High-molecular weight Aβ oligomers and protofibrils are the predominant Aβ species in the native soluble protein fraction of the AD brain

Abstract

Alzheimer’s disease (AD) is characterized by the aggregation and deposition of amyloid β protein (Aβ) in the brain. Soluble Aβ oligomers are thought to be toxic. To investigate the predominant species of Aβ protein that may play a role in AD pathogenesis, we performed biochemical analysis of AD and control brains. Sucrose buffer-soluble brain lysates were characterized in native form using blue native (BN)-PAGE and also in denatured form using SDS-PAGE followed by Western blot analysis. BN-PAGE analysis revealed a high-molecular weight smear (>1000 kD) of Aβ₄₂-positive material in the AD brain, whereas low-molecular weight and monomeric Aβ species were not detected. SDS-PAGE analysis, on the other hand, allowed the detection of prominent Aβ monomer and dimer bands in AD cases but not in controls. Immunoelectron microscopy of immunoprecipitated oligomers and protofibrils/fibrils showed spherical and protofibrillar Aβ-positive material, thereby confirming the presence of high-molecular weight Aβ (hiMWAβ) aggregates in the AD brain. In vitro analysis of synthetic Aβ₄₀- and Aβ₄₂ preparations revealed Aβ fibrils, protofibrils, and hiMWAβ oligomers that were detectable at the electron microscopic level and after BN-PAGE. Further, BN-PAGE analysis exhibited a monomer band and less prominent low-molecular weight Aβ (loMWAβ) oligomers. In contrast, SDS-PAGE showed large amounts of loMWAβ but no hiMWAβ₄₀ and strikingly reduced levels of hiMWAβ₄₂. These results indicate that hiMWAβ aggregates, particularly Aβ₄₂ species, are most prevalent in the soluble fraction of the AD brain. Thus, soluble hiMWAβ aggregates may play an important role in the pathogenesis of AD either independently or as a reservoir for release of loMWAβ oligomers.

Introduction

Alzheimer’s disease (AD) is characterized by the extracellular deposition of amyloid β protein (Aβ) aggregates in the brain [1]. Although high-molecular weight Aβ (hiMWAβ) oligomers, Aβ protofibrils and fibrils, low-molecular weight Aβ (loMWAβ) oligomers, such as dimers, trimers or Aβ*56, have been observed in human AD brain tissue or in mouse models of AD [1–9], it is not entirely clear which Aβ species are the most relevant ones for the development of AD and how these Aβ forms are related to one another in vivo. Some studies have used SDS-PAGE for protein analysis [3–5, 9], which denatures and dissociates proteins into individual polypeptides before determining its molecular weight. By contrast, others have performed only dot blot analysis [6]. Currently, only size exclusion chromatography has been used to study oligomers in non-SDS-treated conditions [3, 9]. However, it is unclear whether interactions with the stationary phase may impact the aggregation state of hiMWAβ species. A detailed analysis of the native Aβ aggregates in the AD brain using blue native-PAGE (BN-PAGE) in comparison with SDS-PAGE analysis that focuses on the identification of the above-mentioned forms of Aβ aggregates is still unavailable.

Antibodies and antibody fragments have been developed to detect specific hiMWAβ oligomers (A11) and protofibrillar/fibrillar conformations (B10AP) [2, 6]. These antibodies and antibody fragments allow isolation of oligomers, protofibrils and fibrils from soluble native protein lysates by immunoprecipitation for further protein analysis. Here, we employed these antibodies and BN-PAGE analysis to clarify whether soluble hiMWAβ oligomers and Aβ protofibrils/fibrils or Aβ dimers and other loMWAβ species represent the predominant Aβ aggregates in the native soluble fraction of the AD brain. SDS-PAGE was used to study the effect of protein denaturation on the spectrum of loMWAβ and hiMWAβ species.

Materials and methods

Neuropathology and human sample characterization

A sample including six AD and four control cases was studied (Table 1). All autopsy brains were collected from individuals who died in the University Hospitals of Bonn or Ulm (Germany). All human tissue was obtained and processed in compliance with German federal laws and with university ethics committee approval.

Table 1.

List of autopsy cases studied

Case no.	Diagnosis	Age	Gender	AD type	Braak-NFT stage	Aβ phase	CERAD plaque score
1	Control	60	m	0	0	0	0
2	Control	66	m	0	I	0	0
3	Control	69	f	0	I	0	0
4	Control	71	f	0	I	0	0
5	AD	79	f	1	IV	3	2
6	AD	78	m	1	IV	4	1
7	AD	62	f	1	VI	4	3
8	AD	91	f	2	IV	3	1
9	AD	84	m	2	VI	4	3
10	AD	64	f	2	VI	4	3

Ten autopsy cases (four controls, including two females and two males, age range 60–71 years; mean age ± S.D.: 66.5 ± 4.8 years and six AD cases, including four females and two males, age range 62–91 years; mean age ± S.D.: 76.3 ± 11.33 years) were analysed. The table shows neuropathological diagnosis of AD according to published criteria [12], age in years, gender, AD type [10], the stage of neurofibrillary tangle (Braak-NFT stage) pathology according to Braak et al.[14, 16], the Aβ-phase representing the distribution of Aβ deposits in subfields of the MTL [18] and the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) score for the frequency of neuritic plaques according to Mirra et al.[17]. m: male; f: female; AD: Alzheimer’s disease.

Demented as well as non-demented patients were examined 1–4 weeks prior to death using standardized protocols for routine clinical examination, including neurological status, upon admission to hospital. These data were used to determine whether individuals clinically fulfilled the DSM-IV criteria for dementia [11]. AD was diagnosed when dementia was observed and when the degree of AD-related neuropathology indicated at least a moderate likelihood for AD according to internationally acknowledged criteria [12].

After assessment of unfixed tissue from one hemisphere for biochemical studies, the brains were fixed in a 4% aqueous formaldehyde solution for at least 3 weeks before undergoing neuropathological screening. Presence or absence of gross infarction, haemorrhage, tumour and other findings were recorded. Tissue blocks from the medial temporal lobe (MTL) were excised at the levels of the (i) anterior limit of the dentate gyrus and (ii) lateral geniculate body [13]. These blocks together with tissue blocks from the occipital cortex (Brodmann areas 17–19) were embedded in paraffin. All sections were cut at 10 μm.

Neurofibrillary changes were detected by immunostaining with an antibody directed against abnormal phosphorylated τ protein (AT-8, Pierce, Rockford, IL, USA, 1/1000) [14]. Neuritic plaques were also diagnosed in sections immunostained with this same antibody. The presence of Aβ deposition was assessed using immunohistochemistry with an antibody raised against Aβ_17–24 (4G8 [15], Covance, Emeryville, CA, USA, 1/5000, formic acid pre-treatment).

Diagnosis of the stages in the development of neurofibrillary changes (Braak NFT stage) and the semi-quantitative assessment of neuritic plaques (CERAD score) were performed in accordance with published and recommended criteria [12, 14, 16, 17]. For staging of Aβ pathology, we used a previously published protocol for four phases of β-amyloidosis in the MTL [18]. This hierarchically based procedure facilitates study of the topographic distribution pattern of Aβ deposition in additional brain regions [18, 19]: phase 1 represents Aβ deposition that is restricted to the temporal neocortex. Phase 2 is characterized by the presence of additional Aβ plaques in the entorhinal cortex and/or in the hippocampal subiculum-CA1 region. The third phase is marked by the presence of Aβ plaques in the outer zone of the molecular layer of the fascia dentata, subpial band-like amyloid and/or presubicular ‘lake-like’ amyloid. The existence of further Aβ plaques in the hippocampal sector CA4 and/or the pre-α layer of the entorhinal cortex characterize the fourth and final phase of Aβ deposition in the MTL. Reference pathology for all cases was performed by one and the same neuropathologist (D.R.T.).

Biochemical analysis of human AD and control brains

Fresh frozen human brain tissue from the six AD and four control cases was used to assess the presence and types of native Aβ aggregates in AD and control brains (Table 1). Protein extraction from 30 mg of fresh frozen human occipital (Brodmann areas 17–19) and temporal cortex (Brodmann areas 35 and 36) was carried out in 2 ml of 0.32 M sucrose dissolved in 1 M Tris-buffer (pH 7.4) with a protease and phosphatase inhibitor cocktail (Complete and PhosSTOP, Roche, Mannheim, Germany). The tissue was homogenized as previously described [20]. The homogenate was placed on ice for 30 min., and the supernatant was clarified by centrifuging for 30 min. at 14,000 ×g at 4°C. To avoid the segregation of high-molecular weight proteins from the soluble into the insoluble fraction, a centrifuging speed in excess of 14,000 ×g was not used. The resultant supernatant, i.e. the sucrose-soluble fraction, was aliquoted into appropriate volumes and stored at –80°C until use. Protein amounts were determined using BCA Protein Assay (Bio-Rad, Hercules, CA, USA).

For immunoprecipitation, 200 μl of brain lysate was incubated with 1 μl anti-Aβ_1–17 (6E10, 1 mg/ml; Covance, Dedham, MA, USA), with 20 μl B10AP antibody fragments coupled to alkaline phosphatase ([2], 0.55 mg/ml) or with 1 μl A11 ([6], 1 mg/ml; Millipore, Temecula, CA, USA) antibodies at 4°C for 4 hrs with gentle agitation. A total of 50 μl of protein G Microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) were added to the mixture and incubated overnight at 4°C on a shaking table with gentle agitation. The mixture was then passed through the μColumns which separate the microbeads by retaining them into the column, while the rest of the lysate flows through. After several mild rinsing steps with 1× tris-buffered saline (TBS) buffer (pH 7.4), the microbead-bound proteins were eluted with 1× Lithium dodecyl sulfate (LDS) sample buffer at 95°C (Invitrogen, Carlsbad, CA, USA).

For BN-PAGE of the sucrose fraction, 50 μg of total protein was prepared with 4× NativePAGE sample buffer (Invitrogen) and subjected to native PAGE 4–16% Bis-Tris gel electrophoresis according to the manufacturer’s protocol (Invitrogen). Native-Mark unstained protein standards (Invitrogen) were used as molecular weight markers. The gel was equilibrated in transfer buffer containing 0.2% SDS for 10 min. After protein transfer onto the nitrocellulose membranes (Bio-Rad), the membrane was boiled in phosphate-buffered saline (PBS) buffer in microwave oven for 6 min. Washing buffer and antibody dilution buffer contained 1 M PBS (pH 7.4) with 0.02% Tween (BioRad). A total of 3% non-fat dry milk (Roth, Karlsruhe, Germany) diluted in antibody-dilution buffer was used to block unspecific binding for 1 hr at room temperature.

For SDS-PAGE, sucrose fractions (50 μg total protein) and immunoprecipitation products were electrophoretically resolved in a precast NuPAGE 4–12% Bis-Tris gel system (Invitrogen). The protein load was controlled either by Ponceau S staining or β-actin (C4, 1/1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) immunoblotting. The proteins were transferred to nitrocellulose membranes and the membranes were boiled with PBS for 6 min. followed by blocking with 5% non-fat dry milk (Roth; diluted in antibody-dilution buffer) for 1 hr at room temperature.

For immunodetection of the blotted proteins, the membranes were incubated for 24 hrs at 4°C with the primary antibodies: anti-Aβ₁₋₁₇ (6E10, 1/1000), anti-Aβ₄₂ (MBC-42, [21] 1/500), anti-Aβ₄₀ (MBC-40, [21] 1/1000) and anti-amyloid precursor protein (APP) (22C11, 1/500; Millipore). The 22C11 anti-APP antibody is directed against an N-terminal part of the APP molecule outside the Aβ region [22]. After washing steps, the corresponding secondary antibodies (EIA grade affinity purified goat antimouse/rabbit IgG-HRP, 1/20000; Bio-Rad) were applied for 2 hrs at room temperature. Blots were developed with an enhanced chemiluminescence (ECL) detection system (Supersignal Pico Western system, ThermoScientific-Pierce, Waltham, MA, USA) and illuminated in ECL Hyperfilm (GE Healthcare, Buckinghamshire, UK). Aβ₄₂- and Aβ₄₀ preparations were used as positive and/or negative controls. All BN-PAGE blots were developed with standard chemiluminescence exposure time of 2–5 min. up to maximum exposure times of 2–3 hrs to detect even minimal amounts of Aβ aggregates. For SDS-PAGE blots, exposure time of 2–5 min. was used except when otherwise indicated.

Electron microscopy of immunoprecipitated oligomeric and fibrillar/protofibrillar proteins from human AD and control brains

For electron microscopy, 5 μl of immunoprecipitated and redissolved A11-positive oligomers or B10AP-positive protofibrils/fibrils were placed on formvar-coated grids. After 1 min. incubation, the excess liquid was wiped off and the grid dried. The grid then was treated with Na-Borhydrite (0.1% in water for 1 min.) followed by blocking with 5% bovine serum albumin, 5% normal goat serum and 0.1% cold-washed fish gelatin in 1 M PBS. The grids were incubated with anti-Aβ_1–17 (6E10, 1/50) for 30 min. After washing, the primary antibody was visualized by 15 nm gold-labelled secondary antibodies (1/30, diluted in 1 M PBS; Aurion Immuno Gold Reagents & Accessories, Wageningen, The Netherlands). Then, the grid was post-fixed in 2% glutaraldehyde and block-stained with a 2% aqueous solution of uranyl acetate (Merck, Darmstadt, Germany) for 1 min. followed by five rinsing steps in H₂O₂. The sections were viewed with a Philips EM400T 120KV (Eindhoven, The Netherlands) and with a Zeiss EM10 (Oberkochen, Germany).

Analysis of synthetic Aβ₄₂ and Aβ₄₀ aggregates in native state and after SDS denaturation

To determine whether synthetic Aβ aggregates primarily form loMWAβ- and hiMWAβ aggregates, we dissolved 15 μmol synthetic Aβ₄₀-peptide (Peptides International, Louisville, KY, USA) in 1 ml cell culture medium (Quantum 263; PAA Laboratories, Pasching, Austria) for 30 min. at 4°C [23]. Aβ₄₂-peptide (Bachem, Bubendorf, Switzerland) was also dissolved in cell culture medium (RPMI1640; GIBCO, Invitrogen) [23]. Aggregation was permitted to occur for 4 hrs at 22°C. To identify oligomers, fibrils and protofibrils structurally we used electron microscopy. For this purpose, 5 μl of the Aβ₄₀- and Aβ₄₂ solutions were placed on a formvar-coated grid for 1 min. before wiping off the excess liquid. The protein-coated grids were block-stained with a 2% aqueous solution of uranyl acetate (Merck).

The protein aggregates were also analysed with BN-PAGE and SDS-PAGE as well as subsequent Western blot analysis using the MBC-40 and MBC-42 antibodies to detect Aβ₄₀ and Aβ₄₂, respectively. This experiment was repeated five times.

Results

High-molecular weight Aβ₄₂ aggregates predominate in native protein preparations of the soluble fraction from human brain lysates

BN-PAGE with subsequent Western blot analysis of the soluble fraction of human AD brain lysates revealed a high-molecular weight anti-Aβ₄₂-positive smear >1000 kD in AD cases (Fig. 1A) that was not found in controls. Aβ monomers, dimers, or other loMWAβ species were not observed in AD cases or in controls (Fig. 1A). The high-molecular weight smear was also seen with anti-Aβ_1–17 at the level of stacking gel in AD cases (Fig. 1C) but not with anti-Aβ40 (Fig. 1B). However, the detection of synthetic Aβ₄₀ but not Aβ₄₂ indicated specific antibody function (Fig. 1B). Anti-Aβ_1–17 stained an additional 150–250 kD band (Fig. 1C) that was also observable with anti-APP antibodies, thereby indicating that this band represents APP-containing material (Fig. 1D). The APP-related band was also present in control cases, whereas the high-molecular weight anti-Aβ_1–17 smear was not seen (Fig. 1C). The BN-PAGE blots from brain samples were developed with a chemiluminescence exposure time of 2–3 hrs to detect even very minimal amounts of proteins.

Fig 1.

Western blot analysis of sucrose soluble proteins from AD and control brains after BN-PAGE (A–D) and after SDS-PAGE (E–H). All BN-PAGE blots were developed 2–3 hrs for chemiluminescence exposure. SDS-PAGE blots were exposed for 2–5 min. (A) The protein lysates from AD brains (cases no. 5–10) in BN-PAGE showed a high-molecular weight anti-Aβ₄₂-positive smear >1000 kD. Such smears were not observed in controls (cases no. 1–4). Synthetic Aβ₄₂ and Aβ₄₀ were loaded as positive and negative controls, respectively. In Aβ₄₂ preparations, long chemiluminescence exposure led to the detection of additional dimer and ∼50 kD bands that were not observed after 2–5 min. exposures, as shown in Figure 3C. (B) The Aβ₄₂-positive material seen in (A) was not detectable in AD (cases no. 5–10) or in the controls (cases no. 1–4) in the native gel blotted with anti-Aβ₄₀ antibodies. Synthetic Aβ₄₂ and Aβ₄₀ were loaded as positive and negative controls, respectively. After 3 hrs of chemiluminescence exposure, synthetic Aβ₄₀ blots display a dimer band at ∼10 kD in addition to the monomer band and the hiMWAβ smear already detected with shorter exposure times as depicted in Figure 3C. (C) The anti-Aβ_1–17 antibody also detected the high-molecular weight protein aggregates >1000 kD in the area of stacking gel in the protein lysate from AD brains (cases no. 5–10), which was not detectable in control brains (cases no. 1–4). In addition, anti-Aβ_1–17 also showed APP bands in AD and control cases (140–240 kD). (D) The APP-positive bands were confirmed with an antibody directed against N-terminal epitope of APP (22C11) in control (cases no. 1–4) and AD cases (cases no. 5–10). (E)–(G) SDS-PAGE analysis of AD brain protein lysates from cases no. 5–10 exhibited Aβ monomer and dimer bands with MBC-42 (E), MBC-40 (F) and anti-Aβ_1–17 (G) that were not detected in control brains (cases no. 1–4). The MBC-42-dimer (E) and 6E10-dimer bands (G) were not seen in all AD cases, whereas anti-Aβ₄₀ consistently detected dimer bands (F). A high-molecular smear was found in most AD cases with all three antibodies directed against Aβ. Interestingly, cases 6 and 10 exhibited nearly no SDS-stable hiMWAβ₄₂ aggregates (E), whereas both cases showed high-molecular anti-Aβ₄₂-positive material in the BN-PAGE (A). (H) With the help of anti-Aβ_1–17 (6E10)-immunoprecipitation, monomer and dimer bands as well as loMWAβ (4–20 kD) smears and hiMWAβ (>160 kD) smears were visible in SDS-PAGE of AD brain lysates (cases no. 5–10) but not in those of controls (cases no. 1–4). The detection of the loMWAβ oligomers required chemiluminescence exposure for 3 hrs (i.e. long exposure times).

SDS-PAGE with subsequent anti-Aβ₄₂, anti-Aβ₄₀ and anti-Aβ_1–17 Western blot analysis showed Aβ monomers and dimers in AD cases (Fig. 1E–G). Aβ aggregates with a molecular weight of >160 kD were observed in four of six cases with anti-Aβ₄₂ and a smear >260 kD was observed in all cases with anti-Aβ_1–17 (6E10) (Fig. 1E–G). After immunoprecipitation with anti-Aβ_1–17, a smear of hiMWAβ- (>160 kD) and loMWAβ aggregates (8–20 kD) as well as dimer bands at ∼10 kD were consistently seen in AD cases (Fig. 1H). LoMWAβ and dimers were detected only after 3 hrs of chemiluminescence exposure but not in controls (Fig. 1H).

A11-antibody and B10AP-antibody fragments precipitate oligomeric and protofibrillar/fibrillar proteins including Aβ oligomers, Aβ protofibrils and Aβ fibrils in AD cases

In controls, immunoelectron microscopy of A11- and B10AP-precipitated proteins revealed a high number of precipitated and aggregated proteins that did not contain Aβ-positive material (Fig. 2A–D). There was no nonspecific labelling with anti-Aβ_1–17 in controls. B10AP-precipitated material exhibited a pattern that showed fibril-/ protofibril-like architectures (Fig. 2A, B) whereas the proteins precipitated by A11 displayed a spherical pattern (Fig. 2C, D).

Fig 2.

Electron microscopic analysis of immunoprecipitated protein aggregates from AD and control brain as precipitated with B10AP-antibody fragments (B10AP-IP) and A11 antibodies (A11-IP). (A), (B) In the control case no. 3, protein aggregates were precipitated with B10AP, but anti-Aβ_1–17 did not show Aβ within these aggregates. There was also no non-specific labelling with anti-Aβ_1–17 because no gold particles were observed. The protein aggregates exhibited protofibril-aggregate-like architecture that is more evident at higher magnification (B). This indicates that B10AP does not specifically bind to Aβ protofibrils or fibrils but to proteins with a distinct protofibrillar/fibrillar conformation, as reported previously [2]. (C), (D) A11-IP from control cases resulted in detection of amorphous to spherical presumably oligomeric protein aggregates, as shown in control case no. 4, but did not exhibit Aβ as a component of these protein aggregates. There was also no non-specific labelling with anti-Aβ_1–17 because no gold particles were seen. The high magnification demonstrates the spherical shape of the precipitated proteins (D). Thus, A11 also binds spherical protein aggregates other than Aβ oligomers, as reported earlier by others [6]. (E), (F) B10AP-IP from AD brain lysate of case no. 7 showed protein aggregates of protofibril-like morphology. Immunogold labelling indicated Aβ_1–17-positive proteins. The frame in E indicates the areas enlarged in (F). At higher magnification, Aβ_1–17-positive material following B10AP-IP exhibited protofibril-like morphology (arrows in F) and less frequently amorphous structures (arrowheads in F). These types of Aβ aggregates prevailed in B10AP precipitates. (G) Only a few precipitated Aβ-positive protein aggregates exhibited fibrillar architecture (case no. 10) resembling synthetic Aβ fibrils (Fig. 3A, B). (H) A11-precipitated protein aggregates from AD case no. 7 exhibited spherical to amorphous morphology. Immunogold particles indicate the presence of Aβ_1–17-positive aggregates. (I)–(J) Similarly, A11-IP extracted mainly spherical and amorphous protein aggregates from AD case no. 10 shown here at higher magnification. Immunogold labelling indicated Aβ_1–17-positive proteins. The frame indicates the area enlarged in (J). The Aβ_1–17-positive aggregates observed after A11-IP showed a spherical shape (arrowheads in J).

In B10AP precipitates of the soluble fraction of AD brain homogenates, we observed protein aggregates with a fibril/protofibril-like pattern similar to that seen in controls. However, in AD brains a high number of Aβ-positive protein aggregates were detected with anti-Aβ_1–17 (Fig. 2E–G). High-magnification analysis of anti-Aβ_1–17-labeled protein aggregates revealed a protofibril-like pattern (Fig. 2F, arrows). However, a few amorphous protein aggregates (Fig. 2F, arrowheads) as well as fibrillar aggregates (Fig. 2G) were seen as well. Spherical Aβ oligomers were not observed following B10AP immunoprecipitation. Amorphous and spherical protein aggregates were observed after A11-immunoprecipitation from AD brain lysates. Anti-Aβ_1–17 antibodies detected protein aggregates of spherical and amorphous morphology (Fig. 2H–J). Protofibril-like structures as seen in B10AP precipitates were not observed in A11 precipitates.

SDS treatment destroys native high-molecular weight Aβ₄₂ and Aβ₄₀ aggregates

Synthetic Aβ₄₂ and Aβ₄₀ formed oligomeric and protofibrillar aggregates as detectable by electron microscopy (Fig. 3A, B). With 2–5 min. chemiluminescence exposure time, BN-PAGE blots revealed a monomer band and an additional prominent smear of Aβ₄₂- and Aβ₄₀ aggregates with a molecular weight >700 and 240 kD, respectively (Fig. 3C). However, after longer exposure time of 2–3 hrs, we observed smeary bands at ∼10 and ∼50 kD in Aβ₄₂ preparations, whereas Aβ₄₀ preparations exhibited an additional dimer band that was not detectable in short chemiluminescence exposure blots (Figs 1A, B, 3C). In SDS-PAGE, we observed few high-molecular weight aggregates above 240 kD in Aβ₄₂ preparations but not in Aβ₄₀ preparations, whereas very prominent Aβ monomer, dimer and trimer bands were observed for both synthetic Aβ₄₂- and Aβ₄₀ preparations. In SDS-treated Aβ₄₀ preparations, tetramer and Aβ*56 bands were present that were not seen in SDS-treated Aβ₄₂ preparations (Fig. 3D).

Fig 3.

Synthetic Aβ₄₂ (A) and synthetic Aβ₄₀ (B) dissolved in cell culture medium aggregated to amorphous oligomers, protofibrils and fibrils (f), as detectable by electron microscopy. (C) In BN-PAGE, a high-molecular weight smear occurred mainly above 700 kD in Aβ₄₂ preparations in addition to a distinct monomeric band at ∼4 kD. Aβ₄₀ preparations produced a smear of Aβ aggregates with a molecular weight above 242 kD in addition to a clear monomeric band at ∼4 kD. The BN-PAGE blots were developed with standard chemiluminescence exposure time of 2–5 min. Longer chemiluminescence exposure for 3 hrs resulted in additional loMWAβ bands depicted in Figure 1A and B, thereby indicating that these preparations contain low levels of these Aβ species as well. (D) In SDS-PAGE, hiMWAβ₄₂ aggregates were strikingly reduced. Instead, the monomer, dimer and trimer bands displayed strong staining. Other loMWAβ₄₂ oligomers were not evident. No hiMWAβ₄₀ aggregates were seen after denaturing SDS-PAGE, but Aβ₄₀ monomer, dimer, trimer and tetramer bands as well as an Aβ*56 band were detectable at 56 kD.

Discussion

Our results show that hiMWAβ₄₂ oligomers and protofibrils with a molecular weight >1000 kD predominate in the soluble fraction of AD brain homogenates when these samples are analysed under native BN-PAGE conditions. Aβ₄₀ aggregates were not detected following BN-PAGE. Immunoelectron microscopy of immunoprecipitated oligomeric, fibrillar and protofibrillar proteins confirmed the presence of protofibrillar and spherical hiMWAβ aggregates in the soluble fraction of AD brain homogenates. These hiMWAβ aggregates were not seen in controls, whereas analysis of control cases revealed that the A11 and B10AP antibodies/antibody fragments precipitate other proteins of similar morphology, and that only a portion of the precipitated proteins from AD cases were Aβ aggregates. Denaturation of the hiMWAβ aggregates by SDS resulted in the detection of Aβ monomers, dimers, and hiMWAβ with a molecular weight >160 kD in SDS-PAGE analysis of AD cases but not in controls. When using immunoprecipitation with anti-Aβ_1–17, a smear of loMWAβ and hiMWAβ was consistently seen following SDS-PAGE and subsequent Western blot analysis, thus indicating that dimers, trimers, tetramers and Aβ*56 are not the only Aβ oligomers that can be detected with SDS-PAGE as shown previously by other groups as well [4, 5]. These results lead us to conclude that, under native conditions, Aβ monomers and loMWAβ aggregates, such as dimers, trimers and Aβ*56, do not represent the major pool of Aβ aggregates in the human AD brain. More likely, loMWAβ aggregates may occur transiently during aggregation or after denaturation of hiMWAβ. The strongest argument in favour of this hypothesis is our finding that hiMWAβ oligomer preparations and Aβ protofibril preparations of synthetic Aβ₄₂- and Aβ₄₀ peptides did not exhibit high levels of loMWAβ oligomers in BN-PAGE but did so in SDS-PAGE. Moreover, subsequent to SDS-induced protein denaturation, hiMWAβ₄₀ aggregates were no longer seen and synthetic hiMWAβ₄₂ aggregates were remarkably reduced. A possible argument against the predominance of hiMWAβ in the soluble fraction is that Aβ monomers tend to aggregate in the presence of oligomers [24] and that this occurs during protein preparation. Nevertheless, in synthetic Aβ preparations with high amounts of aggregated Aβ, we detected a significant monomer band after BN-PAGE. This may indicate that Aβ monomers in the soluble brain lysates remained stable during the process of native protein preparation. As such, it is likely that the hiMWAβ₄₂ aggregates observed in the native soluble fraction indeed represent the major form of soluble Aβ in the human AD brain.

Our finding that Aβ₄₀ was detected in AD cases in SDS-PAGE but not in BN-PAGE could be attributable either to a lower resolution of native gels in comparison to that of denaturing gels or to the fact that a potential smear of Aβ₄₀ aggregates falls far below detectable levels. Presumably, SDS treatment denatures all kinds of Aβ₄₀ oligomers and, in so doing, leads to the accumulation of Aβ₄₀ monomers in a single band. Thus, a non-detectable Aβ₄₀ smear in BN-PAGE might be converted into a detectable well-defined band in the SDS-PAGE. This hypothesis is supported by our finding of a detectable hiMWAβ₄₀ smear in synthetic Aβ₄₀ preparation that disappeared after SDS treatment and converted into strongly stained monomer and loMWAβ oligomer bands. In BN-PAGE, the spectrum of synthetic hiMWAβ₄₀ oligomers was greater (>240 kD) than that of synthetic hiMWAβ₄₂ oligomers (>700 kD). This suggests that the concentrations of distinct hiMWAβ₄₀ oligomers are lower than those of distinct hiMWAβ₄₂ oligomers because of the more widespread distribution of hiMWAβ₄₀ aggregates in the gel. As a result, hiMWAβ₄₀ oligomers and may be less easily detected in native brain lysates. An alternative explanation, on the other hand, could be that Aβ₄₀ interacts with other proteins that hide its C-terminus. In addition, the predominance of hiMWAβ₄₂ in the native soluble fraction of the AD brains investigated here confirms previous reports of a predominant occurrence of Aβ₄₂ in parenchymal soluble and insoluble Aβ aggregates in AD [25, 26].

At first, the results reported here appear to contradict the findings of other authors, who argue that distinct loMWAβ oligomers, such as dimers and Aβ*56, are critical for the development of AD [3, 9, 27]. These authors provide evidence that loMWAβ oligomer preparations received by size-exclusion chromatography are detectable in human as well as transgenic mouse brains, and are capable of inducing cognitive deficits in the rat [9] or altering long-term potentiation [3]. Given our in vitro and in vivo findings, however, one could also speculate that small amounts of loMWAβ oligomers (possibly resulting from the denaturation of hiMWAβ oligomers, protofibrils and fibrils) either are critical for the development of AD or that, upon their administration, loMWAβ oligomers may spontaneously aggregate and form hiMWAβ oligomers, as appears to be the case based upon our native gel analysis of Aβ₄₀- and Aβ₄₂ preparations and the results of Nguyen et al.[24], who showed that Aβ oligomers accommodate added Aβ monomers. Thus, hiMWAβ aggregates contribute to the pathogenesis of AD either on its own or do so indirectly by providing the reservoir of hiMWAβ aggregates that denature and, in so doing, release loMWAβ oligomers.

That Aβ aggregates, including Aβ plaques, dissociate during the pathogenesis of AD is corroborated by the finding that in late-stage AD cases plaque frequency is lower than in earlier stages [28]. The relevance of soluble hiMWAβ for the pathogenesis of AD may be further supported by the finding of neuritic degeneration near Aβ plaques, i.e. in areas with high levels of hiMWAβ presumably dissolved from Aβ plaques, in the APP transgenic mouse brain [7, 8, 29], in aged rhesus monkeys [30] and in the AD brain [31], and also by the finding that dendritic degeneration in another APP-transgenic mouse model begins at the same time as the deposition of initial diffuse Aβ-plaques, i.e. when hiMWAβ aggregates begin to predominate in the cortex [32].

Here, the stability of hiMWAβ₄₂ aggregates was greater than that of Aβ₄₀ aggregates in in vitro experiments, thus confirming previous reports that soluble Aβ₄₂ aggregates are more stable than Aβ₄₀ aggregates [33, 34]. Taken together with our finding that hiMWAβ₄₂ aggregates predominate in the native soluble fraction of the brain, it may be speculated that it is the stability of soluble Aβ₄₂ aggregates in the soluble compartment of the brain that accounts for its predominance in parenchymal Aβ plaque deposition [26].

In conclusion, the results of the present study strongly suggest that hiMWAβ oligomers, protofibrils and fibrils are the predominant soluble Aβ aggregates in the AD brain. loMWAβ oligomers in high concentrations are detectable only after denaturation of hiMWAβ aggregates. In view of the denaturation of hiMWAβ aggregates and fibrils into loMWAβ oligomers, we propose that Aβ plaques consisting of both fibrillar Aβ as well as soluble hiMWAβ aggregates may serve as reservoirs for the release of loMWAβ oligomers.

Conflict of interest

D.R.T. received research grants from the Deutsche Forschungsgemeinschaft (DFG-grant TH624/6–1) and from the Alzheimer Forschung Initiative (AFI Grant #10810). M.F. was supported by the Deutsche Forschungsgemeinschaft (SFB 610) and the Landesexzellenz-Netzwerk Biowissenschaften. There are no other conflicts of interest.