Background

Parkinson's disease (PD) is a neurodegenerative motor disorder affecting up to 2% of the population over the age of 65. Although it is characterised by the presence of bradykinesia, resting tremor and rigidity, up to 88% of patients also show significant psychiatric and autonomic symptoms [1]. The most common of these non-motor symptoms are Parkinson's disease dementia (PDD), with a cumulative prevalence ranging between 50 and 75% of cases [2] and dementia with Lewy Bodies (DLB), a similar condition to PDD except that dementia rather than motor symptoms are primary. DLB is the second most common cause of dementia after Alzheimer's disease (AD) and is characterised by progressive cognitive decline and parkinsonism [3]. Currently, there is no long-term cure for PD, PDD or DLB.

The major histopathological hallmark of PD, PDD and DLB is the alpha-synuclein (αSN) positive intraneuronal inclusion known as a Lewy body (LB). Although the presence of LBs in the substantia nigra is diagnostic for PD, αSN pathology is also seen in multiple extranigral regions and may account for the wide range of non-motor symptoms observed. The detailed mechanisms underlying the pathological changes in PD are not known but αSN is thought to play a central role. αSN is predominantly expressed in central nervous system neurons where it is localised to pre-synaptic terminals, regulates synaptic vesicle formation and neurotransmitter release [4, 5] and can affect synaptic plasticity during learning [6]. However, recent evidence suggests that small oligomer aggregates of αSN accumulate at the pre-synaptic membrane and trigger synapse degeneration in PD and DLB [7–9]. The transfer of αSN to neighbouring neurons [10, 11] may account for the stereotypical progression of αSN pathology through the brain similar to the staging of tau pathology in AD [12]. The loss of synapses in the hippocampus is characteristic of the PD patients that develop dementia [13] and in a rat model of α-synucleinopathy, synaptic degeneration preceded neuronal loss [14]. Thus, synapse degeneration is a common feature observed in PD, PDD and DLB.

There has been little study of the molecular mechanisms underpinning αSN induced synapse degeneration in these disorders. To investigate these mechanisms the effect of αSN on synapses in cultured cortical or hippocampal neurons was determined by quantifying the amount of synaptophysin using an enzyme-linked immunoassay (ELISA) [15]. Synaptophysin is a pre-synaptic membrane protein associated with recycling vesicles that are essential for neurotransmission [16, 17] and the amount of synaptophysin has been used to access synaptic density in the brain [18–20] and cultured neurons [15]. An understanding of the molecular mechanisms that underlie αSN-induced synapse damage may help identify drugs that reduce this process.

Results

αSN causes synapse damage

The synapse degeneration in PD and DLB that is associated with oligomers of αSN [7–9] was modelled in vitro. The addition of recombinant human αSN reduced the synaptophysin content of cortical neurons in a dose-dependent manner (Figure 1A). The synaptophysin content was reduced to 50% of control neurons (EC50) following the addition of 500 nM αSN. This effect of αSN on synapses occurred at concentrations that did not kill neurons; for example, the addition of 10 μM αSN reduced the synaptophysin content of cortical neurons by greater than 80% without affecting their viability as measured by thiazolyl blue tetrazolium (100% cell survival ± 8 compared with 96% ± 6, n = 9, P = 0.22). Similarly, immunoblots showed that αSN reduced the amount of synaptophysin in neuronal extracts without affecting the amount of β-actin (Figure 1B). The addition of βSN, another member of the synuclein family of proteins [6], did not affect the synaptophysin content of cortical neurons. We found that the addition of αSN, but not βSN, also reduced the amount of synaptophysin in hippocampal neurons (Figure 1C), an observation consistent with a report that the loss of synapses in the hippocampus is characteristic of the PD patients that develop dementia [13].

Figure 1
figure 1

αSN triggered the loss of synaptophysin from cultured neurons. (A) The synaptophysin content of cortical neurons incubated for 24 hours with αSN (●) or βSN (○) as shown. Values shown are the mean amount of synaptophysin (units) ± SD, n = 15. (B) Immunoblots showing the amount of synaptophysin and β-actin in extracts from cortical neurons that had been incubated for 24 hours with αSN as shown. (C) The synaptophysin content of hippocampal neurons incubated for 24 hours with αSN (●) or βSN (○) as shown. Values shown are the mean amount of synaptophysin (units) ± SD, n = 12.

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αSN reduced synaptic vesicle recycling

The uptake of FM1-43, a fluorescent dye that is taken up into synaptic vesicles, was used as a measure of synaptic vesicle recycling and hence neurotransmission [21]. Here we report that the uptake of FM1-43 by cortical neurons was reduced following the addition of αSN, but not after the addition of βSN (Figure 2). This effect of αSN was observed at lower concentrations than that required to reduce the synaptophysin content of neurons and the concentration of αSN required to reduce synaptic vesicle recycling by 50% was approximately 30 nM.

Figure 2
figure 2

αSN reduced synaptic vesicle recycling. The amount of the fluorescent dye (FM1-43) taken up by synaptic vesicles in cortical neurons that had been incubated for 24 hours with different concentrations of αSN (●) or βSN (○) as shown and stimulated with ACh for 10 minutes. Values shown are the mean % fluorescence (where 100% = fluorescence in control cortical neurons) ± SD, n = 15.

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βSN reduced αSN-induced synapse damage

Although transgenic mice studies showed that the expression of βSN reduced neurodegeneration in mice expressing human αSN [22, 23], the molecular mechanisms underlying the interactions between αSN and βSN are unknown. We found that pre-mixing αSN with an excess of βSN (1:10) reduced the αSN-induced loss of synaptophysin in cortical neurons, whereas pre-mixing αSN with human serum albumin (1:10) had no affect (Figure 3). As this result may have been caused by a direct effect of βSN on neurons, cortical neurons were pre-treated with 10 μM βSN, washed and then incubated with αSN. Pre-treatment of neurons with βSN did not affect the loss of synaptophysin induced by αSN (data not shown).

Figure 3
figure 3

βSN reduced the αSN-induced loss of synaptophysin. The synaptophysin content of cortical neurons incubated with varying concentrations of αSN (●), αSN premixed with βSN (1:10)(□) or αSN premixed with human serum albumin (1:10)(■) for 24 hours. Values shown are the mean amount of synaptophysin (units) ± SD, n = 12. (*) = amount of synaptophysin significantly higher than in neurons incubated with the same concentration of αSN alone (P < 0.01).

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Aβ1-42enhanced αSN-induced synapse damage

The amyloid hypothesis of Alzheimer's disease (AD) pathogenesis maintains that the primary event is the production of neurotoxic amyloid-β (Aβ) peptides following the proteolytic cleavage of the amyloid precursor protein into different sized fragments [24, 25]. These fragments include Aβ1-42 which is widely regarded as a major pathogenic species in AD [26]. Since recent reports showed that αSN and Aβ1-42 co-exist in heterologous oligomers [27, 28] the effect of Aβ1-42 on αSN-induced loss of synaptophysin was examined by pre-mixing the two peptides. The addition of Aβ1-42 in the ratio (1:50) increased αSN-induced synapse damage (Figure 4A). Thus, while the EC50 of αSN alone was 500 nM, the EC50 of Aβ1-42:αSN (1:50) was 25 nM. These concentrations of Aβ1-42 did not affect synapses when added on their own. In contrast, pre-mixing αSN with the control peptide Aβ42-1 (1:50) did not affect αSN-induced loss of synaptophysin. Since the predominant Aβ species found within the brain is Aβ1-40 [29] the effect of Aβ1-40 on αSN was also tested. However, there was no significant difference in the synaptophysin content of cortical neurons incubated with αSN and neurons incubated with Aβ1-40/αSN (1:50) (data not shown). These results may have been caused by a direct effect of Aβ1-42 on the neurons. Our observation that pre-treatment of cortical neurons with 1 nM Aβ1-42 did not affect αSN-induced loss of synaptophysin (Figure 4B) suggested that Aβ1-42 did not sensitise neurons to the effects of αSN.

Figure 4
figure 4

Aβ 1-42 enhanced the αSN-induced loss of synaptophysin. (A) The synaptophysin content of cortical neurons incubated for 24 hours with varying concentrations of αSN (●), αSN premixed with Aβ1-42 (50:1) (■) or αSN premixed with Aβ42-1 (50:1) (□). Values shown are the mean amount of synaptophysin (units) ± SD, n = 18. (*) = amount of synaptophysin significantly lower than in neurons incubated with the same concentration of αSN alone (P < 0.01). (B) The synaptophysin content of cortical neurons pre-treated for 1 hour with control medium (●) or 1 nM Aβ1-42 (○) and incubated for 24 hours with αSN as shown. Values shown are the mean amount of synaptophysin (units) ± SD, n = 12. (C) The synaptophysin content of cortical neurons incubated for 24 hours with αSN (●), αSN premixed with 7PA2-CM (■) or αSN premixed with CHO-CM (□). Values shown are the mean amount of synaptophysin (units) ± SD, n = 12. (*) = amount of synaptophysin significantly lower than in neurons incubated with the same concentration of αSN alone (P < 0.01).

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The ability of synthetic Aβ peptides to self associate results in a mixture of physical complexes ranging from small soluble oligomers to large fibrils. Since the dynamic nature of Aβ aggregation means that it is difficult to ascribe biological function to specific Aβ assemblies using synthetic peptides, the activity of naturally derived, stable Aβ oligomers was also examined. We found that pre-mixing αSN with 7PA2-conditioned medium (7PA2-CM), which contained naturally secreted stable Aβ oligomers [30, 31], increased the αSN-induced loss of synaptophysin in cortical neurons (Figure 4C). In contrast, pre-mixing αSN with CHO-CM had no effect.

Aβ enhanced αSN-induced inhibition of synaptic vesicle recycling

The addition of Aβ oligomers also affected αSN-induced inhibition of synaptic vesicle recycling. Thus, pre-mixing αSN with 7PA2-CM enhanced αSN-induced inhibition of FM1-43 uptake into synapses. The concentration of αSN alone required to reduce synaptic vesicle recycling by 50% was 30 nM, while the concentration of αSN that had been mixed with 7PA2-CM to have a similar level of effect was 1 nM (Figure 5).

Figure 5
figure 5

Aβ 1-42 enhanced the αSN-induced inhibition of synaptic vesicle recycling. The amount of the fluorescent dye (FM1-43) taken up into synaptic vesicles in cortical neurons that had been incubated for 24 hours with different concentrations of αSN (●), or with αSN that had been premixed with either 7PA2-CM (■) or with CHO-CM (□) as shown and stimulated with ACh for 10 minutes. Values shown are the mean % fluorescence (where 100% = fluorescence in vehicle treated cortical neurons) ± SD, n = 18. (*) = Fluorescence significantly lower than in neurons incubated with the same concentration of αSN alone (P < 0.01).

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αSN enhanced Aβ1-42-induced synapse damage

Next we examined whether non-toxic concentrations of αSN affected Aβ1-42-induced loss of synaptophysin. Here we show that pre-mixing Aβ1-42 with αSN increased the Aβ1-42-induced loss of synaptophysin from neurons (Figure 6). Thus, while the EC50 of Aβ1-42 alone was 50 nM, the EC50 of αSN:Aβ1-42 (2:1) was 5 nM. In contrast, the addition of βSN (2:1) did not affect Aβ1-42-induced loss of synaptophysin. Pre-treatment of cortical neurons with 10 nM αSN did not affect Aβ1-42-induced loss of synaptophysin (data not shown). We also sought to determine if αSN affected another peptide that caused synapse damage. Synapse degeneration is a feature of human and experimental prion diseases [32, 33] which was modelled by the addition of the prion-derived peptide PrP82-146 to cortical neurons [15]. As shown in Figure 6B, there was no difference in the synaptophysin content of cortical neurons incubated with PrP82-146 and those incubated with a combination of αSN/PrP82-146 (2:1).

Figure 6
figure 6

αSN enhanced the Aβ 1-42 -induced loss of synaptophysin. (A) The synaptophysin content of cortical neurons incubated for 24 hours with Aβ1-42 (●) Aβ1-42 premixed with αSN (1:2) (■) or Aβ1-42 premixed with βSN (1:2) (□). Values shown are the mean amount of synaptophysin (units) ± SD, n = 15. (*) = amount of synaptophysin significantly lower than in neurons incubated with the same concentration of Aβ1-42 alone (P < 0.01). (B) The synaptophysin content of cortical neurons incubated for 24 hours with PrP82-146 (●) or PrP82-146 that had been premixed with αSN (1:5) (○). Values shown are the mean amount of synaptophysin (units) ± SD, n = 15.

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αSN did not affect the accumulation of Aβ1-42in synapses

We explored the possibility that αSN increased the binding of Aβ1-42 to synapses as an explanation of the effect of αSN on Aβ1-42-induced loss of synaptophysin. Time course studies showed that Aβ1-42 accumulated in synaptosomes isolated from cortical neurons after 1 hour. Therefore cortical neurons were incubated with 100 nM biotinylated Aβ1-42, or 100 nM biotinylated-Aβ1-42 that had been pre-mixed with 500 nM αSN for 1 hour. The amount of biotinylated Aβ1-42 found in synaptosomes isolated from these neurons was not altered by pre-mixing with αSN (Figure 7A), indicating that αSN did not alter the binding, or trafficking of Aβ1-42 to synapses. The effect of Aβ1-42 on the accumulation of αSN in synapses was also examined. Cortical neurons were incubated with 200 nM αSN, or a combination of 4 nM Aβ1-42 and 200 nM αSN (1:50), for 1 hour and synaptosomes prepared. Immunoblots showed that the amount of αSN found within synapses was not affected by presence of Aβ1-42 (Figure 7B).

Figure 7
figure 7

αSN did not affect the accumulation of Aβ 1-42 in synapses. (A) The amount of biotinylated-Aβ1-42 found in synaptosomes derived from cortical neurons incubated for 1 hour with 100 nM Aβ1-42 (□) or 100 nM Aβ1-42 that had been pre-mixed with 500 nM αSN (■). Values shown are the mean amount of Aβ1-42 expressed as a % of the amount added ± SD, n = 12. (B) Immunoblot showing the amount of αSN in synaptosomes collected from cortical neurons incubated with 200 nM αSN alone or with 200 nM αSN pre-mixed with Aβ1-42 (50:1).

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Discussion

The loss of synapses is a prominent feature of many neurodegenerative diseases including AD, PDD and LBD. The main mediators of neuropathology in PDD and LBD are thought to be oligomers of αSN [7, 9] and in this study the addition of αSN impaired synapse function and triggered a loss of synaptophysin from cortical neurons. These effects occurred at concentrations of αSN that did not affect neuronal survival; an observation consistent with reports that synapse degeneration preceded neuronal loss in a rat model of α-synucleinopathy [14]. A reduction in the synaptophysin content of hippocampal neurons was observed after incubation with αSN, consistent with reports that a loss of synapses in the hippocampus is characteristic of the PD patients that develop dementia [13].

The addition of βSN did not affect synapses indicating that synaptic defects were dependent upon the specific amino acid sequence of αSN. Recent reports from a transgenic mouse model of PD showed that the expression of βSN reduced the accumulation of αSN and neurodegeneration in mice expressing human αSN [22, 23]. Another study showed that βSN formed mixed oligomers with αSN [34]. In this study we showed that mixing with βSN reduced the loss of synaptophysin induced by αSN; results consistent with the idea that molecular interactions between αSN and βSN affect the toxicity of αSN.

Approximately 25% of AD patients develop parkinsonism and 50% of PD patients develop AD-type dementia after 65 years of age [35]. In addition, 70% of patients with sporadic AD display αSN-positive, LB-like inclusions in the amygdala and limbic structures [36–38]. The loss of synapses that occurs in AD is associated with the production of Aβ oligomers [39–41]. Both Aβ and αSN accumulate in the brain in DLB [3] and levels of αSN are increased in AD [42], observations which suggest that interactions between αSN and Aβ affect the pathogenesis of AD, PDD and DLB [43].

The addition of small amounts of Aβ1-42, which had no effect on synapses on their own, increased the effects of αSN on synapses. Critically pre-treatment with Aβ1-42 did not sensitize cortical neurons to the synaptic effects of αSN and while we cannot exclude the possibility of a transient sensitizing effect of Aβ1-42, our results suggest that direct interactions between Aβ1-42 and αSN increased the toxicity of αSN. The studies using synthetic Aβ1-42 peptides were complimented by studies using 7PA2-CM containing naturally secreted, stable Aβ oligomers [30, 31]. Pre-mixing with 7PA2-CM also increased the effects of αSN upon synaptic vesicle recycling and synapse damage. Relatively small amounts of Aβ1-42 (1:50) were required to facilitate the αSN-induced loss of synaptophysin suggesting that Aβ1-42 seeded the formation of toxic αSN oligomers; an observation consistent with reports that Aβ promotes the aggregation of αSN in transgenic mice [44]. Conversely, the addition of non-toxic concentrations of αSN increased Aβ1-42-induced loss of synaptophysin.

Aβ1-42 exists in multiple forms from small soluble toxic oligomers to large insoluble amyloid fibrils. As the toxicity of Aβ1-42 is affected by its state of aggregation [45, 46] the addition of αSN may stabilize Aβ1-42 oligomers in a toxic configuration. Mixing αSN with Aβ1-42 did not increase the amount of αSN or Aβ1-42 found within synapses showing that the increased toxicity of hetero-oligomers was not due to increased binding of Aβ1-42 or αSN to synapses.

Although Aβ1-42 is considered to be the major neurotoxin generated in AD [47–50] other Aβ fragments are produced [24, 25, 51] and since Aβ1-40 is the predominant Aβ species formed in AD [29, 52] the effect of Aβ1-40 on αSN-induced loss of synaptophysin was also tested. We found that αSN did not affect the reduction in synaptophysin in response to Aβ1-40, nor did Aβ1-40 affect the reduction in synaptophysin induced by αSN. Synapse degeneration is also a feature of human and experimental prion diseases [32, 33] and the prion-derived peptide PrP82-146 triggered a reduction in synaptophysin in cortical neurons [15]. Although PrP82-146 has similar biophysical properties to Aβ1-42 in that it adopts a β-helix-rich conformation, forms oligomers and fibrils which are protease resistant [53], αSN did not affect PrP82-146-induced loss of synaptophysin.

Conclusions

We conclude that the addition of αSN reduced the synaptophysin content of cultured cortical and hippocampal neurons, a model that mimics the synapse damage observed in PDD and DLB. The effect of αSN was modified by other proteins found in the central nervous system including βSN which reduced the effects of αSN, and Aβ1-42 which increased the effects of αSN on synapses. Conversely, αSN increased the effects of Aβ1-42 on synapses. Our results suggest that interactions between the synucleins and Aβ peptides may affect synapses in AD, PDD and Lewy body disorders.

Methods

Primary neuronal cultures

Cortical neurons were prepared from the brains of mouse embryos (day 15.5) as described [15]. Neurons were plated at 2 × 105 cells/well in pre-coated 48 well plates (5 μg/ml poly-L-lysine) in Ham's F12 (PAA) containing 5% foetal calf serum (FCS) for 2 hours. Cultures were shaken (600 r.p.m for 5 minutes) and non-adherent cells removed by 3 washes in PBS. Neurons were grown in neurobasal medium (NBM) containing B27 components (PAA) for 10 days. Immunohistochemistry showed that the cells were greater than 97% neurofilament positive. Fewer than 3% of cells stained for glial fibrillary acidic protein (astrocytes) or for F4/80 (microglial cells). Hippocampal neurons were prepared from the brains of adult mice as described [54]. Hippocampi were dissected from brains and triturated in Ham's F12 containing 5% FCS, 0.35% glucose, 0.025% trypsin, and 0.1% type IV collagenase. After 30 minutes at 37°C, the cells were triturated and the cell suspension was passed through a 100 μM cell strainer. Cells were collected, washed twice and plated at 2 × 105 cells/well in 48 well plates (pre-coated with poly-L-lysine). After 24 hours cultures were shaken (600 r.p.m for 5 minutes) to remove non-adherent cells, washed twice and the remaining neurons were cultured in NBM/B27 and 10 ng/ml glial-derived neurotrophic factor (Sigma) for 7 days. Neurons were incubated with peptides for 24 hours and the amount of synaptophysin in cell extracts measured.

Cell extracts

Neurons were washed 3 times with PBS and homogenised in a buffer containing 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 0.2% sodium dodecyl sulphate (SDS) and mixed protease inhibitors (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), Aprotinin, Leupeptin, Bestain, Pepstatin A and E-46) (Sigma) at 106 cells/ml. For immunoblots cells were homogenised in extraction buffer (as above) at 107 cells/ml and digested with DNAse (Sigma) for 1 hour at 37°C. Cell debris was removed by low speed centrifugation (300 × g for 5 minutes).

Synaptophysin ELISA

The amount of synaptophysin in neuronal extracts was measured by ELISA [15]. Briefly, the capture mAb was anti-synaptophysin MAB368 (Millipore). Samples were added for 1 hour 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. Absorbance was measured on a microplate reader at 405 nm and the synaptophysin content of samples was expressed as units where 100 units was defined as the amount of synaptophysin in untreated neurons.

Synaptic vesicle recycling

The fluorescent styryl dye FM1-43 (Biotium) that is readily taken up into synaptic recycling vesicles [55] was used to determine synaptic activity as described [21]. Treated neurons were incubated with 1 μg/ml FM1-43 and 1 μM acetylcholine (ACh) for 10 minutes, washed 5 times in ice cold PBS and solubilised in methanol at 1 × 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 defined as the amount of fluorescence in untreated neurons incubated with FM1-43 and ACh.

Synaptosome preparations

Synaptosomes were prepared on a discontinuous Percoll gradient [56]. Briefly, 106 cortical neurons were homogenized at 4°C in 1 ml of SED solution (0.32 M sucrose, 50 mM Tris-HCl, pH 7.2, 1 mM EDTA, and 1 mM dithiothreitol, and centrifuged at 1000 × g for 10 minutes. 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 minutes at 4°C. The synaptosomes were collected from the interface of the 15% and 23% Percoll steps. The fraction was washed twice (16,000 × g for 30 minutes at 4°C) and suspended in extraction buffer containing 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.2% sodium dodecyl sulphate and mixed protease inhibitors.

Biotinylated Aβ1-42ELISA

The amounts of biotinylated Aβ1-42 in extracts were determined by ELISA. Nunc Maxisorb Immunoplates were coated with 1 μg/ml protein A (Innova) followed by 0.1 μg/ml mAb reactive to amino acids 1 to 16 of β-amyloid (clone 6E10 - Signet) and blocked with 5% milk powder. Samples were boiled in 0.2% SDS, cooled and incubated for 1 hour. Biotinylated Aβ1-42 was detected with extravidin-alkaline phosphatase and 1 mg/ml 4-nitrophenyl phosphate (Sigma). Absorbance was measured on a microplate reader at 405 nm and results were calculated by reference to a standard curve generated form serial dilutions of biotinylated Aβ1-42.

Western analysis

Samples were mixed with an equal volume of Laemmli buffer, boiled, and subjected to electrophoresis on a 15% polyacrylamide gel (Invitrogen). Proteins were transferred onto a Hybond-P PVDF membrane (Amersham Biotech) by semi-dry blotting. Membranes were blocked using 10% milk powder; synaptophysin was detected using a mouse monoclonal antibody (mAb) anti-synaptophysin SY38 (Abcam), β-actin was detected by incubation with a mouse mAb (clone AC-74, Sigma) and human αSN was detected by incubation with mAb 211 raised against amino acids 121 to 125 of human αSN (Santa Cruz Biotech). These were visualised using a combination of biotinylated secondary antibodies (Dako), extravidin-peroxidase and an enhanced chemiluminescence kit.

Peptides

Recombinant human αSN and βSN were purchased from Sigma. Synthetic peptides containing the amino acids 1 to 42 (Aβ1-42) or 1 to 40 (Aβ1-40) of the Aβ protein, biotinylated-Aβ1-42 and a control peptide consisting of amino acids 1 to 42 in reverse order (Aβ42-1) were obtained from Bachem. Peptides containing amino acids 82 to 146 of the human PrP protein (PrP82-146) and was a gift from Professor Salmona (Mario Negri, Milan). Aβ peptides were first dissolved in hexafluoroisopropanol, lyophilised and subsequently solubilised and stored at 1 mM in DMSO. Stock solutions of peptides were stored at 1 mM, thawed on the day of use and diluted/mixed in NBM for 1 hour at 37°C. Dilutions/mixtures were subjected to vigorous shaking (Disruptor Genie, full power for 10 minutes) before they were added to neurons. Chinese hamster ovary (CHO) cells stably transfected with a cDNA encoding APP751 containing the Val717Phe familial AD mutation (referred to as 7PA2 cells) were cultured in DMEM with 10% FCS [30, 31]. Conditioned medium from these cells contains stable Aβ oligomers (7PA2-CM). Conditioned medium from non-transfected CHO cells (CHO-CM) was used as controls. These were mixed with peptides and subjected to vigorous shaking (as above), diluted in NBM and added to neurons.

Statistical Analysis

Differences between treatment groups were determined by 2 sample, paired T-tests. For all statistical tests significance was set at the 1% level.