Curcumin Reverses Metal-induced Ab Aggregation Independent of Metal Chelation Stanley Kwok-Kuen CHEUNG, Larry BAUM Department of Medicine and Therapeutics, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Presentation Number: 171.7 Curcumin disaggregated Ab(1-40):CuCl2 aggregates. Shown below is a western blot of supernatants in reducing sample buffer, at two different film exposures. Amount of monomeric, dimeric and trimeric Ab(1-40) compared with standard Ab(1-40) from the above gel. Curcumin significantly reduces amyloid pathology in Tg2576 mice. However, the mechanism by which curcumin does so is still under investigation. Curcumin inhibits Ab oligomerization and disaggregates Ab fibrils in vitro, and binds to plaques. Alzheimer’s disease amyloid plaques co-localize with metal ions, such as Fe2+ and Zn2+, which enhance the aggregation of synthetic amyloid-b in vitro. Previously, we showed that curcumin has different binding affinities to Fe2+ and Cu2+, in the micromolar range, while Zn2+ binding was not detectable. Thus, the purpose of this experiment is to see whether curcumin can reverse metal-induced Ab aggregates. 10mM of Ab(1-40) and Ab(1-42) were induced to aggregate by either 20mM of CuCl2 or ZnCl2 at pH 6.6 with 20mM of Thioflavin-T for 24 hours at 37oC. The disaggregation assay was done by adding 0, 0.1, 0.5, 1, 5, 10 and 20mM of curcumin, and the mixtures were incubated for 2 hours. The fluorescence signal was measured at Ex:432nm and Em:493nm. The experiments were done in triplicate. After fluorescence measurement, the samples of Ab(1-40):CuCl2 aggregates were centrifuged at 20,000 x g for 10 minutes at either 4oC or room temperature. The supernatant was analysed by 16.5% Tris-Tricine SDS-PAGE. After transferring onto 0.2mm nitrocellulose blotting paper, it was probed with monocloncal Ab antibody 6E10. The developed films were scanned and analysed by densitometer against standard Ab(1-40) peptides in the amount of 1, 5, 10 and 20ng per lane. Introduction 1 Curcumin disaggregated Ab(1-40):CuCl2 aggregates. Shown below is a western blot of supernatants in non-reducing sample buffer: Amount of monomeric and dimeric Ab(1-40) compared with standard Ab(1-40) from the above gel. 20 10 5 1 0.5 0.1 0 1 5 10 20 Curcumin Concentration, mM Ab40, ng Dimer Monomer 4 20 10 5 1 0.5 0.1 0 1 5 10 20 Curcumin Concentration, mM Ab40, ng Monomer Trimer Dimer 5 20 10 5 1 0.5 0.1 0 1 5 10 20 Curcumin Concentration, mM Ab40, ng Dimer Monomer 2 Methods 6 Conclusions 3 Curcumin can disaggregate amyloid-b aggregates induced by metal ions. Curcumin can disaggregate metal-induced amyloid-b aggregates independent of its binding affinities with different metal ions. Such disaggregation may be due to the binding of curcumin directly to Ab at the site(s) which is/are not competing with metal ions. Determination of Ab-curcumin binding would help to develop drugs which can prevent and cure Alzheimer’s disease. Thioflavin-T fluorescence measurement of the disaggregation of Ab aggregates induced by metal ions. EC50s are less than 0.5mM. Results