Anti-Aβ_1–11 Antibody Binds to Different β-Amyloid Species, Inhibits Fibril Formation, and Disaggregates Preformed Fibrils but Not the Most Toxic Oligomers^*

Antigen Preparation and Immunization of Mice

We synthesized two copies of the N terminus of the immunodominant B cell epitope of Aβ_1–11 in tandem with a potent foreign T cell epitope, PADRE, and then conjugated them through the C terminus to MAP, (Invitrogen) as we described previously (33). 5.5–7.5-month-old hemizygous 3xTg-AD mice (32, 34) provided by Dr. LaFerla's laboratory at the University of California (Irvine, CA) were immunized four times biweekly with 2Aβ_1–11-PADRE-MAP vaccine or fibrillar A_β42 (fA_β42). Before immunization, the peptide was mixed with a Th1 type conventional adjuvant, Quil A, and mice were injected subcutaneously with 50 μg of the antigen (35). Sera were collected from individual animals 9 days after the last boost and kept at +4 °C.

Detection of Anti-Aβ Antibodies by Enzyme-linked Immunosorbent Assay

Total anti-Aβ₄₂ antibodies were detected as described previously (35, 36) with small modifications; to develop the reaction, we used the 3,3′,5,5′-tetramethylbenzidine peroxidase substrate (Pierce), and plates were analyzed spectrophotometrically at 450 nm. The concentrations of antibody were calculated with a standard curve generated using 6E10 monoclonal antibodies. To determine the specific isotypes, pooled sera from mice were diluted 1:500 and tested in duplicate using appropriate secondary antibodies specific to IgG1, IgG2a^b, IgG2b, and IgM (35, 36).

T Cell Proliferation and Production of Cytokines by Immune Splenocytes

Using [³H]thymidine, we analyzed T cell proliferation in splenocyte cultures from individual animals as we described previously (35, 36). The same splenocytes used for T cell proliferation were utilized for detection of Th1 (IFNγ) or Th2 (IL-4) lymphokines by ELISPOT technique, also described in detail in Refs. 33 and 35.

Purification of Anti-Aβ_1–11 Antibody

Sera from 3xTg-AD mice immunized with 2Aβ_1–11-PADRE-MAP epitope vaccine were purified by an affinity column (SulfoLink; Pierce) per the manufacturer's instructions. Briefly, 2 mg of Aβ_1–11-Cys peptide (synthesized at NeoMPS, San Diego, CA) was dissolved in 2–3 ml of buffer containing 50 mm Tris, 5 mm EDTA-Na, pH 8.5, and immobilized to the iodoacetyl-activated 6% cross-linked agarose through its free sulfhydryl groups. The remaining active binding sites were blocked with a 0.05 m solution of cysteine. Sera were diluted 1:1 in PBS, added to the column, and allowed to pass through the column. The column was washed with PBS, and then bound material was eluted with glycine buffer (100 mm, pH 2.5–3.0), neutralized, and concentrated by centrifugation using a Centricon filter (cut-off 30 kDa; Millipore Corp.). Purified antibodies were analyzed via 10% Tris-SDS-PAGE, and the concentrations were determined by enzyme-linked immunosorbent assay.

Detection of Aβ Plaques in Human Brain Tissues

Anti-Aβ_1–11 antibodies (1 μg/ml) were screened for the ability to bind to human Aβ plaques using 50-μm brain sections of formalin-fixed hippocampal tissue from a case with neuropathological and behavioral patterns typical of severe AD (33, 37). A digital camera (Olympus) was utilized to capture images of the plaques at ×10 magnification. The binding of anti-Aβ_1–11 antibodies to the β-amyloid plaques was blocked by preabsorption of the antibodies with 2.5 μm of 2Aβ_1–11 peptide as described (33).

Dot Blot Assay, Immunoprecipitation, and Western Blotting

To detect binding of Aβ_1–11 antibody to different forms of Aβ₄₂ peptide, we applied 2 μl of monomeric, oligomeric, or fibrillar forms of Aβ₄₂ (100 μm each) to a nitrocellulose membrane as described (38). After blocking and washing, the membranes were probed with anti-Aβ_1–11 (1 μg/ml). The membranes were incubated with appropriate horseradish peroxidase-conjugated anti-mouse antibody (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Blots were developed using Luminol reagent (Santa Cruz Biotechnology) and exposed to Kodak X-Omat AR film. For identification of species stabilized after disaggregation of preformed fibrils, 2 μl of resulting solutions were applied to a nitrocellulose membrane and probed with biotinylated A11 antibody (1 μg/ml) and streptavidin-conjugated secondary antibody.

To detect APP and different Aβ forms in brain tissue recognized by anti-Aβ_1–11 antibody, we used a combination of immunoprecipitation with Western blotting described previously (37). Briefly, the brain tissue was processed as described previously (39), except that cortices of right hemispheres of brains were used. Frozen cortices were thawed, minced, and then homogenized in 50 mm Tris-HCl buffer containing 150 mm NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS (pH 8.0), and a mixture of protease inhibitors (MP Biomedicals). Homogenates were centrifuged (100,000 × g, 1 h, 4 °C), and the supernatants were utilized for immunoprecipitation with 20.1 monoclonal antibodies of IgG2b isotype recognizing 1–8 aa of Aβ peptide (40). Immunoprecipitated proteins were analyzed in 10% Tris-SDS-polyacrylamide gel, transferred on the nitrocellulose membrane, and visualized after incubation with anti-Aβ_1–11 antibody (1 μg/ml) followed by incubation with horseradish peroxidase-anti-mouse secondary antibodies and Luminol (Santa Cruz Biotechnology).

Surface Plasmon Resonance (SPR) Analysis

Binding studies were performed on the BIAcore 2000 SPR platform (Biacore). Monomeric, oligomeric, or fibrillar forms of Aβ₄₂ peptides were immobilized to the surface of biosensor chip (CM5) via an amine coupling of primary amino groups (N termini or Lys¹⁷ or Lys²⁸ residues) of the appropriate peptide to carboxyl groups in the dextran matrix of the chip. Serial dilutions of anti-Aβ_1–11 antibody, 6E10 antibody, or irrelevant mouse IgG (125, 250, and 500 nm) in the running buffer containing 10 mm HEPES, 150 mm NaCl, 0.05% surfactant P20, pH 7.4, were injected at 5 μl/min over each immobilized form of peptide, and the kinetics of binding/dissociation was measured as change of the SPR signal (in resonance units). Each injection was followed by a regeneration step of a 30-s pulse of 1 m NaCl, 50 mm NaOH. Fitting of experimental data was done with BIAevaluation 3.0 software.

Intrahippocampal Injection of Antibodies and Quantitative Image Analysis

Anti-Aβ_1–11 antibody was administered into the hippocampus of 18-month-old homozygous 3xTg-AD mice via injections, as described previously (41). Mouse IgG to irrelevant antigen (mouse anti-osteoprotegerin antibody) was used as a negative control. Anti-Aβ_1–11 antibody and control IgG were injected at a concentration of 2 μg/2 μl into the left hippocampus over a 3–5-min period. Mice were perfused and sacrificed on the 6th day after surgery, and the brains were fixed in 4% paraformaldehyde for 48 h. Serial coronal sections (50 μm) were cut using the Vibratome slicing system (Pelco) and stored in PBS. Prior to staining, sections were incubated in 90% formic acid for 4 min at room temperature. Amyloid plaques were stained using 6E10 biotinylated monoclonal antibody (Signet Laboratories) and developed using an immunodetection kit (Vector Laboratories), as we previously described (33, 37). To detect specific areas in which intrahippocampally injected anti-Aβ or irrelevant antibodies were diffused, we stained the adjacent brain sections using anti-mouse biotinylated IgG as recommended by the manufacturer (Vector Laboratories). Images were captured by an Olympus microscope. Immunostainings were observed by the means of a Sony high resolution CCD video camera (XC-77) and NIH Image version 1.59b5 software. For every animal, 10 images (525 × 410 μm each) in the hippocampus area of two adjacent sections were captured with a ×20 objective. Samples consisted of five images from the superficial layer and five from the deep layer. NIH imaging was used to analyze the area occupied by β-amyloid (Aβ load) relative to the background and expressed in the percentage units at a threshold of 125 that was established and held constant throughout the image analysis.

Inhibition of Aβ₄₂ Fibrillization

Aβ stock solutions (2 mm) were obtained by dissolving the lyophilized peptide in 100 mm NaOH followed by water bath sonication for 30 s. The aggregation of Aβ₄₂ into fibrils (25 μm final concentration) was initiated in 10 mm HEPES, 100 mm NaCl, 0.02% sodium azide, pH 7.4, at room temperature with stirring for up to 180 h, in the presence and absence of 0.5 μm purified anti-Aβ_1–11 antibody diluted in PBS buffer. The reactions were monitored as a function of time via thioflavin T (ThT) fluorescence and at initial and final time points by transmission electron microscopy (TEM), as described below. Fluorescence spectroscopy data were fit to integral logistic equations using the Sigmaplot software (Systat Software Inc., Point Richmond, CA), and the apparent rate constant for the fibril growth, $k_{\text{app}}^a$, and the lag time were determined as described before (42–47). Although $k_{\text{app}}^a$ is not an elementary association rate constant, it constitutes a useful parameter for comparison of fibrillization kinetics (43, 44, 48).

Disaggregation of Preformed Aβ₄₂ Fibrils

Aβ₄₂ fibrils were prepared as described above in the absence of any additive. The aggregation reaction was allowed to proceed until equilibrium was reached (∼7 days). Then PBS vehicle or 0.5 μm purified anti-Aβ_1–11 antibody (25 μm final Aβ₄₂ concentration) was added to the preformed fibrils. The reactions were monitored as a function of time via ThT fluorescence and at initial and final time points by TEM, as described below. Fluorescence spectroscopy data in the presence of the anti-Aβ_1–11 antibody were fit to a complex first order-exponential decay equation,

where Ft represents the ThT fluorescence at time t, Fi and Ff are the initial and plateau levels of fluorescence, and $k_{\text{app}}^d$is the pseudo-first order rate constant of disaggregation. This fit was obtained using the Table Curve 2D software trial version (Systat Software Inc., Point Richmond, CA), which allows automated modeling of data to the best fit. This model describes the dissociation of fibers into two different species and assumes that the initial concentration of these species is zero.

Inhibition of Aβ₄₂ Oligomerization

Aβ₄₂ stock solutions (2 mm) were obtained by dissolving the lyophilized peptide in 100 mm NaOH. The aggregation reaction was initiated by diluting the stock solution in PBS, pH 7.4, 0.02% sodium azide (45 μm final Aβ₄₂ concentration). These assembly conditions facilitate visualization of Aβ₄₂ oligomers. The reactions were maintained at room temperature without stirring for up to 10 days in the presence of 45 μm purified anti-Aβ_1–11 antibody, 6E10 monoclonal antibody, or mouse IgG to irrelevant antigen, anti-osteoprotegerin antibody (BD PharMingen) diluted in PBS buffer. The reactions were monitored via transmission electron microscopy as described below.

ThT Fluorescence Assay

To investigate the time course of Aβ₄₂ fibril formation, 10-μl aliquots were removed at various time points during aggregation and mixed with 120 μl of ThT (3 μm, dissolved in the assembly buffer). ThT fluorescence was measured at λ_ex = 442 nm and λ_ex = 482 nm until equilibrium was reached in the control reactions, using a Gemini XPS plate reader (Molecular Devices, Sunnyvale CA). To monitor the time course of Aβ₄₂ fibril disaggregation, ThT (10 μm final concentration) was added to preformed fibrils in the presence or absence of the anti-Aβ_1–11 antibody. Fluorescence was measured in the plate reader in real time. Control reactions were carried out in the presence of PBS vehicle and were corrected by subtracting the buffer blank. Aggregation and disaggregation reactions in the presence of the anti-Aβ_1–11 antibody were corrected using antibody-only control reactions. This assay assumes that ThT fluorescence is a measure of filament mass (49, 50).

Electron Microscopy

1-μl aliquots of the aggregation and disaggregation reactions were adsorbed onto 200-mesh Formvar/carbon-coated nickel grids (Electron Microscopy Sciences; Ft. Washington, PA) until dry. The grids were then washed with water, stained with 2% uranyl acetate, and washed again. The grids were allowed to dry between all steps and were viewed using a Phillips CM 12 microscope operated at 65 kV.

Neurotoxicity Assay

Mouse primary neurons were prepared from cortices of embryonic day 17–18 mouse embryos. Briefly, cortices were dissected and incubated in trypsin (37 °C, 7 min). The cortices were pelleted at 1000 rpm for 5 min and were resuspended in NB-N2 medium (5 μg/ml insulin, 20 nm progesterone, 100 μm putresine, 30 nm selenium, 100 μg/ml transferring) (Sigma). After 10–15 min, the cells were passed through a glass pipette four times and then through a cell strainer and washed with NB-N2. Cells were plated on polylysine-coated 96-well plates. Aβ oligomers and fibrils were prepared as described (38). Aβ₄₂ oligomers and fibrils were incubated alone or with antibodies (anti-Aβ_1–11 and irrelevant anti-osteoprotegerin) for 50 min at room temperature with occasional mixing to ensure the maximal interaction. After incubation, the peptide/antibody mixtures were diluted into culture medium to a final concentration of peptide and antibodies of 2.5 and 0.5 μm, respectively. This medium was added (100 μl) to mouse primary neurons plated at ∼10⁴/well. The treatment time was 4 h. Untreated controls were run in parallel. Following incubation, the neurotoxicity was assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay per the manufacturer's instructions.

Statistical Analysis

All statistical parameters (average values, S.D., and significant differences) used in these studies were calculated using Prism 3.03 software (GraphPad Software, Inc.). Statistically significant differences were obtained using a t test or an analysis of variance following Tukey's or Bonferroni's multiple comparison post-test, and a p value of <0.05 was considered as significantly different.

Epitope Vaccine Induced High Titers of Polyclonal Anti-Aβ Antibodies Specific to the N-terminal Region of Aβ₄₂ without Generation of Autoreactive T Cells

Immunization of 3xTg-AD mice of H-2^b immune haplotype with the second generation peptide epitope vaccine induced strong humoral response with an average concentration of anti-Aβ antibody of 205 μg/ml (titer ∼1:180,000) (Fig. 1a) of IgG1, IgG2a^b, and IgG2b isotypes (Fig. 1b). Antibody isotyping has previously been used as an indirect measure of the contribution of Th1 (IgG2a) and Th2 (IgG1) cytokines to the immune response (51). In our experiments, the ratio of IgG1/IgG2a^b was close to 1, implying that predominantly Th1 type immune responses were induced after immunizations with epitope vaccine formulated in a Th1-type adjuvant, Quil A. This result was confirmed by analysis of cytokine production. Vaccinated animals induced predominantly IFNγ production along with moderate levels of IL-4. Fewer splenocytes from control mice immunized with fAβ₄₂ generated production of IFNγ or IL-4 (Fig. 1c), whereas splenocytes from naive animals did not produce Th1 and Th2 cytokines (data not shown). Immunizations with epitope vaccine also induced a robust T cell proliferation after restimulation of the immune splenocytes with PADRE but not self-Aβ₄₀ peptide (Fig. 1d). This finding offers an indication that PADRE-specific but not autoreactive T cells provide a necessary support for production of high titers of anti-Aβ_1–11 antibody in 3xTg-AD mice.

Anti-Aβ_1–11 Antibody Generated by the Epitope Vaccine Binds to β-Amyloid Plaques and to Aβ₄₂ Monomeric and Aggregated Species

Therapeutic feasibility of anti-Aβ_1–11 antibody was tested after isolation and purification from the sera of vaccinated 3xTg-AD mice. Purified polyclonal anti-Aβ_1–11 antibody stained Aβ plaques in brain tissue from an AD clinical case (Fig. 2a). This binding was specific, since it was blocked by preabsorption with 2Aβ_1–11 peptide (Fig. 2b) or Aβ₄₂ (data not shown). Importantly, anti-Aβ_1–11 antibody binds to APP, Aβ monomers, and different forms of oligomers, as shown via analysis of soluble fraction of brain extracts from aged APP/Tg 2576 mice by a combination of immunoprecipitation and Western blot (see “Experimental Procedures”). In these experiments, binding of anti-Aβ_1–11 antibody to 12-mer oligomers cannot be detected due to its molecular weight similarity to the heavy chain of immunoglobulins (Fig. 2c).

Anti-Aβ_1–11 antibody bound equally well to monomeric, oligomeric (38), and fibrillar forms of Aβ₄₂, as demonstrated by dot blot assay (Fig. 2d). Notably, the oligomers that were used for this study included octamers, tetramers, and trimers besides higher order aggregates as detected by Western blot analysis with A11 rabbit antibodies (38 and data not shown). Next, for the first time to our knowledge, the binding capability of anti-Aβ_1–11 antibody to different Aβ species has been determined using an SPR assay. This experiment revealed that anti-Aβ_1–11 antibody (Fig. 2, e and f) as well as control 6E10 antibody (Fig. 2, e and g) specifically bind to immobilized monomers, oligomers, and fibrils.

Both anti-Aβ_1–11 and 6E10 antibodies bound more strongly to monomeric and oligomeric forms, rather than to fibrillar Aβ₄₂. Dissociation constants (K_D) of anti-Aβ_1–11-peptide complexes for monomeric, oligomeric, and fibrillar Aβ₄₂ are 1.7 × 10⁻⁸, 1.4 × 10⁻⁸, and 5.1 × 10⁻⁸ m, respectively. The binding of anti-Aβ_1–11 antibodies to all Aβ species was significantly stronger than that of 6E10, for which K_D values correspond to 1.8 × 10⁻⁷, 1.2 × 10⁻⁷, and 4.3 × 10⁻⁷ m for monomeric, oligomeric, and fibrillar Aβ₄₂, respectively (Fig. 2, e–g). Of note, irrelevant mouse IgG interacted with Aβ₄₂ peptide nonspecifically on the base-line level (data not shown).

Intracranial Administration of Anti-Aβ_1–11 Antibodies Reduces the Aβ Deposits in Brains of 3xTg-AD Mice

To further characterize in vivo therapeutic utility, anti-Aβ_1–11 antibody was injected into the brains of aged 18-month-old 3xTg-AD mice (n = 4) with developed AD-like pathology. Antibodies (2 μg/2 μl) were administered via intrahippocampal injections, and mice were sacrificed on the 6th day after antibody administration. As controls, we used mice (n = 4) injected into the hippocampus with the same concentration of irrelevant mouse IgG. Quantitative image analysis shows individual variations of Aβ deposition in hippocampus of 18-month-old homozygous 3xTg-AD mice. In both experimental and control groups, there were mice with a low, medium, or high number of Aβ deposits (Table 1). A significant reduction (83%, p = 0.030) in β-amyloid load has been observed after comparison of ipsilateral (injected) hippocampus of mice treated with the anti-Aβ_1–11 antibody with that of controls administrated with irrelevant antibody (Fig. 3, a–c). Comparing the Aβ ratios of immunoreactivity between the ipsilateral/contralateral (uninjected) areas also revealed a substantial (77%) and statistically significant decrease (p < 0.05) of amyloid load in mice injected with anti-Aβ_1–11 antibody versus the control group (Fig. 3, d–f). This comparison has been made after quantitative image analysis of areas of the hippocampus in which antibodies/IgG diffused after the administration. These areas were determined by additional staining for mouse IgG in the adjacent brain sections of experimental and control animals (data not shown). These data suggest that anti-Aβ_1–11 antibody is able to significantly reduce Aβ deposition in the brains of aged 3xTg-AD mice after intracranial administration.

Anti-Aβ_1–11 Antibody Inhibits Aβ₄₂ Assembly into Fibrils

An increasing body of evidence suggests that antibodies raised against Aβ peptide could be useful for clearance of Aβ deposits in vivo (18, 21, 23, 24, 26, 52) possibly by inhibiting or delaying Aβ aggregation (26, 27, 30). Similar data were also detected here using the anti-Aβ_1–11 antibody. To understand the underlying mechanism and to further investigate the therapeutic utility of the anti-Aβ_1–11 antibody, we first tested whether the anti-Aβ_1–11 antibody can inhibit Aβ₄₂ fibrillization. The aggregation of Aβ₄₂ into fibrils initiated in the presence and absence of anti-Aβ_1–11 antibody was monitored using TEM. At time 0, no aggregated species were detected either in the control (Fig. 4a) or in the antibody-treated samples (data not shown). After a 7-day incubation in the absence of antibody, Aβ₄₂ formed fibrils with classic morphology (Fig. 4b). In contrast, in the presence of the anti-Aβ_1–11 antibody, Aβ₄₂ formed a heterogeneous population of nonfilamentous aggregates (Fig. 4c), which resembled oligomers of 3–10 nm. Occasionally, clusters of bigger oligomers up to 20 nm in length could also be detected. These clusters had a beaded appearance and did resemble mature fibrils. To confirm this observation, the time course of Aβ₄₂ fibrillization was monitored using ThT fluorescence, in the absence or presence of the anti-Aβ_1–11 antibody. The control reaction followed nucleation-dependent polymerization kinetics, with a lag time of 54.28 ± 1.67 h and an apparent rate constant of assembly, $k_{\text{app}}^a=0.13\pm 0.02{\text{h}}^{-1}$(Fig. 4d). Anti-Aβ_1–11 antibody, at substoichiometric concentration, inhibited the formation of Aβ₄₂ ThT-positive species. Although this reaction was followed for 200 h, the ThT signal maintained at levels less than 10% of the plateau signal of the control reaction. In the presence of anti-Aβ_1–11 antibody, the lag time of Aβ₄₂ aggregation increased to ∼80 h, and the $k_{\text{app}}^d$decreased to 0.04 ± 0.02 h⁻¹ (Fig. 4d). The low ThT signal detected in antibody-treated reactions could be attributed to small amounts of short fibrillar material or nonfibrillar species that have been shown before to react with ThT (30, 48). These results, generated in two independent assays, suggest that anti-Aβ_1–11 antibody is a potent inhibitor of Aβ₄₂ fibrillization at substoichiometric concentrations relative to Aβ₄₂ protomer (1:50, antibody/peptide molar ratio). The inhibitory activity could be attributed to both inhibition of filament nucleation and extension.

Anti-Aβ_1–11 Antibody Mediates Aβ₄₂ Fibril Disaggregation

To determine whether plaque clearance observed in vivo could be recapitulated in in vitro models of disease, we extended our studies to investigate whether anti-Aβ_1–11 antibody can disaggregate preformed Aβ₄₂ fibrils. Aβ₄₂ was allowed to assemble until equilibrium was reached based on ThT fluorescence (7–8 days of incubation), and preformed fibrils were treated either with the PBS vehicle or with the anti-Aβ_1–11 antibody. Aliquots were removed at time 0 and 24 h after the initiation of disaggregation and imaged by TEM. At time 0, both the control (Fig. 5a) and the antibody-treated reactions (data not shown) revealed a classical fibrillar morphology. Control reactions maintained fibrillar morphology 24 h after the initiation of disaggregation (Fig. 5b). In contrast, no fibrils were detected in the antibody-treated sample at the end of the same time period (Fig. 5c). Round structures resembling oligomers ranging in size from 3 to 10 nm and oligomer clusters populated this reaction. To confirm these results and investigate the mechanism of disaggregation, the kinetics of the disaggregation reactions were monitored in real time by ThT fluorescence. In the absence of the antibody, the ThT signal continued to increase slowly over this period of time, consistent with Aβ₄₂ fibrils being in the equilibrium phase of assembly (Fig. 5d). In contrast, in the presence of anti-Aβ_1–11 antibody, ThT fluorescence decreased over the same time period down to 35 ± 0.43% of the starting level of fluorescence (Fig. 5d), consistent with Aβ₄₂ filament disaggregation. The disaggregation reaction followed complex first-order kinetics with a first order rate constant of disaggregation, $k_{\text{app}}^d$of 0.10 ± 1.4 × 10⁻³ h⁻¹ (Fig. 5d). This pattern is consistent with filament dissociation into two different species, and the low ThT fluorescence and the lack of mature fibrils by TEM at equilibrium suggest that at least one of these species is ThT-reactive without showing classical fibrillar morphology. These data confirm that anti-Aβ_1–11 antibody can destabilize Aβ₄₂ fibrils at low concentrations relative to the peptide (1:50, antibody/peptide molar ratio).

To identify the nature of molecules stabilized by the anti-Aβ_1–11 antibody, the resulting species were probed with the anti-oligomer-specific antibody A11 (38). No immunoreactivity with the A11 antibody was observed (Fig. 5e, lane 1), whereas freshly prepared oligomers used as a positive control showed strong immunoreactivity with the same antibody (Fig. 5e, lane 2). These data suggest that the anti-Aβ_1–11 antibody specific to linear peptide disaggregates Aβ₄₂ preformed fibrils to small aggregates, which are not A11-immunopositive, although their size (3–10 nm) and shape resembles the oligomers when imaged by TEM.

Anti-Aβ_1–11 Antibody Delays Aβ₄₂ Oligomerization but Does Not Disaggregate Preformed Aβ₄₂ Oligomers

It was suggested that the most toxic forms of Aβ₄₂ peptide in brains are soluble oligomers of various size (13–17). Thus, it was important to test the efficacy of polyclonal anti-Aβ_1–11 antibody in inhibition of amyloid oligomerization or disaggregation of preformed oligomers. Aβ₄₂ was subjected to aggregation under conditions that facilitate visualization of Aβ₄₂ oligomers at physiological pH. Aliquots of the Aβ₄₂ assembly reactions treated with the anti-Aβ_1–11 antibody, 6E10 antibody, or irrelevant mouse IgG were removed at various time points and imaged by TEM. At time 0, few aggregated species were detected. (Fig. 6, a, d, and g). In the presence of irrelevant mouse IgG, Aβ₄₂ aggregated into a heterogenous population of oligomers with sizes ranging from 3 to 10 nm (Fig. 6b). In the presence of anti-Aβ_1–11 antibody, fewer oligomers formed, and oligomerization appeared to be delayed (Fig. 6h). In contrast, 6E10-treated Aβ₄₂ assembled into abundant oligomers within 4 days of the initiation of the aggregation reaction (Fig. 6e). 10 days after the initiation of aggregation, the irrelevant mouse IgG (Fig. 6c) and 6E10-treated (Fig. 6f) reactions showed amyloid fibers with classical appearance. At the same time point during aggregation, and in the presence of the anti-Aβ_1–11 antibody, Aβ₄₂ showed a nonfibrillar structure, dominated by aggregates resembling oligomers and oligomeric assemblies of beaded appearance. The size of the most abundant oligomer population was ∼10 nm, and clustering of as many as seven oligomers yielded extended nonfibrillar structures of beaded appearance (Fig. 6i). Neither of the antibodies disaggregated preformed Aβ₄₂ oligomers (data not shown).

Together, the results described above confirm that anti-Aβ_1–11 antibody is a potent Aβ₄₂ fibrillization inhibitor and suggest that the mechanism underlying this inhibitory activity is stabilization of nonfibrillar structures resembling oligomers and oligomer assemblies. However, the formation of Aβ₄₂ oligomers in the presence of anti-Aβ_1–11 antibody is greatly delayed compared with control reactions or Aβ₄₂ supplemented with 6E10 antibody. In addition, consistent with its activity in vivo, anti-Aβ_1–11 antibody is equally potent in destabilizing preformed, mature amyloid fibers down to the structures that do not react with anti-oligomer-specific antibodies.

Anti-Aβ_1–11 Antibody Protects Neuronal Cells from Aβ₄₂ Oligomer and Fiber-mediated Toxicity

The data presented above suggested that anti-Aβ_1–11 antibody is therapeutic and might exhibit a protective effect on Aβ-induced neurotoxicity. To test that possibility, we performed in vitro toxicity studies using mouse primary neurons. The viability of cells exposed to Aβ₄₂ oligomeric and fibrillar species preincubated with or without anti-Aβ_1–11 antibody was measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide toxicity assay. The data showed that both Aβ₄₂ fibrils and oligomers are cytotoxic, reducing cell viability to about 62 and 24%, respectively (Fig. 7). Preincubation of Aβ₄₂ fibrils with anti-Aβ_1–11 antibody (1:5, antibody/peptide molar ratio) resulted in the rescue of cell viability to maximum level. Similarly, preincubation of Aβ₄₂ oligomers with anti-Aβ_1–11 antibody increased cell viability to ∼46%. In contrast, preincubation of both Aβ₄₂ species with a control mouse IgG specific to irrelevant antigen did not rescue cells from oligomer or fiber-mediated cell death. These data suggest that anti-Aβ_1–11 antibody inhibits Aβ₄₂ fiber-mediated neurotoxicity and alleviates oligomer toxicity in vitro.