Reciprocal modulation of Aβ42 aggregation by copper and homocysteine

Abstract

Hyperhomocysteinemia is a risk factor for Alzheimer's disease (AD). Both homocysteine (Hcy) and amyloid β (Aβ), which accumulates in the brain of AD patients, bind copper. Aim of this study was to test the hypothesis that the association of Hcy and AD results from a molecular interaction between Hcy and Aβ that is mediated by copper. We established a microtiter plate format thioflavin T aggregation assay to monitor Aβ42 fibrillization. Copper (5 μM) completely prevented Aβ42 (5 μM) fibrillization. Homocysteine in the absence of copper did not impact Aβ42 fibrillization, but physiological concentrations of Hcy (10-100 μM) attenuated the inhibitory effect of copper on Aβ42 fibril formation. These results were qualitatively confirmed by electron microscopy, which did not reveal morphological differences. To compare the toxicity of fibrillar and non-fibrillar Aβ42 exposed to copper or Hcy, rat primary cortical neurons were treated in vitro with 5 μM Aβ42 for 72 h. After incubation with 5 μM Aβ42 that had been aggregating in the absence of Hcy or copper, cell viability was reduced to 40%. Incubation with 5 μM Aβ42, in which fibril formation had been prevented or reverted by the addition of 5 μM copper, resulted in cell viability of approximately 25%. Accordingly, viability was reduced to 25% after incubation with 5 μM monomeric, i.e., non-fibrillized, Aβ42. The addition of Hcy plus copper to 5 μM Aβ42 yielded 50% viability. In conclusion, copper prevents and reverts Aβ fibril formation leading rather to formation of lower order oligomers or amorphous aggregates, and Hcy reduces these effects. Such mechanisms may explain the association of hyperhomocysteinemia and AD, leading to novel therapeutic strategies in the prevention and treatment of this disease.

Keywords: Alzheimer’s disease; Aβ; copper; cytotoxicity; homocysteine; primary neurons.

Figure 1

Effect of metal ions and homocysteine (Hcy) on Aβ42 fibrillization as observed in ThT-assay. Fibrillization of 5 μM Aβ42 (light gray) in the presence of (A) 5 μM ZnCl₂ (light blue) or 5 μM CuCl₂ (light green), (B) increasing concentrations of Hcy (5 μM—red, 10 μM—orange, 25 μM—dark green, 50 μM—pink, and 100 μM—dark blue), (C) 5 μM Hcy (red) or 5 μM CuCl₂ (light green), or 5 μM CuCl₂ together with increasing Hcy concentration (10 μM—orange, 25 μM—dark green, 50 μM—pink, and 100 μM—dark blue), and (D) 5 μM Hcy (red) or 5 μM ZnCl₂ (light blue), or 5 μM ZnCl₂ together with increasing Hcy concentration (25 μM—dark green, 50 μM—pink, and 100 μM—dark blue). All components were added to the reaction mixture directly at the beginning of the fibrillization reaction.

Figure 2

Visualization of Aβ42 fibrils. 110,000x transmission electron microscopy images of the end point products of Aβ42 fibrillization after 4 h of aggregation at 30°C. (A) 5 μM Aβ42 alone, (B) 5 μM Aβ42 + 50 μM Hcy and (C) 5 μM Aβ42 + 5 μM CuCl₂. Aβ42, Hcy and CuCl₂ were all added at the beginning of the ThT-assay. Scale bar represents 100 nm.

Figure 3

Cell viability and morphology of rat primary cortical neurons after treatment with CuCl₂ and Hcy in the absence or presence of Aβ42. Aggregations for viability and morphology studies were performed identically to previous aggregation assays, but lacking ThT due to its cytotoxicity. After 4 h agitation, samples were subsequently collected for the assessments. (A) Primary neurons were incubated for 72 h with increasing μM concentrations of CuCl₂ (dark gray), CuCl₂ + Hcy (light gray), or Hcy (stripes) without Aβ42, to study their individual cytotoxicity. Control sample (aggregation assay reaction mixture) is visualized in the white column. (B) Effect of CuCl₂ and Hcy on Aβ42-induced toxicity. 5 μM Aβ42; 5 μM Aβ42 + 5 μM CuCl₂; 5 μM Aβ42 + 5 μM CuCl₂ + 50 μM Hcy; 5 μM Aβ42 + 50 μM Hcy were incubated on the cells for 72 h. As controls non-fibrillar 5 μM Aβ42 (MonoAb) and aggregation assay reaction mixture without Aβ42 were used. All values are relative to reaction mixture control sample ± S.D. (C) Immunofluorescent staining of primary cortical neurons after 24 h incubation. Antibody against neuronal marker, MAP2 (green), visualizes the changes of neuronal morphology; whereas anti-human APP (red) shows the Aβ aggregates and DAPI (blue) the cell nuclei. Concentrations were as indicated in (B). Scale bar represents 100 μm.

Figure 4

Fibrillization of H6A mutated Aβ42 and scrambled Aβ42 in ThT-aggregation assay, and their cytotoxicity in rat primary cortical neurons. Fibrillization of 5 μM H6A mutated Aβ42 (H6A; light blue) (A) in the presence of increasing concentrations of Hcy (10 μM—orange, 25 μM—dark green, 50 μM—pink, and 100 μM—dark blue), (B) with 5 μM CuCl₂ and increasing concentrations of Hcy (10 μM—orange, 25 μM—dark green, 50 μM—pink, and 100 μM—dark blue). (C) 5 μM scrambled Aβ42 (ScAβ; light blue) does not form fibrils when incubated alone or together with 5 μM CuCl₂ or 5 μM CuCl₂ + 50 μM Hcy or 50 μM Hcy. (D) Cell viability of rat primary neurons after 72 h incubation with H6A fibrils or ScAβ incubated under same conditions. Samples from aggregation assay without ThT, but with CuCl₂, Hcy or both, were collected at the plateau after 4 h incubation. Concentrations were 5 μM H6A or ScAβ; 5 μM H6A or ScAβ + 5 μM CuCl₂; 5 μM H6A or ScAβ + 5 μM CuCl₂ + 50 μM Hcy; 5 μM H6A or ScAβ + 50 μM Hcy. As a control non-fibrillar 5 μM H6A or ScAβ (Mono) and aggregation assay reaction mixture without H6A or ScAβ were used. All values are relative to reaction mixture control sample ± S.D.

Figure 5

Aβ42 fibrils untangle by addition of CuCl₂ at timepoint 120 min in ThT aggregation assay. (A–C) CuCl₂ was added at the point indicated by an asterisk. (A) Addition of 5 μM CuCl₂ caused a drastic reduction in ThT fluorescence when added to an aggregation reaction of 5 μM Aβ42 (dark green). Water, in equal volume, (H₂O, dark gray) did not induce reduction in ThT fluorescence. As controls, aggregation of 5 μM Aβ42 (light gray) and 5 μM Aβ42 + 5 μM CuCl₂, added at the beginning of the reaction (light green), are shown. (B) When Aβ42 fibrils are formed in the presence of 15 μM Hcy (orange), their untangling after CuCl₂ addition depends on the molar ratio between CuCl₂ and Hcy (5 μM CuCl₂—light green and 15 μM CuCl₂—dark green). Water added in equal volumes to CuCl₂ did not change ThT fluorescence (light and dark blue). As a control aggregation of 5 μM Aβ42 (light gray) is shown. (C) After CuCl₂ addition Aβ42 fibrils that were previously formed in high Hcy concentration (5 μM Aβ42+ 50 μM Hcy) untangled. Degree of untangling was dependent on the molar ratio of Aβ42 to added CuCl₂ and on the molar ratio of Hcy to added CuCl₂ concentration (5 CuCl—light green, 15 μM CuCl₂—dark green, 20 μM CuCl₂—purple, 25 μM CuCl₂—light pink, 30 μM CuCl₂—light blue, and 50 μM CuCl₂—dark blue). (D) Water addition at timepoint 120 min did not induce changes in the ThT fluorescence of the reactions of 5 μM Aβ42 + 50 μM Hcy. Water was added in equal volumes as CuCl₂ in panel (C).