Monoclonal antibodies against Aβ42 fibrils distinguish multiple aggregation state polymorphisms in vitro and in Alzheimer disease brain

Abstract

Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process.

Keywords: Alzheimer Disease; Amyloid; Aβ; Monoclonal Antibody; Peptide Conformation; Protein Aggregation.

FIGURE 1.

SPOT assay. Left, epitope mapping results for the 19 mOC antibodies that reacted with Aβ segments in the SPOT assay, along with 6E10 and 4G8. Right, interpretation of the SPOT assay results. The sequence of the Aβ peptide is shown beginning with the −3 position. The apparent epitope is the sequence contained in common by all positive spots and is shown in red.

FIGURE 2.

Representative dot blot results. Aβ40 (a) and Aβ42 (b) were aggregated under three different conditions over a 10-day time period. The immunological reactivities of the aggregates from time 0, and the 3- and 10-day time points were then tested with the 23 mOC antibodies, along with 6E10 and 4G8. Results obtained with antibodies 6E10, 4G8, and mOC 3, 9, 23, 31, 76, and 87 are displayed as representative examples. The three aggregation conditions were as follows: Condition A, peptide resuspended in 100 mm NaOH and diluted in phosphate buffer; Condition B, peptide resuspended in HFIP and diluted in water; Condition C, peptide resuspended in 100 mm NaOH and diluted in HEPES/NaCl buffer.

FIGURE 3.

Complete dot blot data. Aβ40 (a) and Aβ42 (b) were aggregated under three different conditions over a 10-day time period. 1-μl aliquots were pipetted onto nitrocellulose membranes at time 0 and at the 3- and 10-day time points. The membranes were then probed with the 23 mOC antibodies, along with 6E10 and 4G8. The three aggregation conditions were as follows: Condition A, peptide resuspended in 100 mm NaOH and diluted in phosphate buffer; Condition B, peptide resuspended in HFIP and diluted in water; Condition C, peptide resuspended in 100 mm NaOH and diluted in HEPES/NaCl buffer.

FIGURE 4.

Representative Western blots results. Aβ40 (a) and Aβ42 (b) were aggregated under three different conditions over a 10-day time period. Aliquots from time 0 and the 3- and 10-day time points were used for Western blotting using the 23 mOC antibodies, along with 4G8 and 6E10. Results obtained using 6E10, 4G8, and antibodies mOC 3, 9, 23, 31, 76, and 87 are displayed here as representative examples. The three aggregation conditions were as follows: Condition (Cond.) A, peptide resuspended in 100 mm NaOH and diluted in phosphate buffer; Condition B, peptide resuspended in HFIP and diluted in water; Condition C, peptide resuspended in 100 mm NaOH and diluted in HEPES/NaCl buffer.

FIGURE 5.

Complete Western blot data. Aβ40 (a) and Aβ42 (b) were aggregated under three different conditions over a 10-day time period. Aliquots from time 0 and the 3- and 7-day time points were used for Western blotting using the 23 mOC antibodies, along with 4G8 and 6E10. The three aggregation conditions were as follows: Condition (Cond.) A, peptide resuspended in 100 mm NaOH and diluted in phosphate buffer; Condition B, peptide resuspended in HFIP and diluted in water; Condition C, peptide resuspended in 100 mm NaOH and diluted in HEPES/NaCl buffer.

FIGURE 6.

mOC antibodies differ in their binding to Aβ fibrils after heat denaturation. Four different fibrillar preparations of Aβ42 were subjected to Western blotting using the 23 mOC antibodies with and without heat denaturation of the membrane prior to the blocking step. Here, we present three representative examples of mOC antibodies with differing responses to the heat denaturation of Aβ42 fibrils.

FIGURE 7.

Dot blot assays of IAPP and α-synuclein aggregates. α-Synculein (left) and IAPP (right) were aggregated over a 6-day time period. The immunoreactivities of samples from time 0 and the 1-, 2-, and 4–6-day time points were tested using the 23 mOC antibodies in a dot blot assay. Antibodies that showed positive reactivity with the α-synuclein and IAPP preparations are shown. IAPP was resuspended in 100 mm NaOH and diluted in PBS, and α-synculein was resuspended in HFIP and diluted in water.

FIGURE 8.

Differential staining of human AD and 3×Tg-AD mouse brain. Sections were stained with the indicated mOC antibodies. Top panels, human AD brain. Bottom panels, 14-month-old 3×Tg-AD mouse brain, except as indicated. Bars, 100 μm.

FIGURE 9.

Immunostaining of human AD brain (a) and 3×TgAD mouse brain (b) with mOC antibodies. The 40-μm-thick serial sections from 14-month-old 3×TgAD mice were stained with the 23 mOC antibodies and 6E10. Some of the antibodies reacted with intracellular aggregates; some were specific to extracellular plaques, and some recognized vascular amyloid. For antibodies showing reactivity with more than one type of aggregate, we have included images representative of reactivity with each type of aggregate. Magnification: ×40.