One of the binding proteins for administered amyloid-β appears to be anti-Aβ IgG antibody in amyloid plaques

Abstract

Background

Amyloid-β peptides (Aβ) applied to Alzheimer's disease (AD) brain bind to amyloid plaques (APs) and this binding is saturable. Meanwhile, anti-immunoglobulin G (IgG) antibodies label APs. Considering the saturable binding of Aβ to APs and the localization of IgGs in APs, specific binding proteins (BPs) for the applied Aβ could be anti-Aβ IgG antibodies (anti-AβAbs) in APs.

Objective

Our objective is to demonstrate whether anti-AβAbs are the BPs for administered Aβ in APs.

Methods

To identify the binding specificity, we established a tissue competition assay utilizing the competitiveness of ligand specificity to BPs. Biotinylated Aβ₄₂ (Bio42) was applied as ligand to AD brain sections. Second, Bio42 and IgG (anti-AβAbs) in APs were observed by double immunofluorescent labeling with ultrahigh-resolution imaging. Finally, to demonstrate the direct binding of Bio42 to anti-AβAbs we employed immunoprecipitation-western blotting.

Results

With the tissue competition assay, binding of Bio42 to APs was competitively blocked by coincubation with competitive Aβ₄₂ (Pep42) in a dose-dependent manner but not with non-competitive Aβ₄₀ (Pep40). Colocalization of Bio42 and anti-AβAbs was clearly detected with ultrahigh-resolution imaging. Anti-AβAbs were immunoprecipitated by Bio42 and immunoblotted by anti-IgG antibodies.

Conclusions

We have provided evidence that the applied Aβ specifically binds to anti-AβAbs rather than aggregating with Aβ in APs. Our results support the scenario that anti-AβAbs surround and/or sequester Aβ from biological fluids to APs, thereby affecting the amount of Aβ in body fluids. It might be important to consider anti-AβAbs in APs for more effective diagnosis and therapy for AD.

Keywords

Alzheimer's disease amyloid-β peptide amyloid plaques anti-Aβ IgG antibody competition assay ultrahigh-resolution imaging

Introduction

Alzheimer's disease (AD) is the primary neurodegenerative disorder causing dementia. Neuropathological hallmarks of AD are characterized by the abnormal aggregation of amyloid-β peptides (Aβ) as amyloid plaques (APs) and of hyperphosphorylated tau as neurofibrillary tangles (NFTs). The preclinical stage of AD is extensive and is initiated 15 to 20 years prior to the emergence of clinical symptoms detected by conventional examination.^1–3 The asymptomatic preclinical phase of AD is heterogenous, while APs and NFTs are also observed in older people with no cognitive impairment or mild cognitive impairment. For these reasons it is difficult to diagnose patients in the early stages of AD.^4–6

APs are the hallmarks of AD and are mainly composed by aggregated Aβ. Several groups have demonstrated the deposition of administered synthetic Aβ to APs and the saturability of the binding.^7–10 Considering saturable binding, binding proteins (BPs) for the applied Aβ may be present in APs. Furthermore, if the applied Aβ is aggregated into APs nonspecifically, the binding of the applied Aβ to APs would not be saturable, and APs would also be larger after applied Aβ.

On the other hand, anti-immunoglobulin G (IgG) antibodies reportedly labeled APs,^11–13 and were also detected as anti-Aβ antibodies in cerebrospinal fluid (CSF) from AD patients, where they may function as autoantibodies to prevent the development of AD.^14–19

Considering the presence of BPs for applied Aβ and the localization of IgG in APs, we hypothesized that anti-Aβ IgG antibodies (anti-AβAbs) are localized in APs as BPs. To demonstrate specific and direct binding of Aβ to anti-AβAbs in APs, we developed a tissue competition assay utilizing the competitiveness of ligand specificity to BPs.

With this assay, we for the first time determined the binding specificity of applied biotinylated Aβ₄₂ peptide (Bio42) to APs in addition to the previously reported localization of anti-IgG antibodies in APs in brain tissue sections. We then showed the colocalization of Bio42 and anti-IgG antibodies in APs of AD brain sections by dual immunofluorescent labeling with an ultrahigh-resolution imaging system. Finally, we employed dot blot and immunoprecipitation-western blotting (IP-WB) to demonstrate the direct binding of Bio42 to anti-IgG antibodies in AD brain tissue lysates. We concluded that the anti-IgG antibodies bound to Bio42 is anti-AβAbs.

Methods

Antibodies

The following well-characterized antibodies were used at the appropriate concentrations for immunohistochemical, immunofluorescence staining, or WB analyses: biotinylated anti-human IgG antibody (Vector Laboratories), anti-biotin antibody (Jackson ImmunoResearch), goat anti-human IgG antibody (Merck). The widely used and well-established antibody 6E10 directed at amino acid residues 3–8 within the N-terminus of human Aβ was employed for WB analyses (BioLegend).

Human brain tissue

We examined 7 biopsy specimens from patients with APs for the tissue competitive assay (age, 74–84 years old). Information on each specimen is described in Supplemental Table 1. Among the patients with APs, two were clinically diagnosed with AD (Patient nos. 1, 2), three were diagnosed with or suspected of having dementia without AD (Patient nos. 3–5), and two were not diagnosed with either AD or dementia (Patient nos. 6–7). Brain tissues were embedded in paraffin and cut into 4-μm-thick sections for the tissue competition assay, absorption assay, and immunofluorescence evaluation.

For dot blot and IP-WB methods, an autopsy specimen from a 92-year-old female patient with AD and systemic ischemia was examined. Brain tissue was lysed in sarkosyl detergent buffer, and the concentration was adjusted to 10 mg/ml with a BCA assay kit (Thermo Fisher Scientific). The use of brain samples was approved by the ethics committee of Tokyo Medical University (approval number: T2020-0230).

Tissue competition assays

To identify the specific BPs for biotinylated synthetic Aβ₄₂ peptide (Bio42) (ANASPEC), a tissue competition assay was performed with nonbiotinylated synthetic Aβ₄₂ peptide (Pep42) (ANASPEC) and nonbiotinylated synthetic Aβ₄₀ peptide (Pep40) (ANASPEC), as depicted in Figure 3.

Figure 1.

Labeling of amyloid plaques (APs) by biotinylated synthetic Aβ₄₂ peptide (Bio42) visualized by diaminobenzidine (DAB). (A-D) Brain sections including APs and cerebral amyloid angiopathy (CAA) were incubated with 0.1 μg/ml, 1 μg/ml, 10 μg/ml, and 100 μg/ml of Bio42 and stained by DAB. Three representative arteries are marked as landmarks (asterisks) that appear in all serial sections. (A) APs and artery walls were labeled clearly with 0.1 μg/ml of applied Bio42. (B) With 1 μg/ml of applied Bio42 the same APs and artery walls were labeled more densely with higher background. In addition, a larger number of APs were observed (arrowheads). (C) In contrast, with 10 μg/ml of applied Bio42 the labelling density was markedly decreased. (D) Moreover, the labeling was absent with 100 μg/ml of applied Bio42. There is an optimal concentration of binding for applied Bio42 to APs (scale bar, 100 μm).

In brief, each peptide was dissolved in a small volume of 1.0% NH₄OH as solvent, followed by 2% dimethyl sulfoxide (DMSO) to a concentration of 100 μg/ml. As a positive control, 15 μl of Bio42 was dissolved in 1485 μl of blocking buffer (Buffer F), resulting in a total volume of 1500 μl, 1 μg/ml, as Buffer A for Figure 2A. Twenty microliters of Pep42 (100 μg/ml) and 2 μl of Bio42 (100 μg/ml) were mixed in 178 μl of blocking buffer (Buffer F) and 1% bovine serum albumin (BSA) in phosphate-buffered physiological saline (PBS) as Buffer E. The total volume of Buffer E for Figure 2E was 200 μl. Next, to produce Buffer D, 20 μl of Buffer E was dissolved in 180 μl of Buffer A. For Buffer C, 20 μl of Buffer D and 180 μl of Buffer A were combined, and for Buffer B, 20 μl of Buffer C and 180 μl of Buffer A were combined. A total of 0.2 μg of Bio42 was thus continuously contained in 200 μl of blocking buffer, and the ratio of Pep42 was altered by the addition of Buffer A to each other buffer. The details for preparing each buffer were described in Supplemental Table 2.

Figure 2.

Specific binding of applied Bio42 to APs demonstrated by a tissue competition assay (A-I) and schematic summary of the results (J, K). (A) Labeling of APs (arrowheads) with Bio42 in a brain tissue from a dementia and CAA patient is presented. Binding of Bio42 to APs and artery walls (asterisks) was specific to Aβ₄₂, as coincubation with non-biotinylated Aβ₄₂ peptide (Pep42) progressively attenuated the binding in dose-dependent manner (Pep42/Bio42 ratio: 0.01 for (B); 0.1 for (C); 1 for (D); and 10 for (E)). (F) No labeling was observed without Bio42 as a negative control. In contrast, coincubation with non-biotinylated Aβ₄₀ peptide (Pep40) had little effect on Bio42 binding (Pep40/Bio42 ratio: 0.1 for (G); 1 for (H); and 10 for (I)), unlike Pep42 (scale bar, 100 μm). A fixed amount of Bio42 (1 μg/ml) was coincubated stepwise with Pep42 (J) or Pep40 (K) in various ratios. The corresponding AP labeling results shown in (A-E) with Pep42 and in (G-I) with Pep40 are summarized in tables. AP labeling with Bio42 was dense in the absence of Pep42 but gradually became fainter as the Pep42/Bio42 ratio increased. Given that Bio42 and Pep42 specifically bind to the BP and that Pep40 is noncompetitive, dense AP labeling is expected to be invariably preserved, regardless of the Pep40/Bio42 ratio.

For immunohistochemical labeling, brain sections were deparaffinized, and endogenous peroxidase was blocked. Antigenicity was retrieved by autoclave treatment at 121°C in antigen retrieval buffer (Nichirei Biosciences Inc.) for 30 min. The sections were treated with 10% BSA in PBS for blocking, and then endogenous avidin/biotin was also blocked (Vector Laboratories). Brain sections were incubated with the above-described buffers at 4°C overnight. After subsequent incubation with streptavidin biotinylated horseradish peroxidase (HRP) complex (Vector Laboratories), development was performed with DAB.

Absorption assays

To demonstrate the specific labeling by biotinylated anti-IgG antibody, absorption assays were performed. The blocking buffer (1% BSA in PBS-Tween) including biotinylated anti-human IgG antibody (1:1,000, Vector Laboratories) was incubated with or without native human IgG protein (Abcam) with respective weight ratios from 1/100,000 to 1/100 compared to biotinylated IgG antibody.

For immunohistochemistry, brain sections were deparaffinized and subjected to pretreatment with 100% formic acid for 5 min at room temperature (RT). Endogenous peroxidase was blocked by 1% hydrogen peroxide in PBS for 30 min at RT. The sections were then treated with 1% BSA with PBS-Tween and incubated with the biotinylated anti-IgG antibody with or without native IgG protein for 2 h at RT. The brain sections were next incubated with streptavidin biotinylated HRP complex and stained by DAB in the presence of imidazole and nickel ammonium chloride.

Immunofluorescence analyses

For dual immunofluorescent labeling for microscopy and STED imaging, the brain sections were deparaffinized, retrieved by 0.1% trypsin, and treated with 1% BSA. For dual labeling, the sections were incubated with Bio42 (1:60) and anti-IgG antibody (1:150), followed by exposure to fluorescence-labeled streptavidin (green) and anti-goat secondary antibody (red) (1:100, 1:60; Molecular Probes), respectively.

Ultrahigh-resolution imaging with a confocal microscopy-equipped STED system

Prior to the observation with the confocal microscopy-equipped STED system, the colocalization of Bio42 and anti-IgG antibody was preliminarily observed by fluorescence microscopy (KEYENCE BZ-X810) in six different brain regions from seven brain specimens (n = 7). Five to seven areas were observed in each brain section. Confocal and ultrahigh-resolution imaging with STED was performed with a Leica TCS SP8 STED 3X (Leica Microsystems) equipped with a HCPLAPO100x/1.40 Oil STED WHITE objective. For two-color STED imaging, the fluorophores Alexa Fluor 488 streptavidin (1:100, Molecular Probes) and Alexa Fluor 555 goat anti-rabbit IgG H&L (1:60, Abcam) were excited by a pulsed white light laser with lines at 488 and 552 nm, respectively. The spectral detector window was set from 495–550 nm. The STED laser at 660 nm was used for both imaging channels. The 660-nm STED laser was set to 50% for Alexa Fluor 488 (green) and 20% for Alexa Fluor 555 (red), and hybrid detectors were used. The channels were recorded sequentially with a pixel size of 11 nm at line scan speeds of 400 Hz, and each acquisition was performed 8 times on average per line. Finally, these settings provided a set of images 2048 × 2048 effective pixels in size. Acquired images were adjusted using the Leica Application Suite X (LAS X) software program (Leica Microsystems).

Dot blots

AD brain tissue (2.5 µl) lysed in sarkosyl detergent buffer (10 mg/ml) was applied to a nitrocellulose membrane (Thermo Fisher Scientific) and air dried. After brief activation in 10% methanol in Tris-buffered saline (TBS), the membrane was boiled in PBS to enhance antigenicity. The membranes were then blocked with 5% skim milk in TBS for 60 min at RT, followed by overnight incubation with Bio42 (1:600), without Bio42, or with anti-IgG antibody (1:15, Merck). The membranes with and without Bio42 were washed in TBS with 0.05% Tween 20 (TBS-T) and then incubated with 1:5000 anti-biotin antibody (Jackson ImmunoResearch) for 90 min at RT. HRP-conjugated secondary antibody against mouse (Nichirei Biosciences Inc.) for anti-biotin antibody or against goat (Nichirei Biosciences Inc.) for anti-IgG antibody was applied to the relevant membrane for 30 min at RT followed by DAB development.

IP-WB

Pull-down of the biotinylated peptide Bio42 by streptavidin magnetic beads (Thermo Fisher Scientific) and WB analysis were performed as depicted in Figure 5B.

Figure 3.

Schematic summary and Bio42 labeling of APs in the tissue competition assay. (A) The specificity of a ligand protein to the binding site in the binding protein (BP) by competitiveness is illustrated. A ligand protein (Aβ₄₂) with biotin (: Bio42) is constitutively bound to the binding site () of the BP (A-1), but with the appearance of another ligand protein without biotin as a competitive protein (: Pep42), Bio42 was no longer invariably bound to the binding site by competitiveness (A-2). When there was more Pep42 than Bio42, the binding was inhibited entirely () (A-3); however, this binding was not affected () by the presence of noncompetitive protein (Aβ₄₀) without biotin (: Pep40) (A-4). (B) To demonstrate that the binding of applied Aβ₄₂ to APs is specific and not nonspecific aggregation, we performed a tissue competitive assay as diagrammed. Bio42 () were probed to brain tissue with amyloid plaques (APs) (), and the predictive labeling of APs with Bio42 by DAB staining was depicted inside of APs as the shape (). Without Pep42, Bio42 binds directly to BPs (anti-Aβ IgG antibody) () on APs, with dense labeling () (B-1). Co-incubation with competitive Pep42 partially reduces labeling () (B-2). Excess amount of Pep42 completely abolishes labeling () (B-3), while excess of non-competitive Pep40 preserves labeling () (B-4). If Bio42 aggregates nonspecifically with Aβ₄₂ containing β-pleated sheets in AP, the AP labeling would remain dense (B-5), or the APs would enlarge in size (B-6). However, neither of these occurs in the presence of excess competitive Pep42 (: B-5) nor Bio42 (: B-6).

For immunoprecipitation, Bio42 was incubated with streptavidin beads in RIPA lysis buffer (Thermo Fisher Scientific) for 60 min in a cold room. Streptavidin magnet beads and 0.5 μl of brain tissue lysed in detergent buffer (10 mg/ml) were then incubated in RIPA buffer containing a protease inhibitor cocktail (Thermo Fisher Scientific) for 60 min in a cold room to remove nonspecific proteins bound to the beads. The purified AD brain lysis buffer was next incubated with beads absorbing Bio42 and rotated in a cold room overnight. The beads were then washed and collected as a pellet by magnet.

For the WB analysis, the pellet was eluted by boiling in SDS buffer (Thermo Fisher Scientific) and separated using a 10–20% Tricine gel (Thermo Fisher Scientific). The proteins were then transferred to a nitrocellulose membrane and blotted with anti-IgG (1:15), 6E10 (1:1000), and anti-biotin (1:500) antibodies, followed by incubation with secondary HRP-conjugated antibodies and visualization with enhanced chemiluminescence (GE Healthcare).

Results

APs were labeled by biotinylated synthetic Aβ₄₂ peptide (Bio42) and were visualized by diaminobenzidine (DAB) after amplification with avidin-biotin-peroxidase method. The brain tissue was obtained from an 83-year-old patient with dementia and cerebral amyloid angiopathy (CAA) as a representative case (Supplemental Table 1, No. 3). APs and artery walls with β-amyloid deposition in brain tissue were incubated with 0.1 μg/ml, 1 μg/ml, 10 μg/ml, and 100 μg/ml at 4°C overnight, respectively (Figure 1). APs and artery walls were labeled with 0.1 μg/ml of applied Bio42 (Figure 1A). Moreover, with 1 μg/ml of applied Bio42 the same APs and artery walls were labeled more densely although with higher background (Figure 1B). Additionally, a larger number of APs were detected (Figure 1B, arrowheads). However, with the higher concentration of applied Bio42 of 10 μg/ml, the labelling density was markedly decreased (Figure 1C). The labeling was completely abolished with 100 μg/ml of applied Bio42 (Figure 1D). Therefore, the optimal binding condition for applied Bio42 to APs was identified with 1 μg/ml as the best concentration of Bio42 for binding, suggesting the presence of specific binding proteins (BPs) to Bio42 in APs as some of the aggregated Bio42 at higher concentration could not bind to APs. Bio42 was not aggregated in buffers at 4°C overnight for incubation under the lower concentration (Supplemental Figure 1).

Figure 4.

Colocalization of Bio42 and anti-AβAb in APs by ultrahigh-resolution imaging. (A) Bio42 and anti-IgG antibody were coincubated with brain tissue from an AD and CAA patient and colocalization of Bio42 and anti-IgG antibody in the APs of was confirmed by double immunofluorescent labeling on confocal microscopy. Bio42 was localized mainly to the amyloid core of the AP and partially to the dystrophic neurites (Bio42, green). Anti-IgG antibody was localized to the core and the whole surrounding dystrophic neurites (IgG, red). Bio42 and anti-IgG antibody (anti-Aβ IgG antibody: anti-AβAb) were colocalized to the core and part of the dystrophic neurites (Merge, yellow). The yellow color was poorly defined, and the outline of the respective color was unclear. (B) In contrast to the observation by confocal microscopy, the outline of the same AP was detected much more clearly and sharply with ultrahigh-resolution imaging using a STED system. Fluorescent labeling of Bio42 (Bio42, green) and anti-IgG antibody (IgG, red) were detected more distinctly separated with the STED system than without it. In the merged image (Merge), the green and red color profiles were separated clearly, especially on the amyloid core; however, many sites of colocalization, which appeared as yellowish dots, were observed, also with the STED system (scale bar, 5 μm). (C) With higher magnification of another AP (inset, square at right upper quarter), the colocalization of Bio42 and anti-IgG antibody (anti-AβAb) was also observed as yellowish particles, and we thus morphologically confirm the binding of Bio42 and anti-IgG antibody (anti-AβAb) by rigorous conditions with the STED system (scale bar, 5 μm).

To identify the presence of specific BPs to the applied Aβ₄₂ in APs, we developed a tissue competition assay utilizing the binding specificity of a ligand to a BP. All the cases of patients for whom we examined brain specimens for the tissue competition assay (Supplemental Table 1) and protocols for preparing each labeling buffer (Supplemental Table 2) are described in the tables. Bio42 was shown to be bound to APs and labeled by DAB in brain tissue from an 83-year-old patient with dementia and CAA, as a representative case (Supplemental Table 1, No. 3) (Figure 2A, arrowheads). Further, the artery walls were also densely labeled with Bio42 (Figure 2A, asterisks). Coincubation of Bio42 with a small amount of nonbiotinylated synthetic Aβ₄₂ peptide (Pep42) (Pep42/Bio42 ratio: 0.01) slightly decreased the number and density of the Bio42 labeled APs and artery walls (Figure 2B). Coincubation with more Pep42 (Pep42/Bio42 ratio: 0.1) resulted in more of a decrease in labeled APs (Figure 2C). Coincubation of Bio42 with the same amount of Pep42 (Pep42/Bio42 ratio: 1) resulted in a significant decrease in the number and density of AP labeling (Figure 2D), and the labeling of the artery walls decreased markedly (Figure 2D, asterisks). Coincubation of Bio42 with 10 times the amount of Pep42 (Pep42/Bio42 ratio: 10) resulted in most labeling of APs and artery walls being abolished (Figure 2E). As a negative control, no labeling was observed without any Bio42 (Figure 2F). In contrast to Pep42, the number of APs labeled with Bio42 was preserved by coincubation with nonbiotinylated synthetic Aβ₄₀ peptide (Pep40) (Pep40/Bio42 ratios: 0.1 and 1; Figure 2G and H). The labeling density of APs and artery walls was also preserved. However, coincubation of Bio42 with 10 times the amount of Pep40 (Pep40/Bio42 ratio: 10) resulted in a decrease in the density and number of labeling (Figure 2I). Similar results were observed in another representative case of brain sections from a 78-year-old female patient clinically diagnosed with cerebral hemorrhage and dementia (Supplemental Table 1, No.4) as presented in Supplemental Figure 2.

We also prepared schematic representative results of AP labeling with Bio42 coincubated with Pep42 (Figure 2J) and Pep40 (Figure 2K). A fixed amount of Bio42, 1 μg/ml, was coincubated stepwise with various amounts of Pep42. The AP labeling coinciding with the respective Pep42/Bio42 ratio in Figure 2A-E is depicted at the bottom of the diagram (Figure 2J). AP labeling with Bio42 was dense without Pep42, but grew gradually fainter as the Pep42/Bio42 ratio increased. Since Pep40 is a noncompetitive protein, dense AP labeling is preserved (Figure 2G, H), regardless of the Pep40/Bio42 ratio (Figure 2K). The density and number of labeling were slightly attenuated when Bio42 was coincubated with a larger amount of Pep40 (Pep40/Bio42 ratio: 10) (Figure 2I).

The model of competitiveness is illustrated in Figure 3A. In general, a ligand usually binds to the binding site of the BP (Figure 3A-1), but when a competitive protein appears, the ligand is no longer invariably bound to the BP due to competitiveness (Figure 3A-2), as the ligand and competitive protein compete for the binding site. When there is a larger amount of competitive protein than the ligand, the binding of the ligand to the BP is inhibited, and the binding site thus becomes occupied by the competitive protein (Figure 3A-3). However, the binding is not affected by a noncompetitive protein (Figure 3A-4).

Since it was previously reported that applied Aβ accumulates in APs and that this is saturable,^7–10 BPs for Aβ may be present in APs. Bio42 was therefore applied as a ligand to brain tissue containing APs, and the labeling of APs by DAB staining was performed (Figure 3B). In the absence of competitive proteins, the applied Bio42 is expected to bind directly to the BPs on the AP, which is labeled densely (Figure 3B-1). When Bio42 is coincubated with competitive Pep42, the binding of Bio42 to the BPs will be partly inhibited by Pep42, resulting in a reduction in the AP labeling density (Figure 3B-2). Coincubation with a larger amount of Pep42 than Bio42 can thus completely inhibit the binding of Bio42 to the BPs, i.e., cause the disappearance of Bio42 labeling of the AP (Figure 3B-3). In contrast, AP labeling is expected to remain densely preserved when coincubated with a larger amount of noncompetitive Pep40 (Figure 3B-4). If Bio42 were to aggregate nonspecifically with Aβ₄₂ in APs containing β-pleated sheets, the binding of Bio42 to APs would not be saturable, and the AP labelling would remain dense even in the presence of a large excess of Pep42 in the tissue competition assay (Figure 3B-5). Alternatively, APs might increase in size due to an excessive amount of Bio42 aggregated with β-pleated Aβ₄₂ in APs (Figure 3B-6). However, neither of these outcomes was observed in Figures 1 and 2.

Figure 5.

Direct binding of Bio42 and anti-aβAb by dot blots and IP-WB. (A) Dot blots of AD brain homogenate were duplicated, and 25 μg of total protein was spotted per dot on a PVDF membrane. Bio42 was incubated with dots of the brain homogenate on the membrane at 4°C overnight (+Bio42, right side) (1). The membrane was then immunoblotted with anti-biotin antibody labeled on the left side of the membrane (Bio, left side) (1, 2) and stained by DAB. The dots of the brain homogenate appeared as larger circles, with Bio42 added as smaller circles (1). The amount of endogenous biotin in AD brain homogenate was below the limit of detection and can therefore be ignored (2). The dots of the brain homogenate immunoblotted with anti-IgG antibody, the left side of the membrane (IgG, left side), also appeared as a circle (3). The area around the dots with infiltration of brain homogenate was also stained in a poorly defined way (3). (B) AD brain homogenate was immunoprecipitated by Bio42. Bio42 was added to immunoprecipitation (IP) buffer containing streptavidin-coated magnetic beads (1). The biotin compartment of Bio42 is bound to streptavidin by a biotin-streptavidin reaction (2). AD brain homogenate was then incubated with Bio42 and the magnetic bead complexes (3). Specific proteins contained in the AD brain homogenate bind to Bio42 (4). The Bio42 and magnetic bead complex, including the specific binding protein (anti-Aβ IgG antibody: anti-AβAb), was collected by magnetic force (5) and then decomposed by detergent buffer for Western blotting (WB). To confirm the specific protein (asterisk: *) bound to Bio42 and the magnetic bead complex in decomposed total proteins, the detergent buffer was immunoblotted by anti-IgG antibody following WB (6). C The detergent buffer containing the decomposed proteins illustrated in (B-6) was loaded in lane 1 for WB. A total of 100 μg of AD brain homogenate was loaded in lane 2. Lanes 1 and 2 were then immunoblotted with an anti-IgG antibody (IgG, bottom). Bio42 was also immunoblotted with the anti-Aβ antibody 6E10 (Aβ, bottom) and anti-biotin antibody (Bio, bottom) in lanes 3 and 4, respectively. With short exposure, only the IgG band in the AD brain homogenate was visible; however, with a longer exposure, a band equivalent to the molecular weight of IgG protein was also visible in lane 1’ (*). The specific protein detected by anti-IgG antibody is indicated by the same asterisk (*) in (B-6). The molecular weight is the same as that of the IgG protein in the AD brain homogenate in lane 2’ with a longer exposure.

As previously demonstrated,^11–13 localization of IgG in APs was observed by anti-IgG antibody with DAB-nickel labeling (Supplemental Figure 3A). In addition, the artery walls and serum were also labeled. To confirm the specificity of labeling, native human IgG protein was coincubated with anti-IgG antibody. Coincubation with 1/100,000 and 1/1000 native human IgG protein decreased the density of labeling, while that with 1/100 abolished the labeling completely (Supplemental Figure 3B-D).

Next, dual immunofluorescent labelling of Bio42 and anti-AβAbs was performed on seven brain specimens (Supplemental Table 1, No. 1–7). The colocalization of Bio42 (Bio42, green) and anti-AβAbs (IgG, red) in APs was confirmed by fluorescence microscopy (Supplemental Figure 4). To rigorously observe the colocalization of Bio42 and anti-AβAbs in APs, ultrahigh-resolution imaging was performed with a stimulated emission depletion (STED) system for dual immunofluorescent labeling. Bio42 and anti-IgG antibody were coincubated with brain tissue from a 77-year-old patient with AD and CAA (Supplemental Table 1, No. 2). On confocal microscopy, localization of Bio42 is evident mainly in the amyloid core and partially in the dystrophic neurites of an AP (Figure 4A, Bio42, green). Anti-IgG antibody was detected both in the core and in the whole surrounding dystrophic neurites (Figure 4A, IgG, red). Bio42 and anti-IgG antibody, anti-Aβ IgG antibody (anti-AβAb), colocalized remarkably in the core, appearing yellowish with a poorly defined outline on confocal microscopy (Figure 4A, Merge, yellow), and partially also in the dystrophic neurites. In contrast, observing the same AP using ultrahigh-resolution imaging with this STED system showed that the profiles of the labeled color were sharpened as dots (Figure 4B, Bio42 and IgG). Most of the Bio42 and anti-AβAb were colocalized in the core of the AP and observed as yellowish dots; however, independent green and red dots were observed in the core due to the STED effect (Figure 4B, Merge). On observing the top right quarter of another AP with higher magnification (Figure 4C, Bio42, square of inset), colocalization was also observed as yellowish particles, suggesting binding of Bio42 and anti-AβAbs (Figure 4C, Merge).

Finally, the presence of anti-AβAbs in AD brain tissue was investigated by dot blots (Figure 5A). AD brain homogenate was spotted on a nitrocellulose membrane and duplicated, and then Bio42 was applied to some spots (+Bio42, Figure 5A-1) and not to other spots. The spots with Bio42 were detected by immunoblotting with anti-biotin antibody and developed by DAB (Figure 5A-1), while the spots without Bio42 were not (Figure 5A-2). The dots of the brain homogenate appeared as larger circles, with Bio42 added as smaller circles (Figure 5A-1). The endogenous biotin level in the brain homogenate was too low to be detected (Figure 5A-2). The spots of the AD brain homogenate were also immunoblotted with an anti-IgG antibody (Figure 5A-3). The anti-IgG antibody is extremely sensitive for detecting permeated IgG around spots. These results suggest that Bio42 bind to anti-AβAbs in AD brain tissue.

Furthermore, to demonstrate the direct binding of Bio42 to anti-AβAbs, IP-WB was performed, with the procedure shown in Figure 5B. In summary, Bio42 were incubated with AD brain homogenate (Figure 5B-1, 2, 3) and then immunoprecipitated by magnetic beads coated with streptavidin (Figure 5B-4, 5). The protein bound to Bio42 was immunoblotted with an anti-IgG antibody (Figure 5B-6, asterisk). Although human native IgG in AD brain tissue was detected by anti-IgG antibody (Figure 5C, lane 2), the protein bound to Bio42 was not detected with short exposure WB (Figure 5C, lane 1). At the same time, Bio42 was immunoblotted with anti-Aβ antibody (6E10) (Figure 5C, lane 3) and anti-biotin antibody (Figure 5C, lane 4), and as predicted, bands equivalent to the molecular weight of Aβ appeared. However, with longer exposure, proteins with the same molecular weight as human native IgG, detected with short exposure (Figure 5C, lane 2), were observed (Figure 5C, lane 1’, asterisk), consistent with the conclusion of direct binding of Bio42 to anti-AβAbs. Other proteins of native human IgG in the brain homogenate were also observed with longer exposure (Figure 5C, lane 2’). Original images of gel membranes were also presented (Supplemental Figure 5A, B).

Discussion

In the present study, we demonstrated that Aβ₄₂ applied to AD brain sections specifically bound to APs and did not nonspecifically aggregate with Aβ in APs based on a tissue competitive assay. In addition, we also observed the specific binding of applied Aβ₄₂ to artery walls with amyloid deposition. Furthermore, we demonstrated that Aβ₄₂ applied to AD brain tissue colocalized in APs according to ultrahigh-resolution imaging and directly bound to anti-AβAbs by IP-WB.

These results are consistent with previous reports that the binding of applied synthetic Aβ to APs is saturable^7–10 and that anti-IgG antibodies show preferential affinity to APs.^11–13 Therefore, it is probable that one of the BPs for Aβ₄₂ is anti-AβAb. It is noteworthy that the density and number of AP and artery wall labeling by Bio42 were slightly attenuated when Bio42 was coincubated with a larger amount of Pep40. This result may suggest that the antibodies bound to Bio42 are not only against Aβ₄₂ but also partially against Aβ₄₀. The antibodies may therefore be against Aβ₄₂ and Aβ₄₀ compound proteins.

Aβ compound proteins and anti-AβAbs form complexes as Aβ-anti-AβAb complexes in biological fluids and may ultimately aggregate into APs. The presence and formation of these complexes might distort the levels of Aβ covered by the anti-AβAbs or sequester Aβ from the biological fluids to APs. The Aβ-anti-AβAb complexes may be involved in the apparent decrease in Aβ₄₂ levels in the CSF preceding the onset of cognitive impairment related to AD.^20–24 We also cannot exclude the possibility that both Aβ₄₂ and Aβ₄₀ bind to unknown proteins in APs with a lower affinity than anti-AβAbs. Another possibility is that applied Bio42 and Pep40, or likewise Bio42 and Pep42, could aggregate together immediately with higher concentration in the buffer as soon as prepared before they reach the APs.

Since a reduction in APs is observed with the administration of immunotherapy, immunotherapies have been approved for clinical use^25,26 and repeatedly have been in clinical trials.^27–29 However, even if the numbers of APs are reduced, the large amounts of preexisting Aβ-anti-AβAb complexes in APs might be redistributed in biological fluids without detection. A previous report found that serum and CSF levels of Aβ-anti-AβAb complexes were negatively correlated with cognitive performance.³⁰ Furthermore, despite the clearance of APs by immunotherapy, the rate of CAA as a side effect appears increased in immunized patients.^31–34 It was previously demonstrated that immune complexes accumulated within the artery walls, interfering with perivascular drainage and the elimination of soluble Aβ.³¹ It was also reported that microglia and macrophages are activated by the binding of the Fc receptor to IgG immune complexes,^35–37 which initiates an inflammatory response close to the blood vessels as meningoencephalitis.^31,38 Our observation of Aβ₄₂ and anti-AβAbs in the artery walls suggests the accumulation of immune complexes at these sites.

Thus, anti-AβAbs could cover Aβ or sequester Aβ from biological fluids to APs, such as primitive plaques, as we showed in our Figure 4, and also could distort the amount of Aβ in body fluids. Since there is a possibility that many other proteins in body fluids are absorbed into APs, biomarkers not associated with primitive plaques might be available for the precise evaluation of proteins in body fluids for early diagnosis of AD.

However, all our experimental results were obtained under limited in vitro conditions as immunolabelling and immunoblotting. Specifically, Bio42 was administered exogenously to brain tissues with APs under static conditions. This method differs from the administration of therapeutic antibodies used for clinical therapy. To further elucidate the relationship between Aβ and anti-AβAbs, it will be essential to observe their interaction under dynamic in vivo conditions over time using a larger number of samples. Recently, the use of human tissues in experiments has become increasingly difficult owing to the restrictions imposed by institutional review board (IRB) regulations. Therefore, our findings are necessary to validate using AD transgenic mouse models in future studies. Furthermore, treatment with immunotherapeutic anti-AβAbs or Aβ should be applied in these models to further investigate the relationship between anti-AβAbs and Aβ in body fluids, in the brain parenchyma as AP formation, and in artery walls as immune complexes.

In the past, the binding proteins for the administered Aβ have not been sufficiently considered. Therefore, considering the presence of Aβ-anti AβAb complexes in APs and artery walls with amyloid deposition, they may aid in the development of novel strategies for establishing effective biomarkers and safer immunotherapies for AD in future trials.

Supplemental Material

sj-docx-1-alz-10.1177_13872877251389631 - Supplemental material for One of the binding proteins for administered amyloid-β appears to be anti-Aβ IgG antibody in amyloid plaques

Supplemental material, sj-docx-1-alz-10.1177_13872877251389631 for One of the binding proteins for administered amyloid-β appears to be anti-Aβ IgG antibody in amyloid plaques by Reisuke H Takahashi, Mayumi Yokotsuka, Ayako Nakamura, Toshitaka Nagao, Gunnar K Gouras and Toshiki Uchihara in Journal of Alzheimer's Disease

Footnotes

Acknowledgements

We thank Dr Ebashi for providing AD brain tissue for the dot blot and IP-WB analyses to the Tokyo Metropolitan Institute of Medical Science, to which R.H.T., A.N., and T.U. previously belonged. We also thank Ms. Kato from Leica Microsystems for offering instruction and details concerning the manipulation of a Leica TCS SP8 STED 3X equipped with an HCPLAPO100x/1.40 Oil STED WHITE objective. Images were acquired by confocal microscopy and analyzed with the Leica Application Suite X (LAS X) software program. Images were adjusted using LAS X following Ms. Kato's advice.

ORCID iDs

Toshitaka Nagao

Toshiki Uchihara

Ethical considerations

This research received approval from the Institutional Review Board of Tokyo Medical University (approval number: T2020-0230) following the ethical standards outlined in the Declaration of Helsinki. Consent to participate in this study is officially announced in the web site of Tokyo Medical University Hospital.

Consent to participate

All materials obtained for brain surgery and pathological diagnosis of patients were anonymized and retrospectively reviewed. Extra human brain tissue was not obtained for this study.

Author contribution(s)

Reisuke H Takahashi: Conceptualization; Investigation; Project administration; Resources; Supervision; Writing – original draft; Writing – review & editing.

Mayumi Yokotsuka: Data curation; Methodology.

Ayako Nakamura: Methodology.

Toshitaka Nagao: Funding acquisition; Resources.

Gunnar K Gouras: Formal analysis; Supervision; Writing – review & editing.

Toshiki Uchihara: Conceptualization; Formal analysis; Project administration; Resources; Supervision; Writing – review & editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Support for this study was provided by Grants-in-Aid for Scientific Research C (Grant No. 23K06811).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability statement

Requests for resources and information should be direct to R.H.T. (takaharh@tokyo-med.ac.jp). and will be reviewed by the authors as soon as possible. All individual-level data are available under a manipulated condition for other researchers. Requests for access to the data will be granted for all research compatible with the consent provided by the patients or their families, i.e., any research in terms of health and disease that does not involve identifying the patients in any manner. Detailed information with anonymization can be provided by request from any qualified investigator for the purpose of recapitulating the findings presented in the article.

Supplemental material

Supplemental material for this article is available online.

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