Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Jan 19;25(3):672-9.
doi: 10.1523/JNEUROSCI.4276-04.2005.

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

Peter J Crouch  1 , Rachel Blake, James A Duce, Giuseppe D Ciccotosto, Qiao-Xin Li, Kevin J Barnham, Cyril C Curtain, Robert A Cherny, Roberto Cappai, Thomas Dyrks, Colin L Masters, Ian A Trounce
Affiliations

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

Peter J Crouch et al. J Neurosci. .

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Inhibition of COX activity by Aβ1-42 solutions aged in the presence of Cu2+. A, Aβ1-42/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 Aβ1-42/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.
Figure 2.
Effects of Aβ1-42/Cu2+ and Aβ42-1/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+ (▤), Aβ1-42/Cu2+ (□), or Aβ42-1/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.
Figure 3.
The requirement for Cu2+ in Aβ1-42-mediated inhibition of COX activity. Mitochondria isolates were incubated for 5 min with vehicle/Cu2+ (▤) or Aβ1-42/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 Aβ1-42 and vehicle solutions. C, Aβ1-42 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.
Figure 4.
SDS-PAGE and Western blot analysis of Aβ1-42/Cu2+ and the correlation between the abundance of Aβ1-42/Cu2+ species and Aβ-mediated inhibition of COX activity. Aβ1-42/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 Aβ1-42/Cu2+ solution, and the correlation (R2) between the two was determined. Five Aβ1-42/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.
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Anandatheerthavarada HK, Biswas G, Robin M-A, Avadhani NG (2003) Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells. J Cell Biol 161: 41-54. - PMC - PubMed
    1. Anderson MF, Sims NR (2000) Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples. Brain Res Protoc 5: 95-101. - PubMed
    1. Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NME, Romano DM, Hartshorn MA, Tanzi RE, Bush AI (1998) Dramatic aggregation of Alzheimer Aβ by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem 273: 12817-12826. - PubMed
    1. Atwood CS, Perry G, Zeng H, Kato Y, Jones WD, Ling K-Q, Huang X, Moir RD, Wang D, Sayre LM, Smith MA, Chen SG, Bush AI (2004) Copper mediates dityrosine cross-linking of Alzheimer's amyloid-β. Biochemistry 43: 560-568. - PubMed
    1. Bitan G, Lomakin A, Teplow DB (2001) Amyloid β-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins. J Biol Chem 276: 35176-35184. - PubMed

Publication types

LinkOut - more resources

-