP106-126 had not been previously reported the question whether HuPrP106-126 treatment leads to a potential re-arrangement of the cytoskeleton, as described similarly for PrP^c 32 was investigated. To examine the process by which HuPrP106-126 induced p-cPLA₂ translocation, neurons were labelled with a range of cytoskeletal markers, including actin, vimentin and beta III tubulin. p-cPLA₂ did not colocalise with actin or vimentin prior to or after treatment with HuPrP106-126 (data not shown). However, 4.8% of p-cPLA₂ colocalised with beta III tubulin before peptide treatment, and this increased to 54% (P < 0.001) after 30 minutes exposure to HuPrP106-126 (Figure 6A and B). Colocalisation was predominantly in the neurites of the cells. Cells pre-treated with PACOCF₃ before exposure to peptide had low levels of cPLA₂ phosphorylation and the average colocalisation of p-cPLA₂ with beta III tubulin was 8%. In addition, colocalisation appeared to be in the nuclear region rather than in the neurites. The relationship between p-cPLA₂ and beta III tubulin also appeared to be PrP-specific. 6.3% colocalisation was measured between p-cPLA₂ and beta III tubulin after exposure to a non-toxic scrambled HuPrP106-126 peptide control (Figure 6) and 6.9% colocalisation was noted in PrP null neurons treated with HuPrP106-126 (Figure 7) both comparable to untreated control levels. Interestingly p-cPLA₂ labelling in PrP null neurons was localised to the nucleus after HuPrP106-126 exposure, indicating that PrP could define the localisation of p-cPLA₂ within the cell.

PMA A23187, poly-D-lysine, paraformaldehyde, Tris–HCl, SDS, glycerol, Na₃VO₄ and palmitoyl trifluoromethyl ketone (PACOCF₃) were obtained from Sigma (Dorset, UK). Primary antibodies included mouse monoclonal cPLA₂ (4-4B-3C, sc-454), rabbit polyclonal anti-phosphorylated cPLA₂ (Ser505 residue, sc-34391), goat polyclonal anti-synaptophysin (sc-7568) (Santa Cruz Biotechnology, Santa Cruz, CA) and goat polyclonal beta III tubulin (Abcam, Cambridge). Fluorescently conjugated secondary antibodies included anti-rabbit Alexa Fluor555 and anti-goat Alexa Fluor 488 (Invitrogen, Paisley, UK).

Primary cortical neurons were prepared from day 15.5 gestation CD-1 mice (Charles River Margate, UK), or 129/Ola PrP null mice originally obtained from Jean Manson as previously described 59 . Primary cortical neurons were prepared from the brains of mouse embryos as described previously 60 and seeded into 24-well or 96-well trays and maintained in neurobasal media (NBM) with B27 components (Invitrogen, Paisley, UK), for 7 days prior to use.

Primary cortical neurons were seeded at a density of 1x10⁶ cells per ml in NBM and left in culture for 7 days. Cells were subsequently treated for 5 days with 10 μM – 40 μM HuPrP106-126 or HuPrP106-126 scrambled (Bachem St. Helens, UK). To ensure homogenous distribution in solution, peptides were sonicated 3 x 10 seconds prior to use. In inhibitory experiments, neurons were pre-treated with 1 μM or 10 μM of the PLA₂ inhibitor PACOCF₃ for 3 hours before the addition of peptide or vehicle control. After incubation, medium was removed and cell survival was measured by the addition of 500 μg ml^-1 of 3,[4,5 dimethylthiazol-2yl]-2,5 diphenyltetrazolium bromide (MTT) (Sigma) which is reduced to a formazan dye in metabolically active cells. MTT was left on the cells for 3 hours with the culture tray kept at 37°C. Cells were then lysed and the MTT-formazan salt was solubilised by the addition of 100 μl per well of DMSO. Optical density was quantified using a SpectraMax M2 microplate reader (Molecular Devices, CA, USA) at a wavelength of 550 nm. Absorbance measured in MTT assays was expressed as percent of the untreated controls (defined as 100%).

Primary cortical neurons were seeded at 2 x 10⁵ cells per well in 24-well trays and left in culture for 7 days. Cells were then incubated for 24 hours with 1 μCi per ml (specific activity 212 Ci per mmol) [5,6,8,9,11,12,14,15-³ H]AA (Amersham Biosciences Amersham, UK) in NBM. Cells were washed twice in NBM and new media containing 0.3% fatty acid free BSA (PAA, Somerset, UK) and 40 μM HuPrP106-126 or scrambled peptide was added. Positive controls included a PKC activator, phorbol 12-myristate 13-acetate (PMA) used at 1 μM, with 5 μM of the calcium ionophore, A23187. In some experiments 1 μM PACOCF₃ was added in media for 3 hours before the addition of peptides. Supernatants were taken after 0, 15 or 30 minutes or 24 hours and centrifuged for 5 min at 16000 x g. Cells were lysed in lysis buffer (63.5 mM Tris–HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 1 mM Na₃VO₄, HALT protease inhibitor cocktail (Pierce, Northumberland, UK). Samples were added to 4 ml scintillant and radioactivity was determined by β-scintillation counting (Packard Tri Carb Liquid Scintillation Analyser, Counter B 1990).

Murine cortical neurons were seeded onto poly-D-lysine coated coverslips at 2 x 10⁵ cells per coverslip and left in culture for 7 days. Cells were treated with 40 μM HuPrP106-126 or a scrambled peptide control. In some experiments 1 μM PACOCF₃ was added in NBM for 3 hours before the addition of peptides. The media was removed and cells washed in PBS before being fixed with 4% paraformaldehyde (PFA) pH 7.4 for 15 min at room temperature. Cells were subsequently permeabilised in 0.1% (v/v) Triton-X100 (Sigma Dorset, UK) in PBS for 10 min at room temperature. Cells were washed, and then incubated with 1% (v/v) donkey or rabbit serum (Chemicon) in PBS for 1 hour at room temperature to block. Cells were then exposed to primary antibody in diluent (1% donkey or rabbit serum) in PBS overnight at 4°C. The coverslips were then washed and counterstained with the appropriate AlexaFluor secondary antibody for 1 hour. Coverslips were washed again, incubated with phalloidin (5 U per ml) for 20 min and exposed to 4 μg per ml DAPI in PBS for 10 min at room temperature to label nuclei. Cells were given a final rinse with ultrapure water before being mounted onto Superfrost Plus slides using Vectashield (Vector Labs, Peterborough, UK) and sealed with Eukitt (Agar Scientific, Stansted, UK). Fluorescence was visualised using a Leica SP5 RS confocal microscope (Leica Microsystems, Milton Keynes, UK) with HC PL Fluotar 20x 0.5 dry objective.

Primary cortical neurons were seeded at 5 x 10⁵ cells per well in 24 well-plates and left in culture for 7 days respectively. NBM was removed and replaced with media containing 40 μM HuPrP106-126. Some cells were pretreated with 1 μM PACOCF₃ prior to addition of peptide. After removal of supernatant cells were washed with ice-cold PBS and 0.4 mM sodium orthovanadate (Na₃VO₄) (Sigma). Cells were lysed in 100 μl of ice-cold lysis buffer (63.5 mM Tris–HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 1 mM Na₃VO₄, HALT protease inhibitor cocktail (Pierce, Northumberland)). 20 μg per lane of protein was resolved using a graduated 4-12% Novex Tris-glycine gel (Invitrogen) using the XCell Surelock Mini-Cell apparatus (Invitrogen). Gels were run at 125 V for around 1.5 hours (Bio-Rad, Hemel Hempstead, UK). Proteins were transferred onto nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK) at 25 V for 60 min and then blocked for two hours at room temperature in PBS containing 5% milk powder (w/v) and 0.1% (v/v) Tween 20 (PBST) (Sigma). Membranes were incubated overnight at 4°C in PBST with 5% (w/v) milk powder containing anti-cPLA₂ mAb (1:1000), phosphospecific anti-cPLA₂ polyclonal (p-cPLA₂) (1:1000), (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-β-Actin mAb (1:5000) (Chemicon, Hampshire, UK). Blots were washed in PBST (1 x 15 min, 3 x 5 min) and incubated with goat anti-mouse or rabbit IgG horseradish peroxidase-conjugated antibody (1:2000) (Pierce) for two hours at room temperature. After washing, an enhanced chemiluminescence kit (Amersham Biosciences) was used to detect bound antibody. Blots were analysed using ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997–2007).

Results from at least three independent experiments are expressed as mean ± standard deviation (S.D.) unless stated otherwise. Colocalisation of molecules in confocal microscopy was calculated using Pearson’s correlation coefficient using Leica LAS AF software (Leica, Milton Keynes, UK). The intensity of the fluorescent label of interest was measured using ImageJ software (version 1.41, Maryland, USA). The overall intensity was divided by the number of cells counted in replicate experiments to give an average intensity for all images. Statistical differences between means were calculated using Student’s t-test or one-way analysis of variance (ANOVA) with a Bonferroni post hoc test using SPSS software (version 14.0, Chicago, USA), with significance set at P < 0.05.