Copper-dependent inhibition of human cytochrome c oxidase by a dimeric conformer of amyloid-beta1-42

Abstract

In studies of Alzheimer's disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-beta (Abeta) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid Abeta peptide (Abeta1-42) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic Abeta1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu2+ and specific "aging" of the Abeta1-42 solution. Maximal COX inhibition occurred when using Abeta1-42 solutions aged for 3-6 h at 30 degrees C. The level of Abeta1-42-mediated COX inhibition increased with aging time up to approximately 6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric Abeta as the only Abeta species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimer's model mouse (Tg2576) revealed abundant Abeta immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous Abeta is associated with brain mitochondria and that Abeta1-42, possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu2+. We conclude that Cu2+-dependent Abeta-mediated inhibition of COX may be an important contributor to the neurodegeneration process in Alzheimer's disease.

Figure 1.

Inhibition of COX activity by Aβ_1-42 solutions aged in the presence of Cu²⁺. A, Aβ_1-42/Cu²⁺ (□) and vehicle/Cu²⁺ (⋄) solutions were aged at 30°C for the times shown and then incubated with mitochondria isolates for 5 min before assaying for COX activity. All activities are expressed relative to the vehicle/Cu²⁺ control. Error margins represent the SD of the mean (n = 4). COX activity for vehicle/Cu²⁺-treated mitochondria isolates was 9.38 (±1.12) k · min^-1 · mg^-1 mitochondrial protein. B, Mitochondria isolates were incubated with Aβ_1-42/Cu²⁺ (aged for 5 h at 30°C) at the concentrations shown and then assayed for COX activity. All activities are expressed relative to the 0 μm Aβ control. Error margins represent the SD of the mean (n = 4). COX activity for 0 μm Aβ-treated mitochondria isolates was 7.23 (±0.63) k · min^-1 · mg^-1 mitochondrial protein.

Figure 2.

Effects of Aβ_1-42/Cu²⁺ and Aβ_42-1/Cu²⁺ on activity of the electron transport chain enzyme complexes II, III, and COX, and the mitochondrial matrix enzyme citrate synthase. Mitochondria isolates were incubated for 5 min with vehicle/Cu²⁺ (▤), Aβ_1-42/Cu²⁺ (□), or Aβ_42-1/Cu²⁺ (▤) solutions that had been aged for 5 h at 30°C and then assayed for the activity of COX (A), complex II (B), complex III (C), and citrate synthase (D). All activities are expressed relative to the vehicle/Cu²⁺ control. Error margins represent the SD of the mean (n = 3-5). COX activity in vehicle/Cu²⁺-treated mitochondria was 7.41 (±1.48) k · min^-1 · mg^-1 mitochondrial protein. Specific activities (min^-1 mg^-1 mitochondrial protein) in vehicle/Cu²⁺-treated mitochondria for complex II, complex III, and citrate synthase were 26.1 (±6.3) nmol succinate, 129.5 (±14.3) nmol cytochrome c, and 231.6 (±21.6) nmol acetyl-CoA, respectively. ANOVA; ^*p < 0.05 and ^**p < 0.001 compared with vehicle/Cu²⁺ control; n = 3-5.

Figure 3.

The requirement for Cu²⁺ in Aβ_1-42-mediated inhibition of COX activity. Mitochondria isolates were incubated for 5 min with vehicle/Cu²⁺ (▤) or Aβ_1-42/Cu²⁺ (□) solutions that had been aged for 5 h at 30°C, and subsequent COX activity in the mitochondria isolates was determined. A, EGTA was included in the 5 min incubation at a final concentration of 700 μm, thus providing an EGTA/Cu²⁺ molar ratio of 2:1. B, Cu²⁺ was omitted from the Aβ_1-42 and vehicle solutions. C, Aβ_1-42 and vehicle solutions were prepared and aged with Cu²⁺ (supplied as CuCl₂ or CuSO₄), Zn²⁺ (as ZnCl₂), Fe²⁺ (as FeSO₄), or Fe³⁺ (as FeCl₃). All activities are expressed relative to the vehicle controls. Error margins represent the SD of the mean (n = 3-5). ANOVA; ^*p < 0.001 compared with vehicle controls; n = 3-5.

Figure 4.

SDS-PAGE and Western blot analysis of Aβ_1-42/Cu²⁺ and the correlation between the abundance of Aβ_1-42/Cu²⁺ species and Aβ-mediated inhibition of COX activity. Aβ_1-42/Cu²⁺ solutions were aged at 30°C for 0, 2, 4, 6, 10, and 24 h and then either cross-linked by PICUP or assayed for their effects on COX activity in mitochondrial isolates. A, Different Aβ species present in the cross-linked samples were resolved on 15% Tris-tricine gels, and their relative abundance, as a percentage of total Aβ, was determined by densitometry. B, The relative abundance of each Aβ species was plotted against percentage COX activity in mitochondria isolates treated with the Aβ_1-42/Cu²⁺ solution, and the correlation (R²) between the two was determined. Five Aβ_1-42/Cu²⁺ solutions were aged for the times shown and then assayed for their effects on COX activity, relative to vehicle/Cu²⁺ solutions aged under the same conditions, or cross-linked and then analyzed by SDS-PAGE. One hundred percent COX activity indicates no Aβ-mediated inhibition relative to the vehicle/Cu²⁺ control treatment. Significance of the R² values was determined by linear regression analysis.

Figure 5.

SDS-PAGE and Western blot analysis of APP and Aβ in brain mitochondria of Tg2576 mice. A, Aβ and APP in total homogenate (H) and mitochondria (M) samples from the brain and liver of 18-month-old Tg2576 (Tg) and wild-type (WT) mice were resolved on 10-20% Tris-tricine gels and Western blotted with WO2 for Aβ/APP. Aβ was greatly enriched in the brain mitochondria of Tg2576 mice but nondetectable in brain mitochondria of age-matched WT controls. Fifty micrograms of total protein (for mitochondria samples) or 30 μg of total protein (for homogenate samples) was loaded per lane. The gels were reprobed with Porin for mitochondria (B) or HO-1 for endoplasmic reticulum (C), and this revealed that the mitochondria fractions were relatively free from endoplasmic reticulum.