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. Author manuscript; available in PMC: 2015 Jul 6.
Published in final edited form as: FEBS J. 2010 Dec 1;278(1):167–178. doi: 10.1111/j.1742-4658.2010.07942.x

Anti-amyloid precursor protein antibodies inhibit amyloid-β production by steric hindrance

Rhian S Thomas 1, J Eryl Liddell 2, Emma J Kidd 1
PMCID: PMC4492582  EMSID: EMS63789  PMID: 21122073

Summary

Cleavage of amyloid precursor protein (APP) by β- and γ-secretases results in the production of amyloid-β (Aβ) in Alzheimer’s disease (AD). We raised two monoclonal antibodies, 2B3 and 2B12, that recognise the β-secretase cleavage site on APP but not Aβ. We hypothesised that these antibodies would reduce Aβ levels via steric hindrance of β-secretase. Both antibodies decreased extracellular Aβ levels from astrocytoma cells, but 2B3 was more potent than 2B12. Levels of soluble sAPPα from the non-amyloidogenic α-secretase pathway and intracellular APP were not affected by either antibody nor were there any effects on cell viability. 2B3 exhibited a higher affinity for APP than 2B12 and its epitope appeared to span the cleavage site while 2B12 bound slightly upstream. Both of these factors probably contribute to its greater effect on Aβ levels. After 60 minutes incubation at pH 4.0, most 2B3 and 2B12 remained bound to their antigen, suggesting that the antibodies will remain bound to APP in the acidic endosomes where β-secretase cleavage probably occurs. Only 2B3 and 2B12, but not control antibodies, inhibited the cleavage of sAPPα by β-secretase in a cell-free assay where effects of antibody internalisation and intracellular degradation were excluded. 2B3 virtually abolished this cleavage. In addition, levels of C-terminal APP fragments, βCTF, generated following β-secretase cleavage, were significantly reduced in cells after incubation with 2B3. These results strongly suggest that anti-cleavage site antibodies can generically reduce Aβ levels via inhibition of β-secretase by steric hindrance and may provide a novel alternative therapy for AD.

Keywords: Alzheimer’s disease, monoclonal antibodies, amyloid-β, β-secretase cleavage site, amyloid precursor protein

INTRODUCTION

Alzheimer’s disease (AD) is characterised pathologically by an over-accumulation in the brain of intracellular neurofibrillary tangles, amyloid-β (Aβ)-containing extracellular senile plaques and neuronal loss [1]. The ‘amyloid hypothesis’ suggests that Aβ accumulation in the brain is an initiating event in AD [2] although it does not explain all aspects of AD pathology [3]. Despite this, it is still the dominant theory used to explain the disease and many therapeutic strategies have therefore concentrated on attempting to modify Aβ accumulation in the brain [4].

Aβ is a 38-43-mer peptide that is cleaved from amyloid precursor protein (APP) [5]. APP can be processed by one of two proteolytic pathways. The non-amyloidogenic route involves cleavage of APP by α-secretase [6] within the Aβ region to release sAPPα [7]. In the amyloidogenic pathway, β-secretase, identified as BACE1 (Beta-site APP Cleaving Enzyme) [8-10], first cleaves APP to liberate sAPPβ and C99 [11]. The γ-secretase complex [12-15] then cleaves C99 to produce Aβ and a C-terminal intracellular fragment [11]. Although there is still some debate regarding the co-localisation of the enzymes and substrates involved, it is generally considered that, after synthesis, a proportion of APP is transported to the cell membrane and is then internalised for processing in the endosomal-lysosomal system where it may be further processed to Aβ [16, 17].

Current therapy in the U.K. is limited to symptomatic treatment with acetyl-cholinesterase inhibitors for moderate AD only [http://guidance.nice.org.uk/TA111, 01/07/10]. Recently there has been much attention given to the development of novel immunotherapeutic approaches in AD. Aβ vaccination, both passive and active, has been used successfully in transgenic mice to reduce Aβ plaque deposition [18] and improve cognition [19-23]. This led to a phase IIA clinical trial involving vaccination with aggregated Aβ42, AN1792, but this was halted when several patients developed meningoencephalitis [24]. Alternative immunotherapeutic approaches are therefore required.

In this paper we present data relating to two monoclonal antibodies, 2B12 and 2B3. Unlike the previous approaches, our antibodies do not bind to Aβ but bind to APP in the vicinity of the β-secretase cleavage site. We previously demonstrated that 2B12 reduced levels of extracellular Aβ40 from cell lines endogenously expressing native APP in a time- and concentration-dependent manner. The mode of action was, however, unclear [25]. Here we present data characterising 2B3 and comparing the two antibodies. We demonstrate that 2B3 also reduces Aβ levels from a native cell line and is more potent than 2B12. This effect on Aβ levels was specific to the cleavage site antibodies. We suggest that these antibodies bind to the extracellular region of APP when it is transported to the cell membrane and become internalised with the protein into the endosomal / lysosomal system where they inhibit BACE1 cleavage via steric hindrance. This will drastically reduce levels of Aβ produced, representing an alternative therapeutic strategy to treat AD.

RESULTS

2B3 and 2B12 have different epitopes on APP

Both 2B3, an IgG1 isotype, and 2B12, an IgG2b isotype, detected full length APP from MOG-G-UVW cell lysates in a Western blot (Fig. 1). Both antibodies detected bands of 103kDa (±2.5kDa) and 56kDa (±0.6kDa), however, 2B3 recognised the 56 kDa fragment, possibly a thrombin cleavage fragment [26], more strongly than it did the 103 kDa fragment of APP, while 2B12 recognised the 103 kDa fragment more strongly than it did the 56 kDa fragment. Neither antibody detected Aβ40 in an ELISA or Western blot [25]; data not shown. The antibodies differentially recognised sAPPα and sAPPβ in an ELISA. There was no significant difference in the recognition by 2B12 of sAPPα or sAPPβ. Similarly, there were no significant differences in the amount of 2B3 bound to sAPPα when compared to the amount of 2B12 bound to either sAPPα or sAPPβ. However, significantly less 2B3 bound to sAPPβ than sAPPα (p<0.01). The amount of 2B3 bound to sAPPβ was also significantly less than the binding of 2B12 to either sAPPα (p<0.001) or sAPPβ (p<0.001).

Fig. 1.

Fig. 1

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(A) Representative Western blot of MOG-G-UVW lysate (50μg) detected with either 2B3 or 2B12 at 2μg/ml on a 10% gel. Both antibodies detected full length APP at 103KDa (±2.5 KDa) and a smaller protein at 56KDa (±0.6 KDa), which was most likely a thrombin cleavage fragment of APP. 2B3 preferentially recognised the 56KDa fragment, n=4-5. (B) Differential 2B3 and 2B12 recognition of sAPPα and sAPPβ as determined by indirect ELISA. Data are expressed as a mean (± SEM) % of the standard antibody (6E10) at 0.1μg/ml. 2B12 recognised sAPPα and sAPPβ equally well, however, significantly more 2B3 bound to sAPPα than to sAPPβ. The amount of 2B3 bound to sAPPβ was also significantly less than the amount of 2B12 bound to either sAPPa or sAPPβ. **p<0.01, ***p<0.001 after one way ANOVA and Bonferroni post-hoc tests, n=4.

2B3 recognises full length APP and a peptide spanning the cleavage site on APP more strongly than 2B12

To determine the relative affinities of 2B3 and 2B12 for APP we first compared their binding to a peptide, Kb, which contains the β-secretase cleavage site on APP (Fig. 2). The antibodies differentially bound to the peptide as evidenced by their differential Hill slopes, 0.90 for 2B3 and 0.63 for 2B12 (p<0.001). The antibody concentration at which half-maximal binding was reached was significantly lower for 2B3, 1.279μg/ml (95% CI 1.153 to 1.418), than for 2B12, 2.963μg/ml (95% CI 1.696 to 5.177) (p<0.001) and the maxima reached for 2B3, 103.7% (95% CI 100.7 to 106.7) was significantly greater than that for 2B12, 73.44% (95% CI 62.34 to 84.54) (p<0.001). Furthermore, significantly more 2B3 than 2B12 was detected bound to Kb at all antibody concentrations greater than 0.1μg/ml (p<0.05).

Fig. 2.

Fig. 2

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Half-maximal binding of 2B3 and 2B12 to a peptide, Kb, spanning the β-secretase cleavage site, as determined by ELISA. Data are expressed as a mean (± SEM) % of the standard antibody (6E10) at 0.05μg/ml. The concentration at which half-maximal binding was reached was significantly lower for 2B3 than for 2B12 (p<0.001). *significant differences between 2B3 and 2B12 with two-tailed Student’s t-tests, p<0.05, n=3-4.

We next compared their binding efficiency to full length APP in a competition ELISA using MOG-G-UVW cell lysate as a source of APP. Both 2B3 and 2B12 interfered with the binding of a commercial detection antibody for APP in a concentration-dependent manner (Fig. 3). At 5, 10 and 20μg/ml 2B12 significantly reduced the binding of the commercial antibody to 90.5% (p<0.01), 89.13% (p<0.001) and 82.9% (p<0.01) of control levels, respectively. This was similar to previously reported levels [25]. At 5, 10 and 20μg/ml, 2B3 also significantly reduced the binding of the second commercial antibody to 87.4% (p<0.01), 82.33% (p<0.001) and 72.94% (p<0.001) of control levels. Even at the highest concentration, 20μg/ml, a control IgG did not significantly reduce the binding of the second detection antibody when compared to control levels. The three antibodies differentially inhibited the binding of the APP detection antibody at 1 μg/ml (p<0.05), 5 μg/ml (p<0.001), 10 μg/ml (p<0.001) and 20 μg/ml (p<0.001). 2B3 and 2B12 produced a significantly greater inhibition in the binding of the APP detection antibody than did the control IgG at all concentrations tested (p<0.05). At 10 μg/ml (p<0.01) and 20 μg/ml (p<0.001) 2B3 also significantly inhibited binding of the detection antibody more than 2B12.

Fig. 3.

Fig. 3

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Binding of 2B3, 2B12 or a control IgG to APP as determined by a competition sandwich ELISA for APP. Data are expressed as mean (± SEM) % of media control. One-way ANOVA indicated that the antibodies differentially inhibited the binding of a commercial anti-APP IgG at each antibody concentration. Antibody data points followed by different letters (either a, b, or c) differ significantly from each other in their ability to inhibit the commercial antibody at that particular antibody concentration after one way ANOVA and Bonferroni, (p<0.05). 2B3 and 2B12 also significantly reduced the binding of the commercial antibody in comparison to media controls at 100%. **p<0.01, ***p<0.001, significantly different from media controls using two-tailed Student’s t-tests. The control IgG did not significantly interfere with the binding of the detection antibody, n=4.

The majority of both 2B3 and 2B12 remained bound to their antigen, Kb, after incubation at different pH values

To determine the effects of pH on the antibodies, 2B3 and 2B12 were bound to the Kb peptide at pH 7.4 and then incubated for 0 or 60 min at pH 4.0 or 7.4. There were significant differences between the persistence in binding of the antibodies to Kb under the various conditions tested (p<0.001) (Fig. 4). There was also a significant interaction between the antibody type (2B3 or 2B12) and pH (p<0.05). The complex formed between 2B12 and Kb was not significantly affected by pH or incubation time. The complex formed between 2B3 and Kb was also not significantly affected by the incubation period but there was a significant reduction in 2B3 binding at pH 4.0 after 60 min (p<0.05). Incubation of Kb with PBST alone at pH 4.0 for 1 hour, prior to incubation with antibody at pH 7.4, did not affect the binding of either 2B3 or 2B12 (data not shown) indicating that the above results are not due to degradation of the antigen. Importantly, even at pH 4.0, there was still significantly more 2B3 bound to Kb than 2B12.

Fig. 4.

Fig. 4

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Effect of pH and incubation time on persistence of 2B3 and 2B12 binding to Kb as determined by ELISA. Data are expressed as mean (± SEM) % absorbance of the standard antibody, 6E10 (0.05μg/ml). Antibodies were first allowed to form a complex with Kb at pH 7.4 and the complex was then incubated with buffers of pH 4.0 or 7.4 for 0 and 60 minutes. The complex formed between Kb and 2B3 was not affected by incubation time but was significantly affected by the pH of the buffer. The persistence in binding of 2B12 to Kb was not significantly affected by either incubation time or pH. At all values tested, significantly more 2B3 remained bound to the Kb peptide than 2B12. *p<0.05, ***<0.001, 2B3 significantly different to 2B12. Data were analysed for statistical significance with GLM univariate analysis and Bonferroni post-hoc tests, n=3-4.

2B3 is more effective at reducing extracellular Aβ40 and Aβ42 levels in cell culture media than 2B12

The antibodies tested differentially inhibited levels of extracellular Aβ40 (p<0.001) (Fig. 5a). 2B12 significantly reduced levels of Aβ40 in MOG-G-UVW cell media to 65.3% of media control levels (p<0.05). This was similar to levels previously reported [25]. 2B3 significantly reduced levels of extracellular Aβ40 to 36.8% of media control levels (p<0.001). Neither the control IgG nor the anti-N-terminal APP antibody had any significant effect on Aβ40 levels. Both 2B3 and 2B12 significantly reduced Aβ40 more than the anti-N-terminal APP (p<0.001, p<0.05, respectively) and 2B3 significantly reduced Aβ40 levels more than 2B12 (p<0.05).

Fig. 5.

Fig. 5

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Levels of extracellular Aβ40 (A) and Aβ42 (B) from MOG-G-UVW culture media after incubation with 2B3, 2B12, an irrelevant mouse IgG (Aβ40 only) or an anti-N-terminal APP antibody (N), all at 10μg/ml for 48 hours. Data are expressed as mean (± SEM) % of media control Aβ levels as detected in a sandwich ELISA and corrected for total cell protein concentration. Both 2B3 and 2B12 significantly reduced both forms of Aβ from media controls. Neither of the control antibodies had any significant effect on Aβ levels. *p<0.05, **p<0.01, ***p<0.001, significantly different from media controls (100%) with two-tailed Student’s t-tests. ap<0.05, significantly different from all other groups, bp<0.05 significantly different from 2B3 and N, cp<0.05 significantly different from N after ANOVA and Tukey’s HSD, n= 3-6 (A) and n=4 (B).

The antibodies also differentially inhibited levels of Aβ42 (p<0.05). 2B12 significantly reduced Aβ42 levels to 54.8% of the media control (p<0.01). 2B3 was again more effective and reduced these levels to 21.9% of media control levels (p<0.01) (Fig. 5b). Aβ42 levels remained at 100.1% of control levels after incubation with the anti-N-terminal APP IgG. Again 2B3 significantly reduced Aβ42 levels more than the anti-N-terminal antibody (p<0.05).

Anti-β-secretase cleavage site antibodies do not alter levels of APP, sAPPα, or affect cell viability but reduce βCTF levels

Neither 2B12, 2B3 nor the irrelevant IgG had any effect on levels of intracellular APP as measured in an ELISA (Fig. 6a). Furthermore they did not have any significant effect on the number of viable cells, as measured using an MTS assay (Fig. 6b). The anti-N-terminal APP antibody appeared to either increase the number of cells or alter the levels of metabolic activity above control levels (p<0.01). Neither 2B12 nor the control IgG had any significant effect on the levels of sAPPα (Fig. 7a). However, incubation with 2B3 significantly reduced βCTF levels to 64.6% (p<0.05) of control values in MOG-G-UVW cells (Fig. 7b).

Fig. 6.

Fig. 6

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Levels of intracellular APP as determined by ELISA (A) and cell viability as determined by MTS assay (B) in MOG-G-UVW cells after incubation with 2B3, 2B12, an anti-N-terminal APP antibody (N, MTS only), or a control irrelevant mouse IgG, all at 10μg/ml for 48 hours. Data are expressed as mean (± SEM) % of control (media only). APP levels were corrected for total cell protein concentration. None of the antibodies tested had any significant effect on levels of APP or cell viability, apart from the anti-N-terminal APP antibody which appeared to stimulate growth. **p<0.01, significantly different from media control using two-tailed Student’s t-tests, n=3.

Fig. 7.

Fig. 7

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Levels of extracellular sAPPα (A) and intracellular βCTF (B) as determined by ELISA from MOG-G-UVW cells after incubation with 2B12, 2B3 or a control irrelevant mouse IgG, all at 10μg/ml for 48 hours. Data are expressed as mean (± SEM) % of control (media only). All levels were corrected for total cell protein concentration. Neither 2B12 nor the IgG antibody significantly affected levels of sAPPα, however, 2B3 significantly reduced levels of βCTF from control levels. *p<0.05, significantly different from media controls using two-tailed Student’s t-tests, n=3.

Anti-β-secretase cleavage site antibodies reduce BACE1 cleavage via steric hindrance in a cell-free assay

Neither 2B3, 2B12, the control IgG nor the anti-N-terminal APP IgG had any detectable effects on total levels of BACE1 in MOG-G-UVW cells, as investigated by Western Blotting (data not shown). However, differences were observed in BACE1 activity after in vitro incubation of sAPPα with BACE1 and the antibodies. The antibodies tested differentially inhibited the cleavage of sAPPα by BACE1, as detected by Western Blotting (p<0.001) (Figs. 8a and 8b). There was a decrease in sAPPα levels from control levels (sAPPα alone) after incubation with BACE1 (media control) of −41.91 OD units. Neither the control IgG nor the anti-N-terminal APP IgG had any significant effect on the activity of BACE1 and decreases in sAPPα on addition of BACE1 were similar to those observed in the presence of control media alone (−36.9 and −44.4 OD units, respectively). The addition of both 2B3 and 2B12 significantly inhibited the action of BACE1 from control conditions. 2B12 significantly reduced this decrease in sAPPα levels to only −17.29 OD units (p<0.05). 2B3 virtually abolished the reduction in sAPPα caused by BACE1 and the decrease in sAPPα levels was only −0.05 OD units (p<0.01). Levels of sAPPα were very similar to the levels observed when sAPPα was incubated with 2B3 alone.

Fig. 8.

Fig. 8

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(A) Representative Western blot of sAPPα incubated with or without BACE1 and 2B12, 2B3, control IgG, anti-N terminal APP antibody (N) (all at 1 μg/ml) or media control. 1) sAPPα alone, 2) sAPPα and BACE1 (media control), 3) sAPPα and 2B12, 4) sAPPα, BACE1 and 2B12, 5) sAPPα and 2B3, 6) sAPPα, BACE1 and 2B3, 7) sAPPα and IgG, 8) sAPPα, BACE1 and IgG, 9) sAPPα and N, 10) sAPPα, BACE1 and N. Only 2B12 and 2B3 inhibited the action of BACE1 in this system. (B) Quantification of Western blots showing the mean (± SEM) change in sAPPα from relevant control levels (i.e. sAPPα and relevant antibody alone) after sAPPα incubation with BACE1 and either 2B3, 2B12, control IgG, anti-N terminal APP antibody (N) or media alone. *p<0.05, significantly different from media control and N, **p<0.01 significantly different from media control, IgG and N, after one way ANOVA and Bonferroni post-hoc tests, n=3-6.

DISCUSSION

Both 2B3 and 2B12 were raised to the same immunogen yet they clearly have different epitopes, as evidenced by their binding profile to APP in Western blotting and by their differential binding to sAPPα and sAPPβ. 2B3 binds significantly less to sAPPβ than it does to sAPPα (approximately 25% less) whereas 2B12 recognises sAPPβ and sAPPα equally well. The evidence suggests that 2B12 binds upstream of the β-secretase cleavage site (towards the N-terminal of APP) since it recognises both sAPPα and sAPPβ. Its epitope, therefore, is in a region common to both peptides. While 2B3 also recognises both of these APP fragments, more 2B3 bound to sAPPα than to sAPPβ. However, 2B3 does not recognise the Aβ peptide. This suggests that 2B3 binds across the β-secretase cleavage site, and only slightly into the Aβ region (Fig. 9).

Fig. 9.

Fig. 9

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Hypothesised epitopes of 2B3 and 2B12 on APP. 2B3 binds across the β-secretase cleavage site while 2B12 binds upstream of this. Neither antibody recognises Aβ when cleaved from APP.

Results from a BLAST (http://www.ncbi.nlm.nih.gov) search on the Ka peptide used to raise both antibodies indicated that they should be specific to APP as this sequence of amino acids is highly conserved in APP and not found in other mammalian sequences (data not shown). In addition, we have previously demonstrated that there was no cross-reaction between 2B12 and a range of peptides tested by ELISA or Western blotting [25].

2B3 binds more effectively to APP than 2B12 as demonstrated by a competition assay, in which 2B3 interfered with the binding of a second detection antibody for APP more efficiently than 2B12. Although these results could be due to the differential epitopes or isotypes of 2B3 and 2B12, meaning that 2B3 might interfere more effectively with the binding of the APP detection antibody, they do suggest that 2B3 has a higher relative affinity for APP than 2B12. This is supported by the results of the affinity rankings of the two antibodies for the Kb peptide, a 15-mer fragment spanning the β-secretase cleavage site on APP. Significantly more 2B3 than 2B12 bound to Kb at all concentrations greater than 0.1μg/ml, and the concentration for half-maximal binding was significantly lower for 2B3. All these data suggest that 2B3 has a higher relative affinity for APP than 2B12.

We hypothesise that 2B3 and 2B12 will bind to the APP ectodomain after APP has trafficked to the cell membrane and will be endocytosed into the cell with the protein. Indeed Tampellini et al. [27] demonstrated that antibodies to the N- and mid region of Aβ bound first to the ectodomain of APP and were then internalised. This immunocomplex would be formed at ‘normal physiological pH’, presumably around pH 7.4. Once internalised, however, the complex may enter organelles where it would be subjected to much lower pH values, potentially as low as pH 4.5 [28, 29]. Low pH is well known to affect antibody binding [30]. Therefore we tested the persistence of the immunocomplex, after it had formed at pH 7.4, at two different pH values. The persistence of the immunocomplex formed when 2B12 bound to Kb was not significantly affected by pH or time of incubation. Similarly, the Kb/2B3 immunocomplex was not affected by incubation time but a decrease in pH did significantly reduce its persistence. Nevertheless, significantly more 2B3 than 2B12 remained bound to the Kb peptide at all pH values and time points tested, and 2B3 also retained nearly 70% of its original binding capacity. We therefore suggest that both antibodies would retain a large proportion of their biological activity, even under the low pH conditions found in the endosomal/lysosomal system [28, 29]. Furthermore, the lack of an effect of time suggests that the immunocomplexes formed will persist for a biologically relevant period of time.

Having demonstrated that 2B3 bound more efficiently to APP than 2B12, we then investigated whether it reduced the production of Aβ40 in a similar manner to 2B12. Both 2B3 and 2B12 significantly reduced extracellular levels of Aβ40, however, neither the anti-N-terminal APP antibody nor the control mouse IgG had any effect on Aβ40. This suggests that it is not sufficient to have an antibody that binds to APP in order to reduce Aβ but that the antibodies must bind in the vicinity of the β-secretase cleavage site to accomplish this effect. We also investigated whether either antibody affected the more aggregatory species of Aβ, Aβ42, since there have been suggestions that the majority of this peptide is cleaved from APP within the trans-golgi network or the endoplasmic reticulum, prior to the trafficking of APP to the cell membrane [31]. If this were indeed the case, then our anti-cleavage site antibodies might be ineffective against this species of Aβ. Again, both 2B3 and 2B12 significantly reduced Aβ42 from control levels but the N-terminal antibody had no effect. This would suggest that at least a portion of this peptide is produced elsewhere in the cell rather than in the secretary pathway, and post APP translocation to the cell membrane. It is interesting to note that, in both cases, 2B3 reduced levels of Aβ40 and Aβ42 more than 2B12, although this was only significant in the case of Aβ40. This could be because of the higher relative affinity of 2B3 for APP over 2B12. Alternatively it could be a function of its epitope, as by binding closer to the cleavage site than 2B12, it may block the access of BACE1 to APP via steric hindrance more effectively than 2B12. Clearly, information regarding the mode of action of the antibodies is important.

There are 3 predominant theories that have been used to explain how Aβ-specific antibodies may bring about the clearance of Aβ [32]: disruption of Aβ aggregates or neutralisation of Aβ oligomers, Fc-receptor-mediated phagocytosis of Aβ by microglia and the peripheral sink hypothesis in which the sequestration of circulating Aβ causes an efflux of Aβ from the brain to the plasma [33, 34]. 2B3 and 2B12 are unlikely to exert their effects by any of these mechanisms as neither bind to Aβ. We previously demonstrated that 2B12 was not toxic to cells in culture [25] and neither of the cleavage site antibodies nor the control IgG antibody affected cell viability levels here. The cleavage site antibodies are therefore not reducing levels of Aβ by initiating cell death. In contrast, the anti-N-terminal APP antibody led to increased absorbance levels in the MTS assay. We did not however, explore this finding any further because we had already demonstrated that this commercially available antibody did not affect Aβ levels and was therefore likely to behave in a very different manner to the cleavage site antibodies. Furthermore, 2B12 did not alter detectable cleavage by the non-amyloidogenic pathway in MOG-G-UVW cells as sAPPα levels remained unchanged.

It has been demonstrated that anti-N-terminal Aβ antibodies, which can reduce Aβ pathology in vivo, can reduce intracellular levels of Aβ in vitro only when internalised into cells [27], yet none of the theories described above fully explain how intracellular Aβ levels may be reduced. We have demonstrated that 2B3 and 2B12 can reduce levels of extracellular Aβ. It is therefore probable that our antibodies are also reducing levels of intracellular Aβ. This is particularly likely since neither of the antibodies bind to the Aβ peptide itself and cannot therefore be increasing its extracellular degradation, or interfering with the assay. We were, however, unable to measure intracellular Aβ40 or Aβ42 because of the low levels detectable in our native cell lines.

Internalised anti-Aβ antibodies or anti-cleavage site antibodies may inhibit the action of β-secretase, however, Tampellini et al. [27] saw no evidence of this with anti-Aβ antibodies. Tampellini et al. [27] observed that Aβ and APP ectodomain antibodies induced increased APP internalisation from the cell surface which actually led to enhanced cleavage by β-secretase and subsequently to enhanced clearance of the antibody-bound βCTF fragments in the lysosomal system. 2B3 and 2B12 did not alter intracellular APP levels, therefore it seems unlikely that their mode of action is via increased degradation of APP. Unlike Tampellini et al. [27], we observed a significant decrease in βCTF levels after incubation with 2B3. This would imply that the cleavage site antibodies do not induce increased internalisation of APP leading to enhanced cleavage by β-secretase but that they are reducing Aβ by a different mechanism. It suggests that they are inhibiting the cleavage of APP by β-secretase. The lack of a significant effect after incubation with 2B12 may be a result of the smaller effect that this antibody has on Aβ levels.

We hypothesised that 2B3 and 2B12 were blocking the action of β-secretase by steric hindrance. We therefore devised a simple cell-free system to investigate this hypothesis that avoided complications from other cellular components and overcame the low levels of APP fragments in the native cell lines. Both 2B3 and 2B12 drastically reduced or nearly abolished BACE1 cleavage of sAPPα. This clearly demonstrates that anti-cleavage site antibodies are capable of inhibiting BACE1 in vitro. The presence of a large protein (IgG) did not non-specifically block the action of BACE1. Crucially, our results demonstrate that antibody epitopes are vitally important to this inhibition since the anti N-terminal APP antibody, which binds some distance from the cleavage site, had no such effect on BACE1. In this simple in vitro system any effects of APP internalisation or enzyme/substrate colocalisation are eliminated. In conjunction with the observed decrease in βCTF levels, results from the cell-free assay system suggest that the mode of action of our cleavage site antibodies is most likely via steric hindrance.

Similar effects on Aβ levels were obtained by Arbel et al. [35] who produced monoclonal antibodies using a peptide containing part of the Swedish mutation at the β-secretase cleavage site. They also demonstrated a reduction in both extracellular and intracellular Aβ levels but in cell lines over-expressing APP. These antibodies have also been shown to improve cognition in the Tg2576 Swedish mutation mouse model of AD pathology [40] and reduce Aβ levels in the V717I London mutation mouse model [41]. We believe that our use of model cell lines which do not over-express APP is very important as the majority of cases of AD occur in people with much lower levels of APP than those associated with transfected cells. As far as we are aware, we have demonstrated for the first time that the most likely mode of action for such antibodies is via steric hindrance.

Immunotherapy for AD remains an exciting prospect, despite the failure of the AN1792 clinical trial [36]. Passive immunisation with β-secretase cleavage site antibodies might alleviate some of the problems associated with this trial such as the T-lymphocyte meningoencephalitis and cerebral micro-haemorrhages [37, 38]. These antibodies would not bind to existing Aβ and would not therefore stimulate the T-lymphocyte response or lead to the excessive complement activation that some believe would be a problem with Aβ antibodies [39]. In conjunction with other immunotherapeutic strategies to reduce plaque load, such antibodies may have a considerable impact on the development of disease-modifying treatments for AD.

MATERIALS AND METHODS

Materials and cell culture

All chemicals and reagents were purchased from Sigma-Aldrich, Poole, U.K. or Fisher Scientific, Leicester, U.K. and all reactions were performed at room temperature unless otherwise specified.

Astrocytoma cells, MOG-G-UVW (ECACC, Porton Down, U.K.) were cultured in a 1:1 mix of Ham’s F10 and Dulbecco’s Modified Eagle’s Medium supplemented with 10% foetal bovine serum (Perbio Science U.K. Ltd, Cramlington, U.K.) and 2mM L-glutamine.

Antibody production and isotyping

Full details of the immunisation protocol and hybridoma development are detailed elsewhere [25]. Antibodies (2B12 and 2B3) were raised to a 15-mer peptide spanning the β-secretase cleavage site on APP, EEISEVKMDAEFRHD, termed Ka. Both antibodies were concentrated from culture medium using Amicon Centriplus YM-100 filters (Millipore, Watford, U.K.) with a nominal molecular weight cut off of 100kDa and the isotype determined using the Isostrip mouse monoclonal antibody isotyping kit (Serotec, Oxford, U.K.).

Western Blotting was performed using standard methods. Briefly, samples were resolved on 10% polyacrylamide gels, transferred on to 0.2μm nitrocellulose membranes (Amersham Biosciences, Little Chalfont, U.K.), incubated with the relevant antibody and detected as previously described [25].

Determination of antibody epitopes

The epitopes of 2B3 and 2B12 on APP were investigated by Western blotting, as above, and by comparing the relative binding profiles of the antibodies to cleavage products of APP in an indirect ELISA. Recombinant sAPPα and sAPPβ (Sigma-Aldrich) were adsorbed to a 96 well microtitre plate (Greiner Bio-One, Stonehouse, U.K.) at 5ug/ml in carbonate/bicarbonate buffer (15mM Na2CO3, 35mM NaHCO3, pH9.8) overnight at 4°C. Plates were blocked with 1% non-fat milk powder for 1 hour then 2B3 or 2B12 were subsequently incubated at 1ug/ml for 2 hours. Antibodies were detected with a secondary anti-mouse antibody conjugated to horseradish peroxidise (HRP), 1:2500 (Pierce, Rockford, IL, U.S.A.) for 1 hour, and visualised with the enzyme substrate, o-phenylenediamine (OPD), in a 0.1 M citrate-phosphate buffer, (24mM citric acid, 51mM Na2HPO4, pH 5.0), incubated for 20 minutes. The reaction was stopped with 2.5M H2SO4 and the absorbance determined at 492nm. The diluent used on day 2 was PBS (137mM NaCl, 1.5mM KH2PO4, 8mM Na2HPO4, 2.5mM KCl, pH 7.4) with 0.05% Tween 20 (PBST). All results were expressed as a proportion of the standard antibody (6E10, Cambridge BioScience Ltd, Cambridge, U.K.) at 0.1μg/ml bound to sAPPα, to correct for any inter-plate variation.

Quantification of APP

APP was quantified using the APP DuoSet (R&D Systems, Abingdon, U.K.) following the manufacturer’s guidelines [25]. Briefly, the capture antibody was used at 4μg/ml in PBS overnight. Plates were blocked with 1% bovine serum albumin (BSA) and 5% sucrose in PBS and samples were quantified using a six point standard curve. The biotinylated detection antibody was used at 300ng/ml and detected using streptavidin-HRP and OPD.

Affinity ranking of 2B3 and 2B12 for an APP fragment

Affinity ranking of the two antibodies was accomplished by comparing their binding properties to a peptide, Kb, which spans the β-secretase cleavage site on APP, in an indirect ELISA. This peptide represents a 15-mer sequence (SEVKMDAEFRHDSGY), slightly further into the Aβ region of APP than Ka, the immunising peptide. ELISA methods follow those detailed above with the following exceptions. Kb was adsorbed to a 96 well microtitre plate at a concentration of 10ug/ml in carbonate/bicarbonate buffer overnight at 4°C. 2B3 or 2B12 were incubated for 2 hours at concentrations ranging from 0.00001 to 30μg/ml and detected as above. All results were expressed as a proportion of the standard antibody (6E10) at 0.05μg/ml.

Binding of 2B3 and 2B12 to full length APP

A competition assay, in conjunction with the sandwich ELISA for APP (R&D Systems) described above, was used to determine relative binding of 2B3 and 2B12 to APP from cell lysates. MOG-G-UVW cells were lysed and concentrated through a filter with a nominal cut-off of 100 kDa (Millipore) to provide predominantly full-length APP at a concentration of 30ng/ml, as described previously [25]. After formation of the APP/capture antibody complex on the 96 well plate and prior to incubation with the detection antibody, the test antibodies, 2B3, 2B12 or control IgG (Pierce), were incubated at concentrations ranging from 1 to 20μg/ml for 1 hour. Binding of these antibodies was then inferred by a decrease in binding of the detection antibody compared to the PBST control alone.

Persistence of 2B3 and 2B12 binding at different pH values

The binding persistence of 2B3 and 2B12 to Kb was investigated at two different pH values using an indirect ELISA. Methods followed those detailed above with the following modifications. Kb was adsorbed to a 96 well plate and blocked as above. 2B3 and 2B12 (5μg/ml) were incubated with Kb for 1 hour in PBST (pH 7.4) and the antibody solution was aspirated. The immunocomplex was then incubated for a further hour in PBST at either pH 7.4 or 4.0 for 0 or 60 minutes. To ensure that the antigen was not degraded or dissociated from the plate by the pH treatment, Kb was also incubated with PBST alone at pH 4.0 for 1 hour prior to incubation with the 2B3 or 2B12 at pH 7.4. Binding of both antibodies was detected as above and all results were expressed as a proportion of a standard antibody (6E10) at 0.05μg/ml. PBST was adjusted to the correct pH with H3PO4.

Effects of 2B3 and 2B12 on levels of Aβ40, Aβ42, sAPPα and βCTF

All experiments were performed in 24 well cluster plates, in triplicate, with a starting density of 25,000 MOG-G-UVW cells per well. Cells were allowed to attach overnight and were then incubated with control media, 2B3, 2B12, an anti-N-terminal APP antibody (22C11, Millipore) or an irrelevant control mouse IgG (Sigma-Aldrich) (all at 10μg/ml) for 48 hours at 37°C. This was repeated on a minimum of 3 different passage numbers where each n = 1 passage. For analysis of Aβ40, media was subjected to immunoprecipitation and ELISA as described in Thomas et al. [25]. Briefly, the ELISA employed the N-terminal Aβ antibody 6E10 (5 μg/ml), as the capture antibody and affinity-purified BAM401AP (0.45μg/ml Autogen Bioclear, Calne, U.K.), specific to the C-terminus of human Aβ40, as the detection antibody. For analysis of Aβ42, media was collected after antibody treatment and tested in a sandwich ELISA (Biosource, Invitrogen, Paisley, U.K.). To determine the effect of the antibodies on sAPPα, MOG-G-UVW cells were incubated as before and media was tested in a sandwich ELISA (IBL, Hamburg, Germany). All cells were lysed and intracellular APP and βCTF (IBL) levels were detected by ELISA as described above. This latter ELISA detects levels of C-terminal fragments of APP after cleavage by β-secretase. All data were normalised to total cell protein concentration as determined by BCA (bicinchoninic acid) protein assay (Pierce).

Effect of 2B3 and 2B12 on MOG-G-UVW cell viability

Viability studies were performed on MOG-G-UVW cells after incubation with 2B3, 2B12, control IgG (Sigma-Aldrich), anti-N-terminal APP antibody (22C11, Millipore) or media control, all at 10ug/ml, using the CellTiter 96® MTS Aqueous One Solution Cell Proliferation Assay (Promega, Southampton, U.K.) in 96-well cluster plates. MOG-G-UVW cells were first allowed to adhere overnight after plating at a concentration of 2000 per well, and were then incubated with treatments for 48 hours. Viability was assessed following the manufacturer’s guidelines.

Effect of 2B3 and 2B12 on BACE1 activity in a cell-free assay

The effect of 2B3 and 2B12 on BACE1 levels was investigated, after antibody treatment as detailed above, and analysed by Western blotting. 12.5g total protein was run on the polyacrylamide gel and detected with anti-BACE1 (0.27 μg/ml, Santa Cruz Biotechnology, Santa Cruz, U.S.A.). The effect of the antibodies on BACE1 activity was also investigated in a cell-free assay. Recombinant human sAPPα, (4 μg/ml, R&D Systems), containing the β-secretase cleavage site, was incubated in the presence or absence of BACE1 (82.5 μg/ml, R&D Systems) for 1 hour at 37°C, with the addition of one of the following treatments, 2B3, 2B12, control IgG (Pierce), anti-N-terminal APP antibody or media control. Prior to the addition of BACE1, the antibodies and sAPPα were allowed to form a complex for 3 minutes. Total reaction volume was 20μl, all reactions were performed in 50mM C2H3O2Na (pH 4.5) and all antibodies were used at 1μg/ml. The reaction was stopped by the addition of 3× Laemmli sample buffer [42]. A volume equivalent to 26.67ng starting sAPPα, was analysed in a Western blot and detected with the anti-N-Term APP IgG (33.3 ng/ml).

Statistical analyses

Data generated in ELISA assays were quantified by comparing data to standard curves included on each plate, using Graphpad Prism® 4. Results were first normalised to total protein concentration, where relevant, and expressed as % of media control values. MTS and ELISA results were then analysed using a Student’s t-test at the two-tailed significance level to determine if concentrations were significantly different to media controls (100%). Where relevant, ELISA data were subsequently analysed with one-way ANOVA.

To compare the relative affinities of 2B3 and 2B12 for the Kb peptide, log antibody concentration was plotted against % absorbance (of standard antibody) and a sigmoidal dose-response curve fitted to the data using Graphpad Prism® 4. Curve parameters were compared using the F-test and differences between 2B3 and 2B12 at each concentration were compared using a Student’s t-test. The persistence in binding of 2B3 and 2B12 to Kb at different pH values was investigated using GLM univariate analysis, with absorbance as the dependent variable and pH, antibody and time as factors. Both antibodies were subsequently investigated independently with ANOVA and Bonferroni.

Western blots for the inhibitory effects of 2B3 and 2B12 on BACE1 were quantified using NIH imager. All bands were first normalised to, and expressed as a % of, sAPPα alone, to allow comparisons between blots. The normalised control density of sAPPα and antibody was subtracted from the relevant experimental condition of sAPPα, antibody and BACE1 to determine the change in sAPPα after incubation with BACE1. The resulting change in sAPPα was analysed using one-way ANOVA and Bonferroni.

Where necessary, data were transformed to fulfil the assumptions of normality and homoskedasticity and to therefore allow the use of parametric testing.

ACKNOWLEDGEMENTS

This work was funded by grant number 79 from the Alzheimer’s Society, U.K. We would like to thank Katrin Hack, Pavlina Doubkova, Lynne Murphy and Shahista Jaffer for their technical assistance with this project.

Abbreviations

AD

Alzheimer’s disease

Aβ

amyloid-β

APP

amyloid precursor protein

BACE1

beta-site app cleaving enzyme

REFERENCES

  • 1.Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev. 2001;81:741–766. doi: 10.1152/physrev.2001.81.2.741. [DOI] [PubMed] [Google Scholar]
  • 2.Hardy J, Allsop D. Amyloid deposition as the central event in the aetiology of Alzheimer’s disease. Trends Pharmacol Sci. 1991;12:383–388. doi: 10.1016/0165-6147(91)90609-v. [DOI] [PubMed] [Google Scholar]
  • 3.Bennett DA, Schneider JA, Wilson RS, Bienias JL, Arnold SE. Neurofibrillary tangles mediate the association of amyloid load with clinical Alzheimer disease and level of cognitive function. Arch Neurol. 2004;61:378–384. doi: 10.1001/archneur.61.3.378. [DOI] [PubMed] [Google Scholar]
  • 4.Hamaguchi T, Ono K, Yamada M. Anti-amyloidogenic therapies: strategies for prevention and treatment of Alzheimer’s disease. Cell Mol Life Sci. 2006;63:1538–1552. doi: 10.1007/s00018-005-5599-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Mori H, Takio K, Ogawara M, Selkoe DJ. Mass spectrometry of purified amyloid beta protein in Alzheimer’s disease. J Biol Chem. 1992;267:17082–17086. [PubMed] [Google Scholar]
  • 6.Asai M, Hattori C, Szabo B, Sasagawa N, Maruyama K, Tanuma S, Ishiura S. Putative function of ADAM9, ADAM10, and ADAM17 as APP alpha-secretase. Biochem Biophys Res Commun. 2003;301:231–235. doi: 10.1016/s0006-291x(02)02999-6. [DOI] [PubMed] [Google Scholar]
  • 7.Allinson TM, Parkin ET, Turner AJ, Hooper NM. ADAMs family members as amyloid precursor protein alpha-secretases. J Neurosci Res. 2003;74:342–352. doi: 10.1002/jnr.10737. [DOI] [PubMed] [Google Scholar]
  • 8.Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, Doan M, Dovey HF, Frigon N, Hong J, et al. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature. 1999;402:537–540. doi: 10.1038/990114. [DOI] [PubMed] [Google Scholar]
  • 9.Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, et al. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999;286:735–741. doi: 10.1126/science.286.5440.735. [DOI] [PubMed] [Google Scholar]
  • 10.Yan R, Bienkowski MJ, Shuck ME, Miao H, Tory MC, Pauley AM, Brashier JR, Stratman NC, Mathews WR, Buhl AE, et al. Membrane-anchored aspartyl protease with Alzheimer’s disease beta-secretase activity. Nature. 1999;402:533–537. doi: 10.1038/990107. [DOI] [PubMed] [Google Scholar]
  • 11.Citron M. Strategies for disease modification in Alzheimer’s disease. Nat Rev Neurosci. 2004;5:677–685. doi: 10.1038/nrn1495. [DOI] [PubMed] [Google Scholar]
  • 12.Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C. Reconstitution of gamma-secretase activity. Nat Cell Biol. 2003;5:486–488. doi: 10.1038/ncb960. [DOI] [PubMed] [Google Scholar]
  • 13.Kimberly WT, LaVoie MJ, Ostaszewski BL, Ye W, Wolfe MS, Selkoe DJ. Gamma-secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2. Proc Natl Acad Sci U S A. 2003;100:6382–6387. doi: 10.1073/pnas.1037392100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Takasugi N, Tomita T, Hayashi I, Tsuruoka M, Niimura M, Takahashi Y, Thinakaran G, Iwatsubo T. The role of presenilin cofactors in the gamma-secretase complex. Nature. 2003;422:438–441. doi: 10.1038/nature01506. [DOI] [PubMed] [Google Scholar]
  • 15.Chen F, Hasegawa H, Schmitt-Ulms G, Kawarai T, Bohm C, Katayama T, Gu Y, Sanjo N, Glista M, Rogaeva E, et al. TMP21 is a presenilin complex component that modulates gamma-secretase but not epsilon-secretase activity. Nature. 2006;440:1208–1212. doi: 10.1038/nature04667. [DOI] [PubMed] [Google Scholar]
  • 16.Yamazaki T, Koo EH, Selkoe DJ. Trafficking of cell-surface amyloid beta-protein precursor. II. Endocytosis, recycling and lysosomal targeting detected by immunolocalization. J Cell Sci. 1996;109:999–1008. doi: 10.1242/jcs.109.5.999. [DOI] [PubMed] [Google Scholar]
  • 17.Koo EH, Squazzo SL, Selkoe DJ, Koo CH. Trafficking of cell-surface amyloid beta-protein precursor. I. Secretion, endocytosis and recycling as detected by labeled monoclonal antibody. J Cell Sci. 1996;109:991–998. doi: 10.1242/jcs.109.5.991. [DOI] [PubMed] [Google Scholar]
  • 18.Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang JP, Johnson-Wood K, Khan K, et al. Immunization with amyloid-beta attenuates Alzheimer disease-like pathology in the PDAPP mouse. Nature. 1999;400:173–177. doi: 10.1038/22124. [DOI] [PubMed] [Google Scholar]
  • 19.Buttini M, Masliah E, Barbour R, Grajeda H, Motter R, Johnson-Wood K, Khan K, Seubert P, Freedman S, Schenk D, et al. Beta-amyloid immunotherapy prevents synaptic degeneration in a mouse model of Alzheimer’s disease. J Neurosci. 2005;25:9096–9101. doi: 10.1523/JNEUROSCI.1697-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, Guido T, Hu K, Huang J, Johnson-Wood K, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med. 2000;6:916–919. doi: 10.1038/78682. [DOI] [PubMed] [Google Scholar]
  • 21.Janus C, Pearson J, McLaurin J, Mathews PM, Jiang Y, Schmidt SD, Chishti MA, Horne P, Heslin D, French J, et al. A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease. Nature. 2000;408:979–982. doi: 10.1038/35050110. [DOI] [PubMed] [Google Scholar]
  • 22.Morgan D, Diamond DM, Gottschall PE, Ugen KE, Dickey C, Hardy J, Duff K, Jantzen P, DiCarlo G, Wilcock D, et al. A beta peptide vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature. 2000;408:982–985. doi: 10.1038/35050116. [DOI] [PubMed] [Google Scholar]
  • 23.Lambert MP, Viola KL, Chromy BA, Chang L, Morgan TE, Yu J, Venton DL, Krafft GA, Finch CE, Klein WL. Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies. J Neurochem. 2001;79:595–605. doi: 10.1046/j.1471-4159.2001.00592.x. [DOI] [PubMed] [Google Scholar]
  • 24.Schenk D. Amyloid-beta immunotherapy for Alzheimer’s disease: the end of the beginning. Nat Rev Neurosci. 2002;3:824–828. doi: 10.1038/nrn938. [DOI] [PubMed] [Google Scholar]
  • 25.Thomas RS, Liddell JE, Murphy LS, Pache DM, Kidd EJ. An antibody to the beta-secretase cleavage site on amyloid-beta-protein precursor inhibits amyloid-beta production. J Alzheimers Dis. 2006;10:379–390. doi: 10.3233/jad-2006-10406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Chong YH, Jung JM, Choi W, Park CW, Choi KS, Suh YH. Bacterial expression, purification of full length and carboxyl terminal fragment of Alzheimer amyloid precursor protein and their proteolytic processing by thrombin. Life Sci. 1994;54:1259–1268. doi: 10.1016/0024-3205(94)00853-1. [DOI] [PubMed] [Google Scholar]
  • 27.Tampellini D, Magrane J, Takahashi RH, Li F, Lin MT, Almeida CG, Gouras GK. Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations. J Biol Chem. 2007;282:18895–18906. doi: 10.1074/jbc.M700373200. [DOI] [PubMed] [Google Scholar]
  • 28.Geisow MJ, Evans WH. pH in the endosome. Measurements during pinocytosis and receptor-mediated endocytosis. Exp Cell Res. 1984;150:36–46. doi: 10.1016/0014-4827(84)90699-2. [DOI] [PubMed] [Google Scholar]
  • 29.Murphy RF, Powers S, Cantor CR. Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J Cell Biol. 1984;98:1757–1762. doi: 10.1083/jcb.98.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Dejaegere A, Choulier L, Lafont V, De Genst E, Altschuh D. Variations in antigen-antibody association kinetics as a function of pH and salt concentration: a QSAR and molecular modeling study. Biochemistry. 2005;44:14409–14418. doi: 10.1021/bi050986v. [DOI] [PubMed] [Google Scholar]
  • 31.Haass C, Lemere CA, Capell A, Citron M, Seubert P, Schenk D, Lannfelt L, Selkoe DJ. The Swedish mutation causes early-onset Alzheimer’s disease by beta-secretase cleavage within the secretory pathway. Nat Med. 1995;1:1291–1296. doi: 10.1038/nm1295-1291. [DOI] [PubMed] [Google Scholar]
  • 32.Weiner HL, Frenkel D. Immunology and immunotherapy of Alzheimer’s disease. Nat Rev Immunol. 2006;6:404–416. doi: 10.1038/nri1843. [DOI] [PubMed] [Google Scholar]
  • 33.Wilcock DM, DiCarlo G, Henderson D, Jackson J, Clarke K, Ugen KE, Gordon MN, Morgan D. Intracranially administered anti-A beta antibodies reduce beta-amyloid deposition by mechanisms both independent of and associated with microglial activation. J Neurosci. 2003;23:3745–3751. doi: 10.1523/JNEUROSCI.23-09-03745.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wilcock DM, Rojiani A, Rosenthal A, Levkowitz G, Subbarao S, Alamed J, Wilson D, Wilson N, Freeman MJ, Gordon MN, et al. Passive amyloid immunotherapy clears amyloid and transiently activates microglia in a transgenic mouse model of amyloid deposition. J Neurosci. 2004;24:6144–6151. doi: 10.1523/JNEUROSCI.1090-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Arbel M, Yacoby I, Solomon B. Inhibition of amyloid precursor protein processing by beta-secretase through site-directed antibodies. Proc Natl Acad Sci U S A. 2005;102:7718–7723. doi: 10.1073/pnas.0502427102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Holmes C, Boche D, Wilkinson D, Yadegarfar G, Hopkins V, Bayer A, Jones RW, Bullock R, Love S, Neal JW, et al. Long-term effects of Abeta42 immunisation in Alzheimer’s disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet. 2008;372:216–223. doi: 10.1016/S0140-6736(08)61075-2. [DOI] [PubMed] [Google Scholar]
  • 37.Nicoll JAR, Wilkinson D, Holmes C, Steart P, Markham H, Weller RO. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med. 2003;9:448–452. doi: 10.1038/nm840. [DOI] [PubMed] [Google Scholar]
  • 38.Ferrer I, Boada Rovira M, Sanchez Guerra ML, Rey MJ, Costa-Jussa F. Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer’s disease. Brain Pathol. 2004;14:11–20. doi: 10.1111/j.1750-3639.2004.tb00493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.McGeer PL. Amyloid-beta vaccination for Alzheimer’s dementia. Lancet. 2008;372:1381–1382. doi: 10.1016/S0140-6736(08)61579-2. [DOI] [PubMed] [Google Scholar]
  • 40.Rakover I, Arbel M, Solomon B. Immunotherapy against APP β-secrectase cleavage site improves cognitive function and reduces neuroinflammation in Tg2576 mice without a significant effect on brain Aβ levels. Neurodegenerative Dis. 2007;4:392–402. doi: 10.1159/000103250. [DOI] [PubMed] [Google Scholar]
  • 41.Solomon B, Frenkel D. Immunotherapy for Alzheimer’s disease. Neuropharmacol. 2010;59:303–309. doi: 10.1016/j.neuropharm.2010.04.004. [DOI] [PubMed] [Google Scholar]
  • 42.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]

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