ANTI-AβX-40 ANTIBODIES EXERT NEUROPROTECTIVE EFFECT AGAINST Aβ-OLIGOMER TOXICITY

Abstract

Amyloid-beta (Aβ) plays a critical role in Alzheimer's disease (AD) pathology. Aβ oligomers have been reported to be more neurotoxic than monomeric forms and correlate with AD severity and synapse loss. We have observed that active vaccination of AD patients against the C-terminal end of Aβ1–40 triggers the production of anti-Aβx-40 antibodies, and that these types of antibodies inhibit Aβ1–40 assembly into oligomers in vitro. In this study, we have compared the cytotoxic damage produced by Aβ1–40 oligomers and monomers on PC12 cells in vitro, and have also analyzed the protective effect exerted by anti-Aβx-40 antibodies against that damage. Oligomers were prepared by dissolving lyophilized Aβ1–40 monomers in a solution containing DMSO, PBS and SDS, and incubating for 18 h at 37°C. Aβ monomers were prepared immediately before their use by dissolving lyophilized Aβ1–40 monomers in a solution of the same formula used for the oligomers. Polyclonal anti- Aβx-40 antibodies were produced in our lab. PC12 cells were grown overnight in 96-well plates at 3 x 105 cells/well, and then incubated with (a) Aβ1–40 oligomers, (b) Aβ1–40 monomers, (c) Aβ1–40 oligomers + anti-Aβx-40 antibodies, (d) Aβ1–40 monomers + anti-Aβx-40 antibodies, or (e) vehicle. The different treatments were assayed at different concentrations and co-cultured for an additional 24 hours. To evaluate Aβ1–40-mediated cytotoxicity, PC12 cell viability was quantified by using the MTT assay (colorimetric assay for assessing cell metabolic activity). Aβ1–40 oligomers produced a concentration-dependent cytotoxic effect on PC12 cells whereas Aβ1–40 monomers did not. Pre-incubation of Aβ1–40 monomers with anti-Aβx-40 antibodies prevented the formation of Aβ1–40 oligomers, resulting in increased cell viability in the cultures. The beneficial effect of anti-Aβx-40 antibodies was dependent on the extent of oligomerization, being more protective when present at the beginning of oligomer formation than once the oligomers have been formed.