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

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.

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-␤ (A␤) peptides following the proteolytic cleavage of the amyloid precursor protein (3)(4)(5). Disease-associated mutations result in the increased production of the 42-amino acid peptide fragment (A␤ 42 ) (6, 7), synthetic and recombinant forms of which acts as toxins. A␤ 42 peptides self-aggregate and are found in multiple conformations ranging from small oligomers to larger fibrils and plaques. The soluble A␤ 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 A␤ oligomers suggests that their effects are mediated through spe-cific receptors. The cellular prion protein (PrP C ) that is highly expressed at synapses (15,16) was recently identified as a receptor that mediates A␤-induced inhibition of synaptic plasticity and impaired memory in a model of AD (17). However, the role of PrP C in AD pathogenesis has been challenged by others who reported that A␤ caused memory deficits in mice in the absence of PrP C (18,19).
Such apparently contradictory findings might be explained by the use of synthetic A␤ peptides, which were combined with different models of memory formation as surrogates of dementia. Studies that use synthetic A␤ preparations may be compromised by their propensity to self-aggregate into a wide variety of oligomer sizes and conformations. The polymorphic nature of A␤ aggregates suggests that there exist disease-relevant conformations of A␤, whereas other conformations are less toxic (20,21). It is difficult to control the size and conformation of synthetic A␤ 42 oligomers, and consequently, it is not clear which of the A␤ conformations are responsible for specific biological properties. To overcome this problem, conditioned medium from 7PA2 cells (7PA2-CM), which contains naturally secreted A␤ oligomers (22), were used in this study. The A␤ oligomers secreted by these cells are SDS stable as are the A␤ oligomers found within the cerebrospinal fluid of Alzheimer patients (23)(24)(25). Because the best correlate of dementia in AD is the loss of synaptic proteins such as synaptophysin (26 -28), the effects of A␤ oligomers on synaptic density in cultured neurons was determined by measuring the amount of cell-associated synaptophysin using ELISA. Our studies show that PrP C plays a major role in A␤-induced synapse damage.

EXPERIMENTAL PROCEEDURES
Primary Neuronal Cultures-Cortical neurons were prepared from the brains of day 15.5 embryos from Prnp wildtype (ϩ/ϩ) 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 ϫ 10 5 cells/well in 48-well plates or at 10 6 cells/well in six well plates that had been coated with poly-Llysine. 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 Ͼ95% of cells were neurofilament-positive. For PrP C binding assays, Prnp (0/0) neurons were incubated with PrP C or Thy-1 for between 10 min and 2 h. For other studies, Prnp (ϩ/ϩ) or Prnp (0/0) neurons were pre-treated with PrP C , Thy-1, or control medium for 2 h and then incubated in the presence or 1 To whom correspondence should be addressed.  46), and a phosphatase inhibitor mixture (PP1, PP2A, microcystin LR, cantharidin, and p-bromotetramisole) (Sigma) at 10 6 cells/ml. Nuclei and cell debris were removed by centrifugation (1000 ϫ g for 5 min). Isolation of Thy-1 and PrP C -PrP C 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. PrP C 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 ϫ 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 ϫ 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 ϫ g for 5 min). For some experiments, synaptosomes were incubated with A␤ 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 ϫ 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 10 6 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 g/ml FM1-43 and 1 M ionomycin for 5 min, washed five times in ice-cold PBS, and solubilized in methanol at 10 6 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 10 6 control neurons incubated with FM1-43 and ionomycin.
Cytoplasmic Phospholipase A 2 (cPLA 2 ) ELISA-The amount of cPLA 2 in extracts was measured by ELISA as described (29). Maxisorb immunoplates were coated with 0.5 g/ml of the mouse mAb anti-cPLA 2 , clone CH-7 (Upstate), and blocked with 5% milk powder. Samples were incubated for 1 h, and the amount of cPLA 2 was detected using a goat polyclonal anti-cPLA 2 (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 cPLA 2 present were expressed as "units of cPLA 2 ," where 100 units were defined as the amount of cPLA 2 in synaptosomes derived from10 6 untreated cortical neurons. The activation of cPLA 2 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) cPLA 2 was detected using a rabbit polyclonal antiphospho-cPLA 2 (Cell Signaling Technology), biotinylated antirabbit IgG, extravidin-alkaline phosphatase, and 1 mg/ml 4-nitrophenyl phosphate. Absorbance was measured at 405 nm, and the amount of activated cPLA 2 present were expressed as units of activated cPLA 2 where 100 units were defined as the amount of activated cPLA 2 in synaptosomes derived from 10 6 untreated cortical neurons.
PrP C ELISA-The amount of PrP C 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 ϫ g for 5 min), and the post-nuclear supernatant was incubated with 0.1 g/ml mAb CH-7 (reactive with cPLA 2 ) or isotype controls for 1 h at 4°C on rollers. Protein G microbeads were added (10 l/ml) (Miltenyi Biotech) for 30 min, and protein G bound antibody complexes isolated using a MACS magnetic system (Miltenyi Biotech) at 4°C.
Western Blotting-For denaturing gels, samples were mixed with Laemmli buffer containing ␤-mercaptoethanol and heated to 95°C for 5 min, and proteins were separated by electrophoresis on 15% polyacrylamide gels. Proteins were trans-ferred 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, ␤-actin by clone AC-74 (Sigma), synaptophysin with MAB368 (Abcam), synaptobrevin with mAb 4H302 (Abcam), cPLA 2 with mAb CH-7, and A␤ with mAb 6E10 (Covance). These were visualized using a combination of biotinylated anti-mouse IgG (Dako), extravidin-peroxidase, and enhanced chemiluminescence. For nondenaturing 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. A␤ was detected with mAb 6E10 and PrP C with mAb ICSM18 followed by biotinylated anti-mouse IgG, extravidin-peroxidase, and enhanced chemiluminescence.
Filtration Assays-PrP C 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 PrP C (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 PrP C by ELISA as described above.
Peptides-Phospholipase A 2 -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 A␤-containing Medium-CHO cells stably transfected with a cDNA encoding APP 751 (referred to as 7PA2 cells) were cultured in DMEM with 10% FCS as described (22). Conditioned medium (CM) from these cells contains stable A␤ 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 ϫ g for 4 h at 4°C to remove cell debris and then passed through a 50-kDa filter (Sartorius) before use.
A␤ 42 ELISA-The amounts of A␤ 42 in preparations were determined by ELISA (Amersham Biosciences) according to the manufacturer's instructions. This involves a capture A␤ 42specific murine mAb and detection by rabbit polyclonal anti-A␤ antibodies, horseradish peroxidase-conjugated antirabbit IgG followed by 3,3Ј,5,5Ј-tetramethylbenzidine. Color was measured at 450 nm and compared with serial dilutions of A␤ 42 controls. The limit of detection in this assay was 200 pg/ml. This ELISA was specific for human A␤ 42 and did not react with A␤ 1-38 , A␤ 1-40 , or A␤ 1-43 . The ELISA detected both A␤ monomers (forms of A␤ that passed through a 5-kDa filter) and A␤ oligomers (A␤ 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
PrP C Mediates A␤-induced Synapse Damage-In support of the hypothesis that the accumulation of soluble A␤ oligomers leads to the synapse damage observed in the brains of patients with AD, the addition of 7PA2-CM, containing A␤ monomers, dimers, trimers, and tetramers (Fig. 1A), reduced the synaptophysin content of cultured Prnp (ϩ/ϩ) neurons indicative of synapse damage. However, these A␤ oligomers had a lesser effect upon synapses in cultured Prnp (0/0) neurons (Fig. 1B). Thus, the concentration of A␤ 42 required to reduce the synaptophysin content of Prnp (ϩ/ϩ) neurons by 50% (EC 50 ) was ϳ700 pM, whereas the EC 50 of A␤ 42 in Prnp (0/0) neurons was Ͼ10 nM. Immunoblots confirmed that exposure of Prnp (ϩ/ϩ) neurons to A␤ 42 caused the loss of synaptic proteins, including synaptophysin and synaptobrevin, without affecting ␤-actin content (Fig. 1C). The loss of synaptophysin was seen at concentrations of A␤ 42 that did not affect cell survival as measured by thiazolyl blue tetrazolium (data not shown). PrP C 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 (ϩ/ϩ) and Prnp (0/0) neurons to a similar extent (Fig. 1D).
Experiments were performed to see whether the presence of PrP C affected the accumulation of A␤ within synapses. Cortical neurons from Prnp (ϩ/ϩ) or Prnp (0/0) neurons were pulsed with 10 ng of A␤ 42 for 1 h, and synaptosomes were isolated. The amount of A␤ 42 present in synaptosomes recovered from Prnp (0/0) neurons was not significantly different from the amount of A␤ 42 in synaptosomes derived from Prnp (ϩ/ϩ) neurons (2.18 ng A␤ 42 Ϯ 0.29 compared with 2.34 ng A␤ 42 Ϯ 0.31, n ϭ 9, p ϭ 0.6) indicating that A␤ 42 can accumulate at synapses via a PrP C -independent pathway. Immunoblots showed that there were similar amounts of A␤ monomers, dimers, trimers, and tetramers in each preparation (Fig. 2).
Because PrP C is readily transferred between cells (30,33), the effects of adding PrP C to the responsiveness of Prnp (0/0) neurons to A␤ 42 was examined. PrP C bound to Prnp (0/0) neurons and was targeted to synapses; a time-dependent accumulation of PrP C within synaptosomes was observed following the addition of 20 ng PrP C (Fig. 3A). Critically, pre-treatment of Prnp (0/0) neurons with 20 ng PrP C significantly increased the A␤ 42 -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 A␤ 42 -induced synapse damage.
PrP C Mediates A␤-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). A␤ 42 inhibits the uptake of FM1-42 in cortical neurons, indicating that it suppresses synaptic vesicle recycling and hence neurotransmission (32). Here, we show that A␤ 42 had a greater inhibitory effect on the uptake of FM1-43 by Prnp (ϩ/ϩ) neurons than by Prnp (0/0) neurons (Fig. 4A). Pretreatment of Prnp (0/0) neurons with 20 ng PrP C increased A␤ 42 -induced inhibition of synaptic vesicle recycling to levels comparable with Prnp (ϩ/ϩ) neurons, whereas the addition of 20 ng of Thy-1 had no effect (Fig. 4B). Collectively, these results indicate that PrP C was involved in A␤ 42 -induced suppression of synapse function.
PrP C Mediates Activation of Synaptic cPLA 2 by A␤-Increasing evidence implicates A␤-induced activation of specific cell signaling pathways in the process that leads to synapse degeneration. For example, A␤ peptides activate cPLA 2 (36,37), and pharmacological inhibition of cPLA 2 protects against A␤-induced synapse damage (29) and ameliorates cognitive impairment in a mouse model of AD (38). For these reasons, the effects of A␤ 42 on synaptic cPLA 2 in Prnp (ϩ/ϩ) and Prnp (0/0) neurons were investigated. Although the addition of 2 nM A␤ 42 increased the amount of activated cPLA 2 found in synapses derived from Prnp (ϩ/ϩ) neurons, it had a lesser effect on cPLA 2 in synapses derived from Prnp (0/0) neurons (Fig. 5A). In contrast, the absence of PrP C did not affect the activation of synaptic cPLA 2 induced by 500 ng/ml of PLAP. Pre-treatment of Prnp (0/0) neurons with 20 ng PrP C , but not 20 ng Thy-1, increased A␤-induced activation of cPLA 2 in synapses (Fig. 5B). We conclude that A␤ activates cPLA 2 by a PrP C -dependent pathway rather than that PrP C is an essential component of cPLA 2 activation.
The activation of cPLA 2 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 (ϩ/ϩ) synaptosomes. The addition of 2 nM A␤ 42 caused cPLA 2 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 cPLA 2 occurs within cholesterol-sensitive lipid rafts (40 -42). In contrast, in Prnp (0/0) synaptosomes incubated with 2 nM A␤ 42 , cPLA 2 remained mostly within the cytoplasm, fractions 4 to 7 (Fig. 6A). The interactions between PrP C and cPLA 2 were studied further by immunoprecipitation. When Prnp (ϩ/ϩ) synaptosomes were incubated with 2 nM A␤ 42 , a mAb to cPLA 2 (CH-7) co-precipitated both A␤ and PrP C . In contrast, mAb CH-7 failed to precipitate A␤ from Prnp (0/0) synaptosomes incubated with 2 nM A␤ 42 (Fig. 6B). Notably, mAb CH-7 did not precipitate PrP C from Prnp (ϩ/ϩ) 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-precip-itate either PrP C or A␤, showing that the interactions between cPLA 2 , PrP C , and A␤ occurs within Triton X-100-insoluble, SDS-soluble lipid rafts.
A␤ Oligomers Cross-link PrP C at Synapses-The damaging effects of A␤ on synapses are caused by A␤ oligomers (11,25,43) that have the capacity to cross-link PrP C , whereas A␤ monomers that cannot cross-link PrP C are thought to be nontoxic (23,44). In addition, aggregated PrP C causes synaptic abnormalities (45), and cross-linkage of PrP C by mAbs leads to cell signaling in T cells (46) and neurodegeneration (47). Collectively, these studies suggest that it is the cross-linkage of PrP C by A␤ oligomers that triggers synapse damage. This hypothesis was tested by incubating Prnp (ϩ/ϩ) synaptosomes with 2 nM A␤ 42 and analyzing extracts using non-denaturing gel electrophoresis. We report that PrP C derived from synaptosomes incubated with A␤ oligomers migrated at higher apparent molecular weights than did PrP C derived from synaptosomes incubated with CHO-CM (Fig. 7A). These results suggest that PrP C incubated with A␤ oligomers forms a complex containing at least two PrP C molecules. To complement this study, the effect of A␤ oligomers on the filtration of soluble PrP C was examined. Soluble PrP C that had been incubated with CHO-CM passed through a 50-kDa filter, indicating that it   12). An asterisk represents a significant difference between Prnp (ϩ/ϩ) and Prnp (0/0) synaptosomes (p Ͻ 0.05). B, the amount of activated cPLA 2 in synaptosomes isolated from Prnp (0/0) neurons, which were pre-treated with 20 ng of PrP C (f) or 20 ng of Thy-1 (Ⅺ), and incubated for 1 h with control medium or 2 nM A␤ 42 . Values shown are the mean amount of activated cPLA 2 (units) Ϯ S.D. from triplicate experiments performed four times (n ϭ 12). An asterisk represents a significant difference between treated synaptosomes (p Ͻ 0.05). NOVEMBER 4, 2011 • VOLUME 286 • NUMBER 44 remained as a monomer. In contrast, incubation of soluble PrP C with 7PA2-CM containing A␤ oligomers reduced the amount of PrP C that passed through a 50-kDa filter by ϳ90%, indicating that A␤ oligomers had caused PrP C to form higher molecular weight complexes (Fig. 7B).

DISCUSSION
The identification of specific receptors for A␤ 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 PrP C (17). However, the role of each of these proteins in the biological activity of A␤ remains unclear. Although a recent study showed that synapse failure induced by synthetic A␤ 42 was mediated by PrP C (17), this observation remains controversial as others have reported synthetic A␤ 42 -induced memory defects in Prnp (ϩ/ϩ) mice (18,

19).
To overcome the problem of synthetic A␤ 42 preparations adopting multiple conformations, our experiments were performed with 7PA2-CM containing stable A␤ oligomers (22), which are similar to the A␤ oligomers found within the cerebrospinal fluid of Alzheimer patients (23)(24)(25). We show that Prnp (0/0) neurons are more resistant to synapse damage induced by A␤ oligomers than neurons that expressed PrP C . Moreover, PrP C introduced into Prnp (0/0) neurons rapidly accumulated at synapses and increased their sensitivity to A␤, thus supporting the hypothesis that PrP C plays a role in mediating A␤ oligomer-induced synapse damage.
Reports that A␤ accumulates within synapses suggests that the presence of specific A␤ receptors (13,14). The observation that similar amounts of A␤ 42 were found within synaptosomes from Prnp (ϩ/ϩ) and Prnp (0/0) neurons indicates that PrP C is not the only receptor for A␤ at the synapse. Moreover, the observation that the accumulation of A␤ 42 at synapses in Prnp (0/0) neurons did not lead to synapse damage indicates that the bind-ing of A␤ oligomers to other synaptic receptors does not trigger synapse damage and that PrP C plays a central role in the A␤-induced activation of the molecular pathways that leads to synapse damage.
Although the mechanisms by which A␤ causes synapse damage remain unclear, increasing evidence implicates A␤-induced activation of specific cell signaling pathways in synapse degeneration. Thus, A␤ peptides activate cPLA 2 (36,37), which is concentrated at the pre-synaptic membrane (29,52), and pharmacological inhibition of cPLA 2 protects against A␤-induced synapse damage (29) and ameliorates cognitive impairment in a mouse model of AD (38). Here, we show that A␤ activates synaptic cPLA 2 in Prnp (ϩ/ϩ) neurons but had a lesser effect on synapses in Prnp (0/0) neurons. Moreover, in Prnp (ϩ/ϩ) synaptosomes, the activation of cPLA 2 was associated with its migration to low density membranes; results that support previous observations that A␤-induced activation of cPLA 2 occurrs within cholesterol-sensitive lipid rafts (42). More specifically, our immunoprecipitation studies show that following the addition of A␤ to Prnp (ϩ/ϩ) synaptosomes, cPLA 2 forms a complex with A␤ and PrP C . This complex was sensitive to SDS indicating that it occurred within a lipid raft. In Prnp (0/0) neurons, the addition of A␤ oligomers failed to activate synaptic cPLA 2 which remained in the cytoplasm and did not form a complex with A␤. The addition of PrP C to Prnp (0/0) neurons increased A␤-induced activation of synaptic cPLA 2 . It is notable that the addition of PLAP activates synaptic cPLA 2 and triggers synapse damage in both Prnp (ϩ/ϩ) and Prnp (0/0) neurons, indicating that PrP C was not essential for activation of cPLA 2 . Such results are consistent with the hypothesis that PrP C acts as a receptor that mediates the A␤-induced activation of synaptic cPLA 2 and synapse damage.
It is widely accepted that A␤ oligomers cause synapse dysfunction (11,43), whereas A␤ monomers are non-toxic (23,44). In this study, we used two methods to show that A␤ oligomers cross-link PrP C in synaptosomes. First, we used non-denaturing gels to show that PrP C from synaptosomes incubated with control medium migrated as a monomer, whereas higher molecular weight forms of PrP C were observed from synaptosomes that had been incubated with A␤ oligomers. The ability of A␤ oligomers to cross-link PrP C was also demonstrated using a filtration assay. Although untreated PrP C passed through a 50-kDa filter, most of the PrP C that had been incubated with A␤ oligomers did not, indicating that it had formed higher molecular weight complexes. These observations are consistent with the hypothesis that A␤ oligomers express more than one PrP C -binding site and can cross-link PrP C .
Observations that aggregated PrP C causes synaptic abnormalities (45) and that cross-linkage of PrP C 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 PrP C that leads to synapse damage. This hypothesis was supported by our observation that cross-linkage of PrP C by mAb 4F2 activates cPLA 2 and causes synapse damage in Prnp (ϩ/ϩ) neurons. We propose a model whereby A␤ oligomers cross-link PrP C creating a lipid raft platform in which the activation of cPLA 2 leads to synapse damage (Fig. 9B). We noted that PrP C FIGURE 9. PrP C mediates the activation of cPLA 2 and synapse damage induced by A␤. Shown is a schematic showing the proposed relationship between PrP C , cPLA 2 , lipid rafts (open clouds), and A␤ oligomers (black ink blots). A, in the absence of A␤ oligomers, cPLA 2 and PrP C and are not associated, and there is no activation of cPLA 2 or synapse damage. B, in Prnp (ϩ/ϩ) synapses, A␤ oligomers cross-link PrP C resulting in a complex containing A␤, PrP C , and cPLA 2 , which leads to the activation of cPLA 2 and synapse damage. C, the addition of mAb 4F2 also cross-links PrP C resulting in the association between cPLA 2 and PrP C , activation of cPLA 2 , and synapse damage. and cPLA 2 do not interact in the absence of A␤ oligomers (Fig.  9A) and that the accumulation of A␤ at the synapses of Prnp (0/0) neurons does not lead to the activation of cPLA 2 , or an association between A␤ and cPLA 2 . Thus, an anti-PrP C mAb (4F2) mimics the effects of A␤ on synapses, it cross-links PrP C , activates cPLA 2 , and causes synapse damage (Fig. 9C).
Controversy surrounds the role of PrP C as a receptor for A␤-induced synapse failure (17)(18)(19). Our observations suggest that soluble A␤ oligomers induce the loss of synaptic proteins from cultured neurons by a PrP C -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 A␤ oligomers can cross-link PrP C , leading to aberrant activation of cPLA 2 and synapse damage, and strengthen the hypothesis that modification of A␤-PrP C interactions may provide novel therapeutics to modify the dementia associated with AD.