Inhibition of amyloid-beta (Abeta) peptide-binding alcohol dehydrogenase-Abeta interaction reduces Abeta accumulation and improves mitochondrial function in a mouse model of Alzheimer's disease

Abstract

Amyloid-β (Aβ) peptide-binding alcohol dehydrogenase (ABAD), an enzyme present in neuronal mitochondria, exacerbates Aβ-induced cell stress. The interaction of ABAD with Aβ exacerbates Aβ-induced mitochondrial and neuronal dysfunction. Here, we show that inhibition of the ABAD-Aβ interaction, using a decoy peptide (DP) in vitro and in vivo, protects against aberrant mitochondrial and neuronal function and improves spatial learning/memory. Intraperitoneal administration of ABAD-DP [fused to the transduction of human immunodeficiency virus 1-transactivator (Tat) protein and linked to the mitochondrial targeting sequence (Mito) (TAT-mito-DP) to transgenic APP mice (Tg mAPP)] blocked formation of ABAD-Aβ complex in mitochondria, increased oxygen consumption and enzyme activity associated with the mitochondrial respiratory chain, attenuated mitochondrial oxidative stress, and improved spatial memory. Similar protective effects were observed in Tg mAPP mice overexpressing neuronal ABAD decoy peptide (Tg mAPP/mito-ABAD). Notably, inhibition of the ABAD-Aβ interaction significantly reduced mitochondrial Aβ accumulation. In parallel, the activity of mitochondrial Aβ-degrading enzyme PreP (presequence peptidase) was enhanced in Tg mAPP mitochondria expressing the ABAD decoy peptide. These data indicate that segregating ABAD from Aβ protects mitochondria/neurons from Aβ toxicity; thus, ABAD-Aβ interaction is an important mechanism underlying Aβ-mediated mitochondrial and neuronal perturbation. Inhibitors of ABAD-Aβ interaction may hold promise as targets for the prevention and treatment of Alzheimer's disease.

Figure 1.

Effect of systemic administration of mito-ABAD peptide on the formation of ABAD-Aβ complex in Tg mAPP mice. A, Brain mitochondria isolated from vehicle-treated non-Tg or Tg mAPP mice, and Tg mAPP mice treated with mito-ABAD-DP or mito-ABAD-RP were subjected to immunoprecipitation with 6E10 antibody followed by immunoblotting with anti-ABAD IgG. Top, Two immunoreactive bands (M_r ∼27 and ∼5 kDa) were detected. Bottom, Immunoblotting for COX IV showing an equal amount of mitochondrial protein used for the experiments. B, C, Quantification of intensity of the immunoreactive bands corresponding to M_r ∼27 (B) and ∼5 kDa (C). n = 3–6 mice per group. *p < 0.01 compared with vehicle- or mito-ABAD-RP-treated mAPP mice.

Figure 2.

Effect of systemic administration of mito-ABAD peptide on mitochondrial function and spatial memory. A, B, Effect on mitochondrial function. The respiratory control rate (RCR) (A), and enzyme activities associated with complexes III and IV (B) were determined in the brain mitochondria isolated from the indicated Tg mice. C, Quantification of the area occupied by HNE-4 staining in hippocampus of the indicated Tg mice. n = 3–8 mice per group. D, Spatial learning and memory were tested using a radial arm water maze in Tg mAPP mice treated with vehicle, mito-ABAD-DP (DP), or mito-ABAD-RP (RP), and non-Tg mice or Tg mAPP mice treated with vehicle (n = 7–11 mice per group). A1–A4 denote the acquisition trials, and R denotes the retention trial. *p < 0.01 compared with vehicle- or mito-ABAD-RP-treated Tg mAPP mice. ^#p < 0.05 compared with vehicle-treated non-Tg mice.

Figure 3.

Expression of ABAD(92-120) reduces ABAD-Aβ complex formation, restores mitochondrial function, and attenuates generation of ROS. A, Immunoprecipitation of cortical mitochondria of the indicated Tg mice with 6E10 followed by immunoblotting with anti-ABAD showed two immunoreactive bands, M_r ∼27 (ABAD/Aβ complex) and ∼5 kDa [ABAD(92-120)/Aβ complex)]. Bottom shows immunoblotting of cortical mitochondrial fractions with α-COX IV. B, C, Quantification of intensity of immunoreactive bands corresponding to M_r ∼27 and ∼5 kDa (B) and oxygen consumption (C, respiratory control ratio) in cortical mitochondria from the indicated Tg mice. D, Fluorescence intensity of HEt (indicator of ROS) in brain homogenates. E, F, Quantification of area occupied by HEt staining in the cerebral cortex (E) and hippocampus (F) of the indicated Tg mice. *p < 0.01 vs Tg mAPP mice. n = 3–6 mice per group.

Figure 4.

Neuropathology and behavior in mAPP/mito-ABAD mice. A, AChE-positive neurites were visualized histochemically in the subiculum (Sb) of the indicated Tg mice. B, AChE activity in the subiculum of the indicated Tg mice. *p < 0.01 vs other groups of mice. C, Behavioral studies in Tg mice. Spatial learning and memory was tested in the radial arm water maze in the indicated Tg mice. A1–A4 denote the acquisition trials, and R denotes the retention trial. *p < 0.01 Tg mAPP mice vs other groups of mice. n = 4–8 mice per group.

Figure 5.

Effect of ABAD antagonizing peptide on accumulation of mitochondrial Aβ in Tg mAPP mice. A, B, ELISA for measurement of mitochondrial Aβ40 (A) and -42 (B) levels in Tg mAPP mice treated with Mito-ABAD-DP or -RP at age of 10–11 months. *p < 0.01 vs vehicle- or Mito-ABAD-RP-treated Tg mAPP mice. C, D, ELISA for Aβ40 (C) and Aβ42 (D) in the cortical mitochondria from the indicated Tg mice at 10–11 months old. *p < 0.01 vs Tg mAPP mice. E, PreP activity for degradation of biotin-Aβ. F, Quantification of density of all Aβ-immunoreactive bands incubated with mitochondrial fractions from non-Tg littermates (lanes 4–7), Tg mAPP mice (lanes 8–11), double Tg mAPP/mito-ABAD (lanes 12–15), and Tg mAPP/mito-ABAD plus anti-PreP antibody mice (lanes 16–19) using NIH ImageJ software. *p < 0.01 vs other groups of mice. n = 5–10 mice per group.