Amyloid-β oligomer specificity mediated by the IgM isotype--implications for a specific protective mechanism exerted by endogenous auto-antibodies

Abstract

Background: Alzheimers disease (AD) has been strongly linked to an anomalous self-assembly of the amyloid-β peptide (Aβ). The correlation between clinical symptoms of AD and Aβ depositions is, however, weak. Instead small and soluble Aβ oligomers are suggested to exert the major pathological effects. In strong support of this notion, immunological targeting of Aβ oligomers in AD mice-models shows that memory impairments can be restored without affecting the total burden of Aβ deposits. Consequently a specific immunological targeting of Aβ oligomers is of high therapeutic interest.

Methodology/principal findings: Previously the generation of conformational-dependent oligomer specific anti-Aβ antibodies has been described. However, to avoid the difficult task of identifying a molecular architecture only present on oligomers, we have focused on a more general approach based on the hypothesis that all oligomers expose multiple identical epitopes and therefore would have an increased binding to a multivalent receptor. Using the polyvalent IgM immunoglobulin we have developed a monoclonal anti-Aβ antibody (OMAB). OMAB only demonstrates a weak interaction with Aβ monomers and dimers having fast on and off-rate kinetics. However, as an effect of avidity, its interaction with Aβ-oligomers results in a strong complex with an exceptionally slow off-rate. Through this mechanism a selectivity towards Aβ oligomers is acquired and OMAB fully inhibits the cytotoxic effect exerted by Aβ(1-42) at highly substoichiometric ratios. Anti-Aβ auto-antibodies of IgM isotype are frequently present in the sera of humans. Through a screen of endogenous anti-Aβ IgM auto-antibodies from a group of healthy individuals we show that all displays a preference for oligomeric Aβ.

Conclusions/significance: Taken together we provide a simple and general mechanism for targeting of oligomers without the requirement of conformational-dependent epitopes. In addition, our results suggest that IgM anti-Aβ auto-antibodies may exert a more specific protective mechanism in vivo than previously anticipated.

Figure 1. OMAB binds to the N-terminus of Aβ with an affinity of 0.5 µM.

OMAB was immobilized to a CM5 chip and tested for biding to (A) Aβ peptides of different lengths at a concentration of 5 µM. The responses were normalized by the molecular weight of each peptide. (B) Aβ(1–16) ranging in concentration from 125 nM to 6 µM. All experiments were performed in PBS at 25 °C. The dissociation constant for Aβ(1–16) was determined by fitting the response at the end of each of the association phases to a single-site binding isotherm.

Figure 2. OMAB selectively reacts with oligomeric Aβ.

A sample containing different oligomeric species of Aβ(1–42) was separated by SEC (-). Fractions, collected throughout the SEC, were tested in an indirect ELISA for reactivity with OMAB (---). In connection to the SEC of Aβ(1–42), a sample containing proteins with known molecular weights was analyzed. The exclusion volume of the peak maxima of these proteins is illustrated in the figure (▪).

Figure 3. A complex between Aβ oligomers and OMAB display very slow dissociation rate.

Different samples of Aβ(1–40) and Aβ(1–42) was separated by SEC. (A) The Aβ(1–40) monomeric fraction or (B) Aβ(1–40) the oligomeric or (C) monomeric fraction of Aβ(1–42) or (D) Aβ(1–42) the oligomeric fraction were analyzed for binding to OMAB, immobilized to a CM5 chip. The dissociation of (A) monomeric Aβ(1–40) was fitted to a single exponential decay (red line) with a rate constant of 0.0092 s and a T1/2 of 75 s, and of (B)(1–40) oligomeric Aβ to a double exponential decay (red line) with rate constants of 0.016 and 1.88 * 10-6, and with a T1/2 of 42 s and 4.2 days, respectively. (C) monomeric Aβ(1–42) was fitted to a single exponential decay (red line) with a rate constant of 0.012 s and a T1/2 of 57 s for the oligomeric fraction of Aβ1-42 (D) no dissociation was detected due to a to very strong interaction. All experiments were performed in PBS at 25°C.

Figure 4. OMAB inhibits the cytotoxicity of Aβ(1–42) in a substoichiometric ratio.

OMAB was incubated with SH-SY5Y cells in the presence or absence of Aβ(1–42). After 48 hours, cell viability was detected using resazurin. # = p<0.001 compared to control cells and * = p<0.001 compared to cells treated with only Aβ(1–42).

Figure 5. Human IgM auto-antibodies specifically detect Aβ oligomers.

A sample containing different oligomeric species of Aβ(1–42) was separated by SEC (-). Fractions, collected throughout the SEC, were tested in an indirect ELISA for reactivity with (A) L11.3 (---) or (B) plasma from eight different human donors (---). Before adding L11.3 or plasma, the ELISA plate was coated with an anti-human IgM antibody to ensure that only IgM auto-antibodies were allowed to react with the Aβ species. The level of endogenous anti-Aβ antibodies is low in the plasma and to ensure a measurable response 5X more total Aβ was applied on the SEC column which explains the different elution profiles.