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Keywords:

  • 40,42 peptides;
  • Alzheimer’s disease;
  • anti-N-terminal Aβ antibodies;
  • APP amyloidogenic processing;
  • auto-Aβ antibodies

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

J. Neurochem. (2012) 120, 732–740.

Abstract

Several prior investigations of Alzheimer’s disease (AD) patients have indicated naturally occurring autoantibodies against amyloid-β (Aβ) species are produced. Although many studies have focused on the relative concentrations or binding affinities of autoantibodies against Aβ-related proteins in AD and aging, data regarding their functional properties are limited. It is generally believed that these antibodies act to aid in clearance of Aβ. However, as antibodies which bind to Aβ also typically bind to the parent amyloid precursor protein (APP), we reasoned that certain Aβ-targeting autoantibodies may bind to APP thereby altering its conformation and processing. Here we show for the first time, that naturally occurring Aβ-reactive autoantibodies isolated from AD patients, but not from healthy controls, promote β-secretase activity in cultured cells. Furthermore, using monoclonal antibodies to various regions of Aβ, we found that antibodies generated against the N-terminal region, especially Aβ1-17, dose dependently promoted amyloidogenic processing of APP viaβ-secretase activation. Thus, this property of certain autoantibodies in driving Aβ generation could be of etiological importance in the development of sporadic forms of AD. Furthermore, future passive or active anti-Aβ immunotherapies must consider potential off-target effects resulting from antibodies targeting the N-terminus of Aβ, as co-binding to the corresponding region of APP may actually enhance Aβ generation.

Abbreviations used:
β-CTF

β-C-terminal fragment

AD

Alzheimer’s disease

APP

amyloid precursor protein

CHO

Chinese hamster ovary

PBS

phosphate-buffered saline

Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common cause of dementia. In the brains of AD patients, amyloid-β (Aβ) peptides, derived from the amyloid precursor protein (APP), accumulate into β-amyloid plaques, one of the pathologic hallmarks of the disease. Neurotoxic oligomeric forms of Aβ are hypothesized to play a critical role in AD pathogenesis (Walsh et al. 2002; Lesne et al. 2006; Haass and Selkoe 2007). Previous studies suggest that both endogenous naturally occurring anti-Aβ autoantibodies, or those generated by vaccination against Aβ, may enhance clearance of the peptide from the brain (Schenk et al. 1999; Morgan et al. 2000; Dodel et al. 2004; Morgan 2011). Indeed, active or passive immunization against Aβ peptide has been proposed as a method for preventing and treating AD (Schenk et al. 1999; Morgan 2011). Active immunization in transgenic AD mice reduced fibril formation, enhanced clearance of Aβ plaques, and improved behavioral impairment (Schenk et al. 1999; Morgan et al. 2000; Morgan 2011). In addition, passive immunization with antibodies recognizing the N-terminal and central domains of Aβ peptides was also effective (DeMattos et al. 2001). In patients vaccinated against the N-terminus of Aβ, considerable decreases in plaque load have been reported, but this clearance of pre-formed plaques was not sufficient to improve cognitive function in AD patients (Holmes et al. 2008). Similarly, passive vaccination of transgenic AD mice against the N-terminus of Aβ inhibited fibril formation and disaggregated pre-formed amyloid fibrils; however, it did not disrupt toxic oligomers (Mamikonyan et al. 2007).

A number of past studies have quantified autoantibodies against Aβ in AD. Some investigators found reduced anti-Aβ autoantibodies in AD patients (Du et al. 2001; Weksler et al. 2002) compared with controls. However, a more recent study indicates that such autoantibodies against the most toxic species of Aβ are reduced in both normal elderly and AD patients (Britschgi et al. 2009). Anti-Aβ autoantibodies are generally believed to promote clearance of the peptide from the brain (Dodel et al. 2004; Taguchi et al. 2008; Bacher et al. 2009). Indeed, natural autoantibodies comprise some two-thirds of the total adult human antibody pool and are multifunctional (Shoenfeld et al. 2007). Many autoantibodies targeting Aβ not only bind to Aβ, but bind also to APP, and while the concentrations and binding of anti-Aβ antibodies to Aβ have been extensively studied, knowledge of their functional effects on APP processing is unknown.

Here, we functionally screened serum autoantibodies from individual AD patients and non-demented age-matched controls for their ability to alter APP processing in Chinese hamster ovary (CHO) cells engineered to over-express Swedish mutant APP and human wild-type presenilin-1 (PS1wt) (CHO/APPswe/PS1wt). Importantly, we found those autoantibodies from AD patients significantly promoted β-secretase activity; a phenomenon not seen in the age-matched control serum. To model this phenomenon in vitro, monoclonal antibodies to various regions of Aβ were screened for APP processing effects in CHO/APPswe/PS1wt cells. Those targeting Aβ1-17 strongly promoted amyloidogenic APP processing. Finally, as in vivo validation, we delivered monoclonal antibodies targeting this Aβ1-17 region via intracerebroventricular injection into transgenic AD model mice over-expressing both APPswe and mutant (ΔE9) human PS1 (PSAPP mice). Similar to our in vitro findings, antibodies targeting this N-terminal region of Aβ promoted significant Aβ generation.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Patients

All samples were obtained from ProteoGenex Inc. (Culver City, CA, USA). Ten patients (five males and five females) with probable Alzheimer’s disease diagnosed according to DSM-IV criteria (MMSE, mean 16.6 ± 2 SD) were included in the study if they were 60–80 years old (mean 75.7 ± 5 SD), and did not have a diagnosis of comorbid autoimmune disease. Healthy controls were matched with AD patients (n = 10) solely on the basis of age (mean 65.6 ± 2.1 SD) and gender. Sample collection from clinical sites in Moscow, Russia were approved by an independent ethics committee in accordance with Russian law, US federal law (HIPPA), WHO, ICH, and GCP guidelines. All participating patients gave written informed consent.

Concentration of human serum

Human sera were concentrated under vacuum at ambient temperature (25°C). Auto-Aβ1-17 antibody levels in the concentrated sera were measured by ELISA. Briefly, 96-well ELISA plates were coated with 100 μL Aβ1-17 (1 μg/mL) and incubated overnight at 4°C. Plates were washed five times with washing buffer, and then blocked for 1 h at 37°C. Following blocking, the plates were washed 4 times with washing buffer and the concentrated human serum samples were applied (100 μL/well) in duplicate or triplicate and incubated at 4°C overnight. The plates were then washed 3 times with washing buffer and anti-Human IgG was diluted 1 : 10 000 and incubated for 1 h. After incubation, the plates were washed three times, and developed with tetramethylbenzidine substrate-chromogen (Dako, Carpinteria, CA, USA). The reaction was stopped with 2 N sulfuric acid (50 μL) and the plates were analyzed spectrophotometrically at 450 nm.

Antibodies

Several well characterized Aβ antibodies were used: mouse monoclonal 6E10 (human Aβ residues 1–17; Covance, Emeryville, CA, USA), 4G8 (Aβ residues 17–24; Covance), 1E11 (Aβ residues 1–8; Covance), VPB-203 (Aβ residues 8–17; Vector Laboratories, Burlingame, CA, USA), 9F1 (Aβ residues 32–40; Calbiochem, La Jolla, CA, USA), AB10 (human Aβ residues1–17; Merck Millipore, Billerica, MA, USA), and Aβ1-12 antibody (BAM10, Sigma-Aldrich, St Louis, MO, USA). Mouse IgG1 and IgG2b (Biolegend, La Jolla, CA, USA) were used as controls. Medium was changed to provide fresh medium to cells just prior to each treatment. Final Aβ antibody concentrations in each treatment were 0.63, 1.25, and 2.5 μg/mL. Cells were incubated with individual antibodies for 3 h.

Cell lines and cell culture

Chinese hamster ovary (CHO) cell lines and human neuroblastoma SH-SY5Y cells, both with stable coexpression of human APP bearing the Swedish mutation (APPswe) and wild-type human PSEN1 (PS1wt) were engineered as previously described (Weggen et al. 2003; Hahn et al. 2011). CHO/APPswe/PS1wt cells were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 1 mM sodium pyruvate and 100 units/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA). SH/APPswe/PS1wt cells were cultured in complete Dulbecco’s modified Eagle’s medium/F12 medium supplemented with 10% fetal bovine serum, 1% geneticin (G418; 40 mg/mL, Invitrogen) and hygromycin (50 mg/mL, Invitrogen). Cells were plated in 24-well plates at a density of 1 × 105 cells per well. After overnight incubation, the cells were treated with Aβ-antibodies at dosages of 0.63, 1.25, and 2.5 μg/mL for 3 h.

Mice

All mice were housed and maintained in the College of Medicine Animal Facility at the University of South Florida (USF), and all experiments were conducted in compliance with protocols approved by the USF Institutional Animal Care and Use Committee. Double transgenic ‘Swedish’ mutant APPK595N/M596L (APPswe) + PS1ΔE9 B6C3-Tg 85Dbo/J strain (PSAPP mice), 8 months old were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Because sex differences can impact Aβ deposition (Jankowsky et al. 2001), we used only females in our analyses (n = 3).

Intracerebroventricular (i.c.v.) antibody treatment

Animals were anesthetized using isoflurane (chamber induction at 4–5% isoflurane, intubation and maintenance at 1–2%). After reflexes were checked to ensure that mice were unconscious, they were positioned on a stereotaxic instrument (Stoelting Lab Standard, Wood Dale, IL, USA). The Aβ antibody (6E10) and isotype control IgG1 were dissolved in sterile distilled water at a concentration of 1 μg/μL. Aβ antibody and control IgG1 (5 μL) were injected into the left lateral ventricle with a microsyringe at a rate 1 μL/min with the following coordinates: (coordinates relative to bregma: −0.6 mm anterior/posterior, +1.2 mm medial/lateral, and −3.0 mm dorsal/ventral) per our previous methods (Giunta et al. 2006). The needle was left in place for 5 min after injection before being withdrawn. At 24 and 48 h after the i.c.v. injections, animals were killed with isofluorane and brain tissues were collected. All dissected brain tissues were rapidly frozen for immunoblot analysis.

Immunoblot analysis

Supernatants of the cells were collected and Aβ monomers and oligomers were visualized using immunoblot protocol. Cultured cells were lysed in ice-cold lysis buffer as described previously (Tan et al. 2002). All antibodies were diluted in Tris-buffered saline (TBS) containing 5% (w/v) non-fat dry milk. Blots were developed using the Luminol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Densitometric analysis was performed as described previously (Rezai-Zadeh et al. 2005) using a FluorS Multiimager with Quantity One software (Bio-Rad, Hercules, CA, USA). Antibodies used for immunoblot analysis included rabbit anti-APP C-terminus polyclonal antibody (pAb369, 1 : 1000) generously provided by Dr. Sam Gandy; rabbit anti-APP C-terminus polyclonal antibody (pAb751/770, 1 : 1000, Calbiochem), N-terminal Aβ 6E10 (1 : 1000; Covance), and β-actin (1 : 1500; as an internal reference control; Sigma-Aldrich).

ELISA

To measure Aβ levels with 4G8 and IgG2b antibody treatment, Aβ40, 42 ELISA kits (Invitrogen) were used following the manufacturer’s instructions with modifications. In treatment groups not utilizing N-terminal Aβ antibodies, the manufacturer’s instructions were strictly followed. In treatment groups utilizing N-terminal Aβ antibodies, to avoid interference with N-terminal capture antibodies, 96-well ELISA plates were coated with 100 μL Aβ32–40 (l mg/mL) in phosphate-buffered saline (PBS) and incubated overnight at 4°C. Plates were washed five times with washing buffer (0.05% Tween-20 in PBS), and then blocked (300 μL/well) for 1 h at 37°C with 1% bovine serum albumin + 0.05% Tween-20 in PBS. Following blocking, the plates were washed four times with washing buffer and samples applied (100 μL/well) in duplicate or triplicate and incubated at 4°C overnight. The plates were then washed three times with washing buffer and 6E10 (2 μg/mL) was added for detection of Aβ. Following another wash, goat anti-mouse IgG with horseradish peroxidase conjugation was diluted 1 : 2000 and incubated for 30 min. After incubation, the plates were washed three times, developed with tetramethylbenzidine substrate-chromogen (Dako). The reaction was stopped with 2 N sulfuric acid (50 μL) and the plates were analyzed spectrophotometrically at 450 nm.

Statistical analysis

All data were normally distributed; therefore, in instances of single mean comparisons, Levene’s test for equality of variances followed by the t-test for independent samples were used to assess significance. In instances of multiple mean comparisons, one-way analysis of variance (anova) was used. Alpha was set at 0.05 for all analyses. The statistical package for the social sciences release IBM SPSS 18.0 (IBM, Armonk, NY, USA) was used for all data analyses.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

1-17 autoantibodies from AD patients promote β-secretase APP cleavage

We treated CHO/APPswe/PS1wt cells with sera-derived auto-Aβ1-17 antibodies from AD patients (n = 10) and non-demented controls (n = 10). Neither group contained individuals with a known co-morbid autoimmune disease. Here, we observed an increase in Aβ species in the cells treated with concentrated total auto-Aβ1-17 antibodies from AD patients compared with age-matched controls. Likewise, there was a corresponding increase in the ratio of β-C-terminal fragment (β-CTF) to β-actin in this same group as determined by immunoblot analysis of the cell lysates (Fig. 1).

image

Figure 1.  The concentrated Aβ autoantibodies from AD patients promote β-secretase cleavage of APP in CHO/APPswe/PS1wt cells. The concentrated sera were individually prepared from AD patients and normal aging controls (Ctrl). (a) Autoantibodies against Aβ peptide 1–17 were measured in the concentrated sera by ELISA. Data are presented as mean (±SD) in a dot-plot (anti-Aβ1-17 IgG mg/mL) from 10 Alzheimer’s disease patients and 10 age-matched controls. A t-test did not reveal a significant difference between Alzheimer’s disease and normal aging controls in terms of quantity of auto-Aβ1-17 antibodies (> 0.05). For functional assessment on APP processing, CHO/APPswe/PS1wt cells were treated with AD- or normal age-matched control-derived concentrated auto-Aβ1-17 antibodies at 1.25 μg/mL for 3 h. (b, top panel) Aβ species were analyzed in conditioned media from the CHO/APPswe/PS1wt cells by immunoblot (IB) analysis using Aβ1-17 antibody (6E10). (b, as indicated below top panel) Human IgG heavy chain (IgGH) and IgG light (IgGL) were analyzed by IB as the internal reference using an anti-human IgG antibody (anti-human Aβ). (b, middle panel) Cell lysates were prepared and subjected to IB analysis of APP CTFs pAb751/770 (C-APP). (b, bottom panel) The β-CTF band was further confirmed by the IB using 6E10 following blot striping. As indicated below this panel, an anti-β-actin antibody was used an internal reference control for Fig. 1b both middle and bottom panels). (c) Densitometry analysis shows the ratio of Aβ to sAPP-α (one of the light exposed blots) (top panel) or β-CTF to β-actin. Aβ and β-CTF IB results are representative of results obtained for 10 cases per group. A t-test revealed a significant difference between AD cases and normal aging controls (n = 10) in either ratio of Aβ to sAPP-α or β-CTF to β-actin. **< 0.01.

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Antibody against N-terminal region of Aβ markedly increases Aβ production

To examine the effects of Aβ antibodies raised against various regions of Aβ, on APP processing we first utilized an in vitro system. CHO/APPswe/PS1wt cells were treated with antibodies raised against Aβ’s N-terminal residues: 1–8, 8–17, 1–17, 17–26, or against the C-terminal residues (33–42) of Aβ, at 1.25 μg/mL (based on the upper limit for Aβ1-17 concentration in AD patient serum yielding amyloidogenic processing in vitro; Fig. 1) for 3 h. We found significant differences between Aβ1-17 antibody (6E10) and other antibodies when compared to control IgG1 and Aβ33-42 as demonstrated by immunoblot for Aβ species (Fig. 2a). Furthermore, immunoblot analysis of cell lysates for β-CTF revealed significantly greater β-CTF generation in cells treated with 6E10 compared with control IgG1 and Aβ33-42 antibody (Fig. 2b). This β-CTF was further confirmed by the IB using BAM10 (Fig. 2c). In addition, similar results were also observed in SH/APPswe/PS1wt cells treated with Aβ antibody against Aβ1-17 peptide (6E10) (Fig. 2d and e). Finally, antibody binding to the cell membranes of CHO/APPswe/PS1wt cells after 1 h incubation was examined by confocal microscopy. We detected, higher binding of anti-Aβ1-17 antibody (6E10) compared to control isotype IgG1 on these cell membranes (Fig. 2f and g). In addition, we also employed SH/APPswe/PS1wt cells for this binding assay and observed similar results in SH/APPswe/PS1wt cells stained with fluorescent-dye conjugated 6E10 (data not shown).

image

Figure 2.  Treatment with Aβ antibody against N-terminal 1–17 peptide (6E10) increases Aβ production in cultured cells. CHO/APPswe/PS1wt cells were treated with Aβ antibody (Ab) against N-terminal peptide, 1–8, 8–17, 1–17 (6E10), 17–26 or against C-terminal Aβ peptide 33–42 at 1.25μg/mL for 3 h. (a) Aβ species were analyzed in conditioned media from the CHO/APPswe/PS1wt cells by IB analysis using monoclonal Aβ1-12 antibody (BAM10). (b) Cell lysates were prepared and subjected to IB analysis of APP CTFs using pAb751/770 (C-APP). (c) The β-CTF band was further confirmed by the IB using BAM10 (Aβ1-12 antibody) following blot striping. One-way anova followed by post hoc comparison revealed significant differences between 6E10 and other antibodies; specifically when comparing with control IgG1 and Aβ33-42 antibody. A t-test revealed a significant difference between 6E10 and IgG1 control treatment conditions, or 6E10 and Aβ33-42 treatment conditions (n = 3 for each condition). In parallel, we employed other antibodies against Aβ1-17 peptide (AB10, EMD Millipore) at similar concentrations for 3 h. We observed similar results (data not shown). In addition, similar results were also observed in SH/APPswe/PS1wt cells treated with 6E10 (d, e). Densitometry analysis shows the ratios of Aβ to sAPP-α (a, d), β-CTF to β-actin (b, c, e) as indicated below the figures. In order to examine if 6E10 antibody could bind to the membrane of CHO/APPswe/PS1wt cells, these cells were incubated with fluorescence conjugated 6E10 (f) or control isotype IgG1 (g) at 1.25 μg/mL for 1 h. Antibody binding to cell membrane was examined by an Olympus FV1000 Confocal Microscope. In parallel, we employed SH/APPswe/PS1wt cells for this binding assay. We also observed similar results (data not shown).

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1-17 antibody dose-dependently promotes Aβ production

To determine the dose-response relationship, we again treated CHO/APPswe/PS1wt cells with Aβ1-17 antibody (6E10) at various concentrations as indicated for 3 h. We found significant differences in Aβ40 levels between 6E10 at 2.5 μg/mL and 1.25 or 0.63 μg/mL by ELISA and immunoblot analyses of the cell supernatants (Fig. 3a and b). As expected, there was also a significant dose-dependent increase in β-CTF (Fig. 3c). In addition, we employed Aβ17-26 antibody (4G8) at the similar concentrations for 3 h. We observed similar results as Aβ1-17 antibody (6E10) (Fig. 3d–f).

image

Figure 3.  Treatment with Aβ1-17 antibody dose-dependently increases Aβ production. CHO/APPswe/PS1wt cells were treated with 6E10 at various concentrations as indicated for 3 h. Supernatants were collected and subjected to Aβ ELISA (a) and IB (b) analyses using BAM10. Cell lysates were prepared and subjected to IB analysis (c) for APP processing by pAb751/770 (C-APP). In addition, the β-CTF band was further confirmed by the IB using BAM10 (data not shown). For panel (a), secreted Aβ peptide species were analyzed by ELISA. Aβ levels are presented as relative fold mean (±SD) over IgG1 control. The results are representative of three independent experiments with n = 3 for each condition. A t-test revealed significant differences in Aβ levels between Aβ1-17 antibody at 2.5 μg/mL and 1.25 or 0.63 μg/mL. For panels (d–f), in parallel, we employed Aβ17-26 antibody (4G8) at the same concentrations for 3 h. We observed similar results as Aβ1-17 antibody (6E10). For panel (d), secreted Aβ40, 42 peptides were analyzed by ELISA antibody. Densitometry analysis shows the ratios of Aβ to sAPP-α (b, e), β-CTF to β-actin (c, f) as indicated below the figures. ***< 0.001.

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1-17 antibody dampens α-secretase activity

To determine how the Aβ1-17 antibody may promote Aβ production, we again treated CHO/APPswe/PS1wt cells with Aβ1-17 antibody (6E10), under the same conditions as above, for immunoblot analysis of APP metabolites: Aβ, sAPP-α, and sAPP-β. We found, upon application of anti-N-terminal Aβ1-17 antibody (6E10), a significant decrease in sAPP-α, corresponding with an increase in Aβ in conditioned media (Fig. 4a). Furthermore, there was a relative increase in sAPP-β in conditioned media from the Aβ1-17 antibody (6E10) treated cells compared with controls by immunoblot analysis (Fig. 4b). Finally, cells exposed to the Aβ1-17 antibody (6E10) displayed a higher ratio of β- to α-CTF in the cell lysate by immunoblot analysis (Fig. 4c).

image

Figure 4.  Treatment with Aβ1-17 antibody promotes APP β-secretase cleavage. As indicated, CHO/APPswe/PS1wt cells were treated with Aβ1-17 antibody (6E10) or IgG1 isotype control at 1.25 μg/mL for 3 h. Conditioned media were collected and subjected to immunoblot analysis for Aβ species, sAPP-α (a) and sAPP-β (b). For panel (a), IB analysis using an anti-Aβ1-12 monoclonal antibody (BAM10) shows secreted sAPP-α and Aβ species. Mouse IgG light (IgGL) was also shown up by the IB as indicated. For panel (b), IB analysis using antibody specifically against soluble APP-β of Swedish type cleaved by β-secretase (6A1) shows secreted sAPP-β. Cell lysates were prepared and subjected to IB analysis for APP processing (c). For panel (c), IB analysis using anti-C-terminal APP rabbit antibody 369 (pAb369) shows full-length holo APP and two bands corresponding to β-CTF (C99) and α-CTF (C83). These results are representative of three independent experiments with n = 3 for each condition.

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1-17 antibody promotes amyloidogenic APP processing in vivo

Once it was determined that Aβ1-17 antibody strongly promoted amyloidenic APP processing in vitro through up-regulation of β-secretase activity, we next sought to determine whether this could be replicated in a mouse model of AD. Thus, we subjected PSAPP mice at 8 months of age to intracerebroventricular (i.c.v.) injection with Aβ1-17 antibody (6E10), Aβ33-42 antibody, or IgG1 control at 5 μg/mouse. The mice were killed 24 and 48 h after the treatment. By immunoblot analysis of brain homogenates using a monoclonal anti-Aβ1-12 antibody (BAM10), we found that Aβ species were increased (Fig. 5a) in the 6E10 treated group compared to the Aβ33-42 antibody or IgG1 control groups. Correspondingly, the ratio of β- to α-CTF in this group was significantly higher than the other Aβ33-42 antibody or IgG1 control groups by immunoblot analysis (Fig. 5b).

image

Figure 5.  Aβ1-17 antibody modulates APP processing in vivo. PSAPP mice at 8 months of age were intracerebroventricular (i.c.v.) injected with Aβ1-17 antibody (6E10), Aβ33-42 antibody (9F1) or control IgG1 at 5 μg/mouse and euthanized 24 and 48 h after the treatment. Mouse brain homogenates were prepared [the right half of brain tissues (the non-injection side)] and subjected to IB analysis for APP processing. For panel (a), IB analysis using Aβ1-12 antibody (BAM10) shows total APP and Aβ species. For panel (b), IB analysis using pAb369 shows full-length holo APP and two bands corresponding to β-CTF (C99) and α-CTF (C83). Densitometry analysis shows the ratios of Aβ to β-actin (a), β-CTF to β-actin (b) as indicated below the figures. IB data presented here are representative of results obtained for three female mice per group at each time point.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The mechanisms by which Aβ autoantibodies might act to modify APP processing are not known. Here, we show for the first time, that naturally occurring concentrated autoantibodies from AD patients promote amyloidogenic processing of APP compared with non-demented controls (Fig. 1). It is hypothesized that the breakdown of immune tolerance to self-antigens may lead to production of autoreactive lymphocytes and autoantibody formation in response to various disease processes (Bei et al. 2009). This process may result in autoantibodies with therapeutic and/or pathological functions (Bei et al. 2009). Why pathologic autoantibodies are produced in some AD patients, and whether these autoantibodies can increase amyloidogenic APP processing in human is still unknown.

Despite these limitations, our data suggest that the most potent autoantibody from AD serum for promoting Aβ generation are those targeting the N-terminal extracellular region of Aβ, specifically Aβ1-17; as antibodies against this region increased indicators of β-secretase processing of APP, specifically total Aβ and β-CTF (Fig. 2). This would suggest promotion of amyloidogenic processing is dependent on the region of APP being bound, however, how exactly this antibody interacts with APP structurally is yet to be elucidated. Nevertheless, our findings suggest that it is a regional membrane effect as C-terminal (intracellular) Aβ antibody binding was largely absent (Fig. 2). Importantly, the corresponding Aβ1-17 region of APP contains the α-secretase cleavage site. Therefore, α-secretase activity may putatively be competitively blocked by Aβ1-17 antibody binding. Cell membrane binding occurs (Fig. 2f–g) and appears crucial (Fig. 4a–c) for the actions of the Aβ1-17 antibody. As the ratio of α- to β-secretase cleavage occurs at a dramatically reduced level from both clinical observation (Fig. 1), as well as in vitro (Figs 2–4); we suggest that these observed effects may result from blockade of the α-secretase cleavage. Further molecular studies will be needed in the future to characterize the dynamics of APP processing in the setting of autoantibody blockade of the α-secretase cleavage of APP.

Notably, the first AD vaccine AN1792, was based on a synthetic form of Aβ1-42. In phase II trials (N = 372 with mild-to-moderate AD), about 6% of patients developed meningoencephalitis and leukoencephalopathy, causing the trial to be halted (Orgogozo et al. 2003). Importantly in this study, immunization resulted in generation of anti-Aβ antibodies targeting the N-terminal Aβ. However, previous studies suggested that it is the Aβ15–42, region which initiated T-cell responses that triggered the meningoencephalitis. The B-cell epitope Aβ11–15 is considered to be important for generation of anti-Aβ antibodies (Monsonego et al. 2003; Lee et al. 2005; Pride et al. 2008). Interestingly, Aβ1-17 antibody binds to the portions of both these functional epitopes.

To determine whether the increase β-secretase activity observed in the AD clinical population (Fig. 1) could be modeled in vivo, we treated PSAPP mice at 8 months of age with i.c.v. Aβ1-17 antibody, Aβ33-42 antibody, or IgG1 control at 5 μg/mouse; based on the upper limits of Aβ1-17 patient blood (Fig. 1) and our in vitro (Figs 2 and 3) studies. We found that addition of the anti-Aβ1-17 antibody (6E10) significantly increased Aβ production (Fig. 5a), compared with the Aβ33-42 antibody or IgG1 control. Such soluble Aβ species, including Aβ42 and resulting multimeric aggregates have been shown recently in vitro and in transgenic mice models to be crucial toxic species (Cleary et al. 2005; Klyubin et al. 2005; Lesne et al. 2006; Townsend et al. 2006; Glabe 2008; Shankar et al. 2008; Tomic et al. 2009). Furthermore, small Aβ oligomers may form intracellularly before being released into the extracellular medium, where they can interfere with synaptic activity or act as seeds to promote fibrillization (Selkoe 2004; Khandogin and Brooks 2007). Additionally, as expected also for this group, the ratio of β- to α-CTF was significantly higher by immunoblot analysis; another indicator of amyloidogenic APP processing by β-secretase (Fig. 5b).

While it is believed that autoantibodies aid in the clearance of Aβ here, we show naturally occurring autoantibodies in AD patients, but not non-demented controls, promote amyloidogenic APP processing. This intriguing and straightforward finding is different from most published reports as it suggests that some or subsets of autoreactive Aβ antibodies may indeed be deleterious, rather than salutary due to their previously reported amyloid-clearing capability. We would propose however, that because the amount of Aβ autoreactive antibody was not significantly different between AD and age-matched controls (Fig. 1), that there may be a functional difference in these antibodies between the two populations. That is, those autoantibodies in the AD group posses the capacity to bind, and subsequently alter the processing of APP towards the amyloidogenic processing route, whereas those from the control group did not yield these effects. This points to a need to understand the exact mechanisms of antibody binding to APP in these two patient groups. It may suggest that in sporadic AD, these autoreactive Aβ antibodies are key to disease development because they alter APP processing, while in normal controls, these antibodies have a different function, namely clearance of amyloid from the brain.

Particularly, the anti-N-terminal Aβ antibody (6E10) against Aβ1-17 peptide, as evidence in vitro and in vivo, appeared to dose-dependently promote amyloidogenic processing of APP via blockade of α-secretase APP cleavage. In conclusion, the ability of these autoantibodies to actually promote the production of Aβ at the level of APP processing may be etiologically important in the development of sporadic forms of AD. Furthermore, future vaccine strategies may need to take into account antibody binding in the Aβ1-17 region of APP; as targeting this region may impart deleterious effects in the form of amyloidogenic APP processing. Moreover, this might dilute the Aβ-clearing effects of these autoantibodies. Further studies will be required to determine if this phenomenon occurs in patients vaccinated against Aβ.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by the NIH/NIA (R01AG032432 and R42AG031586, JT) and a Veterans Affairs Merit grant (JT).

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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