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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2011 Sep 7;286(44):37955–37963. doi: 10.1074/jbc.M111.248724

Amyloid-β-induced Synapse Damage Is Mediated via Cross-linkage of Cellular Prion Proteins

Clive Bate ‡,1, Alun Williams §
PMCID: PMC3207431  PMID: 21900234

Abstract

The cellular prion protein (PrPC), which is highly expressed at synapses, was identified as a receptor for the amyloid-β (Aβ) oligomers that are associated with dementia in Alzheimer disease. Here, we report that Aβ oligomers secreted by 7PA2 cells caused synapse damage in cultured neurons via a PrPC-dependent process. Exogenous PrPC added to Prnp knock-out(0/0) neurons was targeted to synapses and significantly increased Aβ-induced synapse damage. In contrast, the synapse damage induced by a phospholipase A2-activating peptide was independent of PrPC. In Prnp wild-type(+/+) neurons Aβ oligomers activated synaptic cytoplasmic phospholipase A2 (cPLA2). In these cells, the addition of Aβ oligomers triggered the translocation of cPLA2 in synapses to cholesterol dense membranes (lipid rafts) where it formed a complex also containing Aβ and PrPC. In contrast, the addition of Aβ to Prnp(0/0) neurons did not activate synaptic cPLA2, which remained in the cytoplasm and was not associated with Aβ. Filtration assays and non-denaturing gels demonstrated that Aβ oligomers cross-link PrPC. We propose that it is the cross-linkage of PrPC by Aβ oligomers that triggers abnormal activation of cPLA2 and synapse damage. This hypothesis was supported by our observation that monoclonal antibody mediated cross-linkage of PrPC also activated synaptic cPLA2 and caused synapse damage.

Keywords: Alzheimer Disease, Amyloid, Phospholipase A, Prions, Synapses, Oligomers, Synaptophysin

Introduction

Alzheimer disease (AD)2 is a complex neurological disorder characterized by a progressive dementia resulting from synaptic failure (1, 2). The amyloid hypothesis maintains that the pivotal event in AD is the production and accumulation of amyloid-β (Aβ) peptides following the proteolytic cleavage of the amyloid precursor protein (3–5). Disease-associated mutations result in the increased production of the 42-amino acid peptide fragment (Aβ42) (6, 7), synthetic and recombinant forms of which acts as toxins. Aβ42 peptides self-aggregate and are found in multiple conformations ranging from small oligomers to larger fibrils and plaques. The soluble Aβ oligomers that are considered to be the principal mediators of neurotoxicity (8–11) demonstrate disease-specific accumulation in the brain (12) and bind to synapses (13, 14). The high potency of Aβ oligomers suggests that their effects are mediated through specific receptors. The cellular prion protein (PrPC) that is highly expressed at synapses (15, 16) was recently identified as a receptor that mediates Aβ-induced inhibition of synaptic plasticity and impaired memory in a model of AD (17). However, the role of PrPC in AD pathogenesis has been challenged by others who reported that Aβ caused memory deficits in mice in the absence of PrPC (18, 19).

Such apparently contradictory findings might be explained by the use of synthetic Aβ peptides, which were combined with different models of memory formation as surrogates of dementia. Studies that use synthetic Aβ preparations may be compromised by their propensity to self-aggregate into a wide variety of oligomer sizes and conformations. The polymorphic nature of Aβ aggregates suggests that there exist disease-relevant conformations of Aβ, whereas other conformations are less toxic (20, 21). It is difficult to control the size and conformation of synthetic Aβ42 oligomers, and consequently, it is not clear which of the Aβ conformations are responsible for specific biological properties. To overcome this problem, conditioned medium from 7PA2 cells (7PA2-CM), which contains naturally secreted Aβ oligomers (22), were used in this study. The Aβ oligomers secreted by these cells are SDS stable as are the Aβ oligomers found within the cerebrospinal fluid of Alzheimer patients (23–25). Because the best correlate of dementia in AD is the loss of synaptic proteins such as synaptophysin (26–28), the effects of Aβ oligomers on synaptic density in cultured neurons was determined by measuring the amount of cell-associated synaptophysin using ELISA. Our studies show that PrPC plays a major role in Aβ-induced synapse damage.

EXPERIMENTAL PROCEEDURES

Primary Neuronal Cultures

Cortical neurons were prepared from the brains of day 15.5 embryos from Prnp wild-type(+/+) and Prnp knock-out(0/0) mice as described (29). Cells were suspended in Ham's F12 medium containing 5% fetal calf serum and seeded at 2 × 105 cells/well in 48-well plates or at 106 cells/well in six well plates that had been coated with poly-l-lysine. After 2 h, cultures were shaken and washed to remove non-adherent cells. Neurons were grown in neurobasal medium containing B27 components and nerve growth factor (5 ng/ml) (Sigma) for 10 days. Immunohistochemistry revealed that >95% of cells were neurofilament-positive. For PrPC binding assays, Prnp(0/0) neurons were incubated with PrPC or Thy-1 for between 10 min and 2 h. For other studies, Prnp(+/+) or Prnp(0/0) neurons were pre-treated with PrPC, Thy-1, or control medium for 2 h and then incubated in the presence or absence of Aβ oligomers or mAb 4F2 for 24 h. Treated neurons were washed three times with PBS and homogenized in a buffer containing 150 mm NaCl, 10 mm Tris-HCl, pH 7.4, 10 mm EDTA, 0.2% SDS, mixed protease inhibitors (4-(2-aminoethyl) benzenesulfonyl fluoride, aprotinin, leupeptin, bestatin, pepstatin A, and E-46), and a phosphatase inhibitor mixture (PP1, PP2A, microcystin LR, cantharidin, and p-bromotetramisole) (Sigma) at 106 cells/ml. Nuclei and cell debris were removed by centrifugation (1000 × g for 5 min).

Isolation of Thy-1 and PrPC

PrPC and Thy-1 were isolated from murine GT1 neuronal cell membranes that had been homogenized and washed repeatedly in 10 mm Tris-HCl, pH 7.4, 100 mm NaCl, 10 mm EDTA, and mixed protease inhibitors. PrPC was isolated using an affinity column loaded with mAb ICSM35 (D-Gen) and eluted using 0.1 m glycine-HCl at pH 2.7, neutralized with 1 m Tris, pH 7.4, isolated by reverse phase chromatography on C18 columns (Waters), and lyophilized as described (30). Thy-1 was isolated using a specific mAb (Serotec) and C18 columns as above and lyophilized. Samples were solubilized in culture medium by sonication.

Isolation of Synaptosomes

Synaptosomes were prepared on a discontinuous Percoll gradient as described (31). Cortical neurons were homogenized in SED solution (0.32 m sucrose, 50 mm Tris-HCl, pH 7.2, 1 mm EDTA, and 1 mm dithiothreitol at 4 °C) and centrifuged at 1000 × g for 10 min. The supernatant was transferred to a gradient of 3, 7, 15 and 23% Percoll in SED solution and centrifuged at 16,000 × g for 30 min at 4 °C. Synaptosomes were collected from the interface of the 15 and 23% Percoll steps and washed twice (16,000 × g for 5 min). For some experiments, synaptosomes were incubated with Aβ or control medium for 1 h on rollers at 37 °C, washed three times with ice-cold PBS, and suspended in ice-cold extraction buffer (150 mm NaCl, 10 mm Tris-HCl, pH 7.4, 10 mm EDTA, 0.2% SDS, and mixed protease/phosphatase inhibitors (as described above)).

Sucrose Density Gradients

Synaptosomes were homogenized in an ice-cold buffer containing 1% Triton X-100, 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 10 mm EDTA, and protease and phosphatase inhibitors (as above). 5–40% sucrose solutions were prepared and layered to produce a gradient. Homogenates were added and centrifuged (50,000 × g for 18 h at 4 °C). Serial fractions were collected from the bottom of gradients.

Synaptophysin ELISA

The amount of synaptophysin in samples was determined by ELISA as described (29). Maxisorb immunoplates (Nunc) were coated with the anti-synaptophysin mAb (MAB368-Chemicon) and blocked with 5% milk powder. Samples were added, and bound synaptophysin was detected using rabbit polyclonal anti-synaptophysin (Abcam) followed by a biotinylated anti-rabbit IgG (Dako), extravidin-alkaline phosphatase, and 1 mg/ml 4-nitrophenol phosphate (Sigma). Absorbance was measured at 405 nm, and the synaptophysin content was calculated. Samples were expressed as “units of synaptophysin” where 100 units was the amount of synaptophysin in control cells/synaptosomes derived from 106 untreated cells.

Synaptic Vesicle Recycling

The fluorescent dye FM1–43, which is taken up into synaptic recycling vesicles, was used to determine synaptic activity as described (32). Treated neurons were incubated with 1 μg/ml FM1–43 and 1 μm ionomycin for 5 min, washed five times in ice-cold PBS, and solubilized in methanol at 106 neurons/ml. Soluble extracts were transferred into Sterlin 96-well black microplates, and fluorescence was measured using excitation at 480 nm and emission at 625 nm. Background fluorescence was subtracted, and samples were expressed as “% fluorescence,” where 100% fluorescence was the amount of fluorescence in synaptosomes from 106 control neurons incubated with FM1-43 and ionomycin.

Cytoplasmic Phospholipase A2 (cPLA2) ELISA

The amount of cPLA2 in extracts was measured by ELISA as described (29). Maxisorb immunoplates were coated with 0.5 μg/ml of the mouse mAb anti-cPLA2, clone CH-7 (Upstate), and blocked with 5% milk powder. Samples were incubated for 1 h, and the amount of cPLA2 was detected using a goat polyclonal anti-cPLA2 (Santa Cruz Biotechnology) followed by biotinylated anti-goat IgG, extravidin-alkaline phosphatase, and 1 mg/ml 4-nitrophenyl phosphate. Absorbance was measured at 405 nm, and the amount of cPLA2 present were expressed as “units of cPLA2,” where 100 units were defined as the amount of cPLA2 in synaptosomes derived from106 untreated cortical neurons. The activation of cPLA2 is accompanied by phosphorylation of the 505-serine residue, which creates a unique epitope and can be measured by ELISA (29). Immunoplates were coated with clone CH-7 and blocked (as above). Samples were incubated for 1 h, and the amount of activated (phosphorylated) cPLA2 was detected using a rabbit polyclonal anti-phospho-cPLA2 (Cell Signaling Technology), biotinylated anti-rabbit IgG, extravidin-alkaline phosphatase, and 1 mg/ml 4-nitrophenyl phosphate. Absorbance was measured at 405 nm, and the amount of activated cPLA2 present were expressed as units of activated cPLA2 where 100 units were defined as the amount of activated cPLA2 in synaptosomes derived from 106 untreated cortical neurons.

PrPC ELISA

The amount of PrPC in samples was determined by ELISA as described (29). Maxisorb immunoplates were coated with mAb ICSM18 (d-Gen). Samples were added, and bound PrP was detected with biotinylated mAb ICSM35 (D-Gen). Biotinylated mAb was detected using extravidin-alkaline phosphatase and 1 mg/ml 4-nitrophenyl phosphate (Sigma). Absorbance was measured on a microplate reader at 405 nm, and the amount of PrP in samples was calculated by reference to a standard curve of recombinant murine PrP (Prionics).

Immunoprecipitations

Synaptosomes were homogenized in ice-cold 1% Triton X-100, 10 mm Tris-HCl, pH 7.2, 100 mm NaCl, 10 mm EDTA, and protease and phosphatase inhibitors. Nuclei and cell debris were removed by centrifugation (1000 × g for 5 min), and the post-nuclear supernatant was incubated with 0.1 μg/ml mAb CH-7 (reactive with cPLA2) or isotype controls for 1 h at 4 °C on rollers. Protein G microbeads were added (10 μl/ml) (Miltenyi Biotech) for 30 min, and protein G bound antibody complexes isolated using a μMACS magnetic system (Miltenyi Biotech) at 4 °C.

Western Blotting

For denaturing gels, samples were mixed with Laemmli buffer containing β-mercaptoethanol and heated to 95 °C for 5 min, and proteins were separated by electrophoresis on 15% polyacrylamide gels. Proteins were transferred onto a Hybond-P PVDF membrane by semi-dry blotting. Membranes were blocked using 10% milk powder; PrP was detected by incubation with mAb ICSM18, β-actin by clone AC-74 (Sigma), synaptophysin with MAB368 (Abcam), synaptobrevin with mAb 4H302 (Abcam), cPLA2 with mAb CH-7, and Aβ with mAb 6E10 (Covance). These were visualized using a combination of biotinylated anti-mouse IgG (Dako), extravidin-peroxidase, and enhanced chemiluminescence. For non-denaturing gels, either concentrated 7PA2-CM or treated synaptosomes were homogenized in 0.5% Nonidet P-40, 5 mm CHAPS, 50 mm Triz, pH 7.4, and mixed protease inhibitors and run under non-denaturing conditions. Proteins were transferred onto a Hybond-P PVDF membrane by semi-dry blotting. Membranes were blocked using 10% milk powder. Aβ was detected with mAb 6E10 and PrPC with mAb ICSM18 followed by biotinylated anti-mouse IgG, extravidin-peroxidase, and enhanced chemiluminescence.

Filtration Assays

PrPC was digested with 0.2 units of phosphatidylinositol-phospholipase C (Bacillus cereus) (Sigma), which removes acyl chains and ensures that it is soluble in PBS. Soluble deacylated PrPC (10 ng/ml) was incubated with 7PA2-CM or CHO-CM for 1 h on rollers and then centrifuged through 50- or 300-kDa filters (Vivaspin, Sartorius). The filtrate was collected and tested for the presence of PrPC by ELISA as described above.

Peptides

Phospholipase A2-activating peptide (PLAP) was obtained from Bachem. Stock solutions of peptides were thawed on the day of use, mixed in neurobasal medium/B27, and shaken.

Preparation of Aβ-containing Medium

CHO cells stably transfected with a cDNA encoding APP751 (referred to as 7PA2 cells) were cultured in DMEM with 10% FCS as described (22). Conditioned medium (CM) from these cells contains stable Aβ oligomers (7PA2-CM). CM from non-transfected CHO cells (CHO-CM) was used as controls. 7PA2-CM and CHO-CM were centrifuged at 100,000 × g for 4 h at 4 °C to remove cell debris and then passed through a 50-kDa filter (Sartorius) before use.

Aβ42 ELISA

The amounts of Aβ42 in preparations were determined by ELISA (Amersham Biosciences) according to the manufacturer's instructions. This involves a capture Aβ42-specific murine mAb and detection by rabbit polyclonal anti-Aβ antibodies, horseradish peroxidase-conjugated anti-rabbit IgG followed by 3,3′,5,5′-tetramethylbenzidine. Color was measured at 450 nm and compared with serial dilutions of Aβ42 controls. The limit of detection in this assay was 200 pg/ml. This ELISA was specific for human Aβ42 and did not react with Aβ1–38, Aβ1–40, or Aβ1–43. The ELISA detected both Aβ monomers (forms of Aβ that passed through a 5-kDa filter) and Aβ oligomers (Aβ that passed through a 50- kDa filter but not through a 5-kDa filter).

Statistical Analysis

Comparison of treatment effects was carried out using Student's two-sample t test and analysis of variance.

RESULTS

PrPC Mediates Aβ-induced Synapse Damage

In support of the hypothesis that the accumulation of soluble Aβ oligomers leads to the synapse damage observed in the brains of patients with AD, the addition of 7PA2-CM, containing Aβ monomers, dimers, trimers, and tetramers (Fig. 1A), reduced the synaptophysin content of cultured Prnp(+/+) neurons indicative of synapse damage. However, these Aβ oligomers had a lesser effect upon synapses in cultured Prnp(0/0) neurons (Fig. 1B). Thus, the concentration of Aβ42 required to reduce the synaptophysin content of Prnp(+/+) neurons by 50% (EC50) was ∼700 pm, whereas the EC50 of Aβ42 in Prnp(0/0) neurons was >10 nm. Immunoblots confirmed that exposure of Prnp(+/+) neurons to Aβ42 caused the loss of synaptic proteins, including synaptophysin and synaptobrevin, without affecting β-actin content (Fig. 1C). The loss of synaptophysin was seen at concentrations of Aβ42 that did not affect cell survival as measured by thiazolyl blue tetrazolium (data not shown). PrPC was not necessary for synapse damage induced by all neurotoxins; the addition of PLAP reduced the synaptophysin content of cortical neurons derived from both Prnp(+/+) and Prnp(0/0) neurons to a similar extent (Fig. 1D).

FIGURE 1.

FIGURE 1.

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PrPC mediates Aβ-induced synapse damage. A, immunoblot showing Aβ monomers, dimers, trimers, and tetramers in concentrated 7PA2-CM (i) or CHO-CM (ii) separated by non-denaturing PAGE and detected with mAb 6E10. B, the amount of synaptophysin in cortical neurons derived from Prnp(+/+) mice (●) or Prnp(0/0) mice (○) and incubated with Aβ42 for 24 h. Values shown are the mean amount of synaptophysin (units) ± S.D. from triplicate experiments performed five times (n = 15). C, immunoblots showing the amount of synaptophysin (i), synaptobrevin (ii), and β-actin (iii) in Prnp(+/+) cortical neurons incubated with Aβ42 as shown for 24 h. D, the amount of synaptophysin in cortical neurons derived from Prnp(+/+) (●) or Prnp(0/0) mice (○) and incubated with PLAP for 24 h. Values shown are the mean amount of synaptophysin (units) ± S.D. from triplicate experiments performed four times (n = 12).

Experiments were performed to see whether the presence of PrPC affected the accumulation of Aβ within synapses. Cortical neurons from Prnp(+/+) or Prnp(0/0) neurons were pulsed with 10 ng of Aβ42 for 1 h, and synaptosomes were isolated. The amount of Aβ42 present in synaptosomes recovered from Prnp(0/0) neurons was not significantly different from the amount of Aβ42 in synaptosomes derived from Prnp(+/+) neurons (2.18 ng Aβ42 ± 0.29 compared with 2.34 ng Aβ42 ± 0.31, n = 9, p = 0.6) indicating that Aβ42 can accumulate at synapses via a PrPC-independent pathway. Immunoblots showed that there were similar amounts of Aβ monomers, dimers, trimers, and tetramers in each preparation (Fig. 2).

FIGURE 2.

FIGURE 2.

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Aβ accumulates at synaptosomes in the absence of PrPC. Immunoblot showing Aβ monomers, dimers, trimers, and tetramers in synaptosomes isolated from cortical neurons derived from Prnp(+/+) mice (i) or Prnp(0/0) mice (ii) and incubated with 10 ng of Aβ42 for 1 h. Synaptosomes were electrophoresed under non-denaturing conditions, and Aβ was detected with mAb 6E10.

Because PrPC is readily transferred between cells (30, 33), the effects of adding PrPC to the responsiveness of Prnp(0/0) neurons to Aβ42 was examined. PrPC bound to Prnp(0/0) neurons and was targeted to synapses; a time-dependent accumulation of PrPC within synaptosomes was observed following the addition of 20 ng PrPC (Fig. 3A). Critically, pre-treatment of Prnp(0/0) neurons with 20 ng PrPC significantly increased the Aβ42-induced synapse damage (Fig. 3B). In contrast, pre-treatment of Prnp(0/0) neurons with 20 ng Thy-1, another GPI-anchored protein that is found within synapses (34), did not affect Aβ42-induced synapse damage.

FIGURE 3.

FIGURE 3.

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PrPC increases Aβ-induced synapse damage in Prnp(0/0) neurons. A, the amount of PrPC found in synaptosomes derived from Prnp(0/0) neurons incubated with 20 ng of PrPC for the time periods as shown (□) or from Prnp(+/+) synaptosomes (■). Values shown are the mean amount of synaptic PrPC (ng/106 cells) ± S.D. from duplicate experiments performed five times (n = 10). B, the synaptophysin content of Prnp(0/0) neurons pre-treated with control medium (○), 20 ng PrPC (□), or 20 ng Thy-1 (■) and incubated for 24 h with Aβ42. Values shown are the mean amount of synaptophysin (units) ± S.D. from triplicate experiments performed four times (n = 12).

PrPC Mediates Aβ-induced Inhibition of Synaptic Vesicle Recycling

The recycling of synaptic vesicles can be studied using the uptake of fluorescent dyes such as FM1-42 (35). Aβ42 inhibits the uptake of FM1-42 in cortical neurons, indicating that it suppresses synaptic vesicle recycling and hence neurotransmission (32). Here, we show that Aβ42 had a greater inhibitory effect on the uptake of FM1-43 by Prnp(+/+) neurons than by Prnp(0/0) neurons (Fig. 4A). Pre-treatment of Prnp(0/0) neurons with 20 ng PrPC increased Aβ42-induced inhibition of synaptic vesicle recycling to levels comparable with Prnp(+/+) neurons, whereas the addition of 20 ng of Thy-1 had no effect (Fig. 4B). Collectively, these results indicate that PrPC was involved in Aβ42-induced suppression of synapse function.

FIGURE 4.

FIGURE 4.

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PrPC mediates Aβ-induced inhibition of synaptic vesicle recycling. A, the amount of FM1–43 in cortical neurons derived from Prnp(+/+) (●) or Prnp(0/0) (○) mice, which were incubated with Aβ42 derived from 7PA2-CM, as shown for 24 h. Cells were then pulsed with 1 μg/ml FM1-43 and 1 μm ionomycin for 5 min. Values shown are the mean amounts of FM1-43 (% fluorescence) ± S.D. from triplicate experiments performed four times (n = 12). B, the amount of FM1-43 in cortical neurons derived from Prnp(0/0) mice that had been pre-treated with control medium (○), 20 ng of PrPC (□), or 20 ng of Thy-1 (■) and incubated with Aβ42 as shown for 24 h. Cells were then pulsed with 1 μg/ml FM1-43 and 1 μm ionomycin for 5 min. Values shown are the mean amounts of FM1-43 (% fluorescence) ± S.D. from triplicate experiments performed four times (n = 12).

PrPC Mediates Activation of Synaptic cPLA2 by Aβ

Increasing evidence implicates Aβ-induced activation of specific cell signaling pathways in the process that leads to synapse degeneration. For example, Aβ peptides activate cPLA2 (36, 37), and pharmacological inhibition of cPLA2 protects against Aβ-induced synapse damage (29) and ameliorates cognitive impairment in a mouse model of AD (38). For these reasons, the effects of Aβ42 on synaptic cPLA2 in Prnp(+/+) and Prnp(0/0) neurons were investigated. Although the addition of 2 nm Aβ42 increased the amount of activated cPLA2 found in synapses derived from Prnp(+/+) neurons, it had a lesser effect on cPLA2 in synapses derived from Prnp(0/0) neurons (Fig. 5A). In contrast, the absence of PrPC did not affect the activation of synaptic cPLA2 induced by 500 ng/ml of PLAP. Pre-treatment of Prnp(0/0) neurons with 20 ng PrPC, but not 20 ng Thy-1, increased Aβ-induced activation of cPLA2 in synapses (Fig. 5B). We conclude that Aβ activates cPLA2 by a PrPC-dependent pathway rather than that PrPC is an essential component of cPLA2 activation.

FIGURE 5.

FIGURE 5.

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PrPC is required for Aβ-induced activation of cPLA2. A, the amount of activated cPLA2 in synaptosomes isolated from neurons derived from Prnp(+/+) (■) or Prnp(0/0) mice (□), which had been incubated for 1 h with control medium, 2 nm Aβ42, or 500 ng/ml PLAP. Values shown are the mean amount of activated cPLA2 (units) ± S.D. from triplicate experiments performed four times (n = 12). An asterisk represents a significant difference between Prnp(+/+) and Prnp(0/0) synaptosomes (p < 0.05). B, the amount of activated cPLA2 in synaptosomes isolated from Prnp(0/0) neurons, which were pre-treated with 20 ng of PrPC (■) or 20 ng of Thy-1 (□), and incubated for 1 h with control medium or 2 nm Aβ42. Values shown are the mean amount of activated cPLA2 (units) ± S.D. from triplicate experiments performed four times (n = 12). An asterisk represents a significant difference between treated synaptosomes (p < 0.05).

The activation of cPLA2 is accompanied by its translocation from the cytoplasm to specific membranes mediated by a lipid-binding N-terminal motif (39). In this study, sucrose density gradients were generated from Prnp(+/+) synaptosomes. The addition of 2 nm Aβ42 caused cPLA2 to migrate from fractions 4 to 7 to low density membranes, fractions 9 to 12. The lipid raft marker FITC-CTxB was found in similar low density membranes (data not shown), indicating that the activation of cPLA2 occurs within cholesterol-sensitive lipid rafts (40–42). In contrast, in Prnp(0/0) synaptosomes incubated with 2 nm Aβ42, cPLA2 remained mostly within the cytoplasm, fractions 4 to 7 (Fig. 6A). The interactions between PrPC and cPLA2 were studied further by immunoprecipitation. When Prnp(+/+) synaptosomes were incubated with 2 nm Aβ42, a mAb to cPLA2 (CH-7) co-precipitated both Aβ and PrPC. In contrast, mAb CH-7 failed to precipitate Aβ from Prnp(0/0) synaptosomes incubated with 2 nm Aβ42 (Fig. 6B). Notably, mAb CH-7 did not precipitate PrPC from Prnp(+/+) synaptosomes incubated with CHO-CM. Initially, immunoprecipitations were carried out on Triton X-100 extracts of synaptosomes at 4 °C, which maintains the integrity of lipid rafts. When these experiments were repeated on synaptosomes solubilized in a detergent containing 0.2% SDS, which disperses lipid rafts, mAb CH-7 failed to co-precipitate either PrPC or Aβ, showing that the interactions between cPLA2, PrPC, and Aβ occurs within Triton X-100-insoluble, SDS-soluble lipid rafts.

FIGURE 6.

FIGURE 6.

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PrPC is required for the Aβ-induced translocation of cPLA2. A, synaptosomes were isolated from Prnp(+/+) (■) or Prnp(0/0) (□) neurons that had been incubated with 2 nm Aβ42 for 1 h. Extracts were prepared and separated on sucrose density gradients and the amount of cPLA2 in each fraction was determined by ELISA. Values shown are the mean amount of cPLA2 (units) ± S.D. from triplicate experiments performed three times (n = 9). B, immunoblot showing the amounts of cPLA2, PrPC, and Aβ precipitated by mAb CH-7 (anti-cPLA2) from Prnp(+/+) synaptosomes incubated with 2 nm Aβ42 (lane 1) or CHO-CM (lane 2). Also shown are precipitates from Prnp(0/0) synaptosomes incubated with 2 nm Aβ42 (lane 3) and precipitates from Prnp(+/+) synaptosomes incubated with 2 nm Aβ42 and homogenized in 0.2% SDS that disperses lipid rafts (lane 4).

Aβ Oligomers Cross-link PrPC at Synapses

The damaging effects of Aβ on synapses are caused by Aβ oligomers (11, 25, 43) that have the capacity to cross-link PrPC, whereas Aβ monomers that cannot cross-link PrPC are thought to be non-toxic (23, 44). In addition, aggregated PrPC causes synaptic abnormalities (45), and cross-linkage of PrPC by mAbs leads to cell signaling in T cells (46) and neurodegeneration (47). Collectively, these studies suggest that it is the cross-linkage of PrPC by Aβ oligomers that triggers synapse damage. This hypothesis was tested by incubating Prnp(+/+) synaptosomes with 2 nm Aβ42 and analyzing extracts using non-denaturing gel electrophoresis. We report that PrPC derived from synaptosomes incubated with Aβ oligomers migrated at higher apparent molecular weights than did PrPC derived from synaptosomes incubated with CHO-CM (Fig. 7A). These results suggest that PrPC incubated with Aβ oligomers forms a complex containing at least two PrPC molecules. To complement this study, the effect of Aβ oligomers on the filtration of soluble PrPC was examined. Soluble PrPC that had been incubated with CHO-CM passed through a 50-kDa filter, indicating that it remained as a monomer. In contrast, incubation of soluble PrPC with 7PA2-CM containing Aβ oligomers reduced the amount of PrPC that passed through a 50-kDa filter by ∼90%, indicating that Aβ oligomers had caused PrPC to form higher molecular weight complexes (Fig. 7B).

FIGURE 7.

FIGURE 7.

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Aβ cross-links PrPC. A, synaptosomes derived from Prnp(+/+) neurons were incubated with 2 nm Aβ42 or CHO-CM for 1 h. Extracts were separated by non-denaturing gel electrophoresis and probed for PrPC. Immunoblot showing PrPC derived from synaptosomes incubated with Aβ oligomers derived from 7PA2 cells (lane 1) or CHO-CM (lane 2). B, soluble PrPC was incubated with CHO-CM (□) or 7PA2-CM containing 2 nm Aβ42 (■) for 1 h and then passed through 50- or 300-kDa filters. The amount of PrPC that passed through each filter was measured. Values shown are the mean amount of PrPC ± S.D. from triplicate experiments performed four times (n = 12).

mAb to PrPC Triggers Synapse Damage

Our hypothesis, that Aβ oligomer-induced cross-linkage of PrPC at the synapse triggers activation of cPLA2 and synapse damage, was tested by incubating neurons with a PrPC-reactive mAb. We report that the anti-PrPC mAb 4F2 triggers the loss of synaptophysin from Prnp(+/+) cortical neurons but had no effect upon synapses in Prnp(0/0) neurons (Fig. 8A). There was no significant loss of synaptophysin from Prnp(+/+) neurons incubated with 1 μg/ml of a mAb to Thy-1 (Serotec) (98 units ± 7 compared with 100 units ± 5, n = 12, p = 0.6). The sensitivity of Prnp(0/0) neurons to mAb 4F2-induced synapse damage was increased by pre-treatment with 20 ng PrPC, but not by pre-treatment with 20 ng Thy-1 (Fig. 8B). The addition of 100 ng/ml mAb 4F2 increased the amount of activated cPLA2 found in synapses derived from Prnp(+/+) neurons (458 units ± 60 compared with 100 units ± 21, n = 12, p < 0.05) but had a lesser affect on synapses from Prnp(0/0) neurons (138 units ± 66 compared with 106 units ± 29, n = 12, p = 0.15).

FIGURE 8.

FIGURE 8.

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A mAb to PrPC causes synapse damage. A, the amount of synaptophysin in cortical neurons derived from Prnp(+/+) mice (●) or Prnp(0/0) mice (○) and incubated with different concentrations of the PrPC-reactive mAb 4F2 for 24 h. Values shown are the mean amount of synaptophysin (units) ± S.D. from triplicate experiments performed five times (n = 15). B, the synaptophysin content of cortical neurons derived from Prnp(0/0) mice pre-treated with control medium (○), 20 ng of PrPC (□), or 20 ng of Thy-1 (■) and incubated for 24 h with different concentrations of mAb 4F2. Values shown are the mean amount of synaptophysin (units) ± S.D. from triplicate experiments performed three times (n = 9).

DISCUSSION

The identification of specific receptors for Aβ has been subject to intensive investigation and several candidate proteins have been identified, including NMDA and mGlu5R receptors (48, 49), the amyloid precursor protein (50), the receptor for advanced glycation end products (51), and PrPC (17). However, the role of each of these proteins in the biological activity of Aβ remains unclear. Although a recent study showed that synapse failure induced by synthetic Aβ42 was mediated by PrPC (17), this observation remains controversial as others have reported synthetic Aβ42-induced memory defects in Prnp(+/+) mice (18, 19). To overcome the problem of synthetic Aβ42 preparations adopting multiple conformations, our experiments were performed with 7PA2-CM containing stable Aβ oligomers (22), which are similar to the Aβ oligomers found within the cerebrospinal fluid of Alzheimer patients (23–25). We show that Prnp(0/0) neurons are more resistant to synapse damage induced by Aβ oligomers than neurons that expressed PrPC. Moreover, PrPC introduced into Prnp(0/0) neurons rapidly accumulated at synapses and increased their sensitivity to Aβ, thus supporting the hypothesis that PrPC plays a role in mediating Aβ oligomer-induced synapse damage.

Reports that Aβ accumulates within synapses suggests that the presence of specific Aβ receptors (13, 14). The observation that similar amounts of Aβ42 were found within synaptosomes from Prnp(+/+) and Prnp(0/0) neurons indicates that PrPC is not the only receptor for Aβ at the synapse. Moreover, the observation that the accumulation of Aβ42 at synapses in Prnp(0/0) neurons did not lead to synapse damage indicates that the binding of Aβ oligomers to other synaptic receptors does not trigger synapse damage and that PrPC plays a central role in the Aβ-induced activation of the molecular pathways that leads to synapse damage.

Although the mechanisms by which Aβ causes synapse damage remain unclear, increasing evidence implicates Aβ-induced activation of specific cell signaling pathways in synapse degeneration. Thus, Aβ peptides activate cPLA2 (36, 37), which is concentrated at the pre-synaptic membrane (29, 52), and pharmacological inhibition of cPLA2 protects against Aβ-induced synapse damage (29) and ameliorates cognitive impairment in a mouse model of AD (38). Here, we show that Aβ activates synaptic cPLA2 in Prnp(+/+) neurons but had a lesser effect on synapses in Prnp(0/0) neurons. Moreover, in Prnp(+/+) synaptosomes, the activation of cPLA2 was associated with its migration to low density membranes; results that support previous observations that Aβ-induced activation of cPLA2 occurrs within cholesterol-sensitive lipid rafts (42). More specifically, our immunoprecipitation studies show that following the addition of Aβ to Prnp(+/+) synaptosomes, cPLA2 forms a complex with Aβ and PrPC. This complex was sensitive to SDS indicating that it occurred within a lipid raft. In Prnp(0/0) neurons, the addition of Aβ oligomers failed to activate synaptic cPLA2 which remained in the cytoplasm and did not form a complex with Aβ. The addition of PrPC to Prnp(0/0) neurons increased Aβ-induced activation of synaptic cPLA2. It is notable that the addition of PLAP activates synaptic cPLA2 and triggers synapse damage in both Prnp(+/+) and Prnp(0/0) neurons, indicating that PrPC was not essential for activation of cPLA2. Such results are consistent with the hypothesis that PrPC acts as a receptor that mediates the Aβ-induced activation of synaptic cPLA2 and synapse damage.

It is widely accepted that Aβ oligomers cause synapse dysfunction (11, 43), whereas Aβ monomers are non-toxic (23, 44). In this study, we used two methods to show that Aβ oligomers cross-link PrPC in synaptosomes. First, we used non-denaturing gels to show that PrPC from synaptosomes incubated with control medium migrated as a monomer, whereas higher molecular weight forms of PrPC were observed from synaptosomes that had been incubated with Aβ oligomers. The ability of Aβ oligomers to cross-link PrPC was also demonstrated using a filtration assay. Although untreated PrPC passed through a 50-kDa filter, most of the PrPC that had been incubated with Aβ oligomers did not, indicating that it had formed higher molecular weight complexes. These observations are consistent with the hypothesis that Aβ oligomers express more than one PrPC-binding site and can cross-link PrPC.

Observations that aggregated PrPC causes synaptic abnormalities (45) and that cross-linkage of PrPC by mAbs leads to the formation of lipid rafts and cell signaling (46) and neurodegeneration (47) raises the possibility that it is the cross-linkage of PrPC that leads to synapse damage. This hypothesis was supported by our observation that cross-linkage of PrPC by mAb 4F2 activates cPLA2 and causes synapse damage in Prnp(+/+) neurons. We propose a model whereby Aβ oligomers cross-link PrPC creating a lipid raft platform in which the activation of cPLA2 leads to synapse damage (Fig. 9B). We noted that PrPC and cPLA2 do not interact in the absence of Aβ oligomers (Fig. 9A) and that the accumulation of Aβ at the synapses of Prnp(0/0) neurons does not lead to the activation of cPLA2, or an association between Aβ and cPLA2. Thus, an anti-PrPC mAb (4F2) mimics the effects of Aβ on synapses, it cross-links PrPC, activates cPLA2, and causes synapse damage (Fig. 9C).

FIGURE 9.

FIGURE 9.

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PrPC mediates the activation of cPLA2 and synapse damage induced by Aβ. Shown is a schematic showing the proposed relationship between PrPC, cPLA2, lipid rafts (open clouds), and Aβ oligomers (black ink blots). A, in the absence of Aβ oligomers, cPLA2 and PrPC and are not associated, and there is no activation of cPLA2 or synapse damage. B, in Prnp(+/+) synapses, Aβ oligomers cross-link PrPC resulting in a complex containing Aβ, PrPC, and cPLA2, which leads to the activation of cPLA2 and synapse damage. C, the addition of mAb 4F2 also cross-links PrPC resulting in the association between cPLA2 and PrPC, activation of cPLA2, and synapse damage.

Controversy surrounds the role of PrPC as a receptor for Aβ-induced synapse failure (17–19). Our observations suggest that soluble Aβ oligomers induce the loss of synaptic proteins from cultured neurons by a PrPC-dependent process, an observation consistent with reports that the loss of synaptic proteins is the best correlate of dementia in AD patients (26–28). Our results indicate that Aβ oligomers can cross-link PrPC, leading to aberrant activation of cPLA2 and synapse damage, and strengthen the hypothesis that modification of Aβ-PrPC interactions may provide novel therapeutics to modify the dementia associated with AD.

Acknowledgments

We thank Drs. Scopes, Nerou, and Treherne (Senexis) for the gift of 7PA2-CM and CHO-CM.

Footnotes

2
The abbreviations used are:
AD
Alzheimer disease
PrPC
cellular prion protein
cPLA2
cytoplasmic phospholipase A2
Aβ
amyloid-β
7PA2-CM
conditioned medium from 7PA2 cells
PLAP
phospholipase A2-activating peptide.

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