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, 106 (47), 20057-62

Amyloid-beta and Tau Synergistically Impair the Oxidative Phosphorylation System in Triple Transgenic Alzheimer's Disease Mice

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Amyloid-beta and Tau Synergistically Impair the Oxidative Phosphorylation System in Triple Transgenic Alzheimer's Disease Mice

Virginie Rhein et al. Proc Natl Acad Sci U S A.

Abstract

Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APP(sw)PS2(N141I) double-transgenic APP152 mice develop Abeta plaques. Cross-breeding generates triple transgenic ((triple)AD) mice that combine both pathologies in one model. To determine functional consequences of the combined Abeta and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from (triple)AD mice, the parental strains, and nontransgenic mice, followed by the quantitative mass-tag labeling proteomic technique iTRAQ and mass spectrometry. Within 1,275 quantified proteins, we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Notably, deregulation of complex I was tau dependent, whereas deregulation of complex IV was Abeta dependent, both at the protein and activity levels. Synergistic effects of Abeta and tau were evident in 8-month-old (triple)AD mice as only they showed a reduction of the mitochondrial membrane potential at this early age. At the age of 12 months, the strongest defects on OXPHOS, synthesis of ATP, and reactive oxygen species were exhibited in the (triple)AD mice, again emphasizing synergistic, age-associated effects of Abeta and tau in perishing mitochondria. Our study establishes a molecular link between Abeta and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
High-resolution respiratory system reveals a heightened defect in the mitochondrial OXPHOS from brains of tripleAD mice. Measurement of oxygen (O2) flux and consumption in freshly isolated mitochondria from cortical brains of age-matched wild-type, APP/PS2, and tripleAD mice. After detection of endogenous respiration (mito), glutamate+malate (g/m) were added to induce state 4 respiration. ADP stimulated state 3 respiration. After determining coupled respiration, FCCP was added and the maximal respiratory capacity measured in the absence of a proton gradient. Cytochrome c (cyt c) demonstrated mitochondrial membrane integrity. To inhibit activities of complexes I–III, rotenone (rot) and antimycine A (AA) were added. Complex IV activity was stimulated by ascorbate/TMPD (A/T) before terminating mitochondrial respiration by adding sodium azide (azide). O2 consumption was normalized to the corresponding citrate synthase (CS) activity. (A) Representative diagrams of O2 flux and consumption in mitochondria from 12-month-old wild-type, APP/PS2, and tripleAD mice in response to titrated substrates and inhibitors of mitochondrial complexes. (B) RCR3/4 (state3/state4 ratio) representing the mitochondrial coupling state was reduced in 8- and 12-month-old APP/PS2 and tripleAD mice. (C) ETS/ROX ratio, which yields an index of the maximum oxygen consumption capacity of the electron transport system (ETS) relative to the magnitude of residual oxygen consumption (ROX), was reduced in 8- and 12-month-old APP/PS2 and tripleAD mice compared with age-matched wild-type mitochondria. (D) Two-way ANOVA revealed a significant effect of the transgene on the respiratory rates of mitochondria between 8-month-old wild-type and APP/PS2 mice (P < 0.001). (E) No difference was observed in respiration between 8-month-old APP/PS2 and tripleAD mice. (F) At 12 months of age, respiration differed again significantly between wild-type and APP/PS2 (P < 0.001) and (G) between APP/PS2 and tripleAD mice (P < 0.001). (B and C) One-way ANOVA post hoc Tukey's. (D–G) Two-way ANOVA post hoc Bonferroni. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. wild-type; ++, P < 0.01; +++, P < 0.001 vs. APP/PS2 (n = 7–12 animals/group).
Fig. 2.
Fig. 2.
Impaired mitochondrial enzyme activities and decreased ATP levels in cortical brain cells from tripleAD mice. (A) Complex I activity (DBQ/HAR ratio) was decreased in 8-month-old pR5 mitochondria. At 12 months, all 3 transgenic mouse models presented a decrease in complex I activity. (B) Citrate synthase (CS) activity was increased in 8-month-old APP/PS2 and tripleAD mice. At 12 months, the increase persisted only in tripleAD mice. (C) Complex IV activity (CIV/CS ratio) was decreased in APP/PS2 and tripleAD mitochondria at 8 months of age. The decrease became more pronounced at the age of 12 months. (D) ATP levels were reduced in 12-month-old APP/PS2 and triple mice. (A–D) One-way ANOVA post hoc Tukey's. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. wild type; +, P < 0.05 vs. pR5 (n = 7–12 animals/group).
Fig. 3.
Fig. 3.
Reduced MMP and increased ROS levels in cortical brain cells from tripleAD mice. (A) MMP (TMRE fluorescence units/mg protein) was reduced in cortical cells from 8-month-old tripleAD mice. At the age of 12 months, MMP was also reduced in cells from APP/PS2 mice. (B) Levels of superoxide anion radicals (DHE fluorescence units/mg protein) and (C) cytosolic ROS (DCF fluorescence units/mg protein) were increased in cells from 12-month-old APP/PS2 and tripleAD mice. (A–C) One-way ANOVA post hoc Tukey's. *, P < 0.05; **, P < 0.01; vs. wild-type, ++, P < 0.01 vs. pR5 (n = 7–12 animals/group).

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