doi:10.1016/j.nbd.2004.09.003 
Copyright © 2004 Elsevier Inc. All rights reserved.
Impaired Cu/Zn-SOD activity contributes to increased oxidative damage in APP transgenic mice
Katrin Schuessela, Stephanie Schäferb, Thomas A. Bayerb, Christian Czechc, d, Laurent Pradierd, Franz Müller-Spahne, Walter E. Müllera and Anne Eckerta, e, 
, 
aDepartment of Pharmacology, Biocentre, J.W. Goethe University of Frankfurt, Germany
bDepartment of Psychiatry, Division of Neurobiology, University of the Saarland Medical Center, D-66421 Homburg, Germany
cF. Hoffmann-La Roche AG, CNS Research, CH-4070 Basel, Switzerland
dAventis Pharma, Research and Development, F-94403 Vitry-sur-Seine, France
eNeurobiology Research Laboratory, Psychiatry University Hospital, CH-4025 Basel, Switzerland
Received 26 July 2004;
revised 7 September 2004;
accepted 13 September 2004.
Available online 20 November 2004.
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Abstract
Oxidative stress plays an important role in the pathogenesis of Alzheimer's disease. To determine which mechanisms cause the origin of oxidative damage, we analyzed enzymatic antioxidant defense (Cu/Zn-superoxide dismutase Cu/Zn-SOD, glutathione peroxidase GPx and glutathione reductase GR) and lipid peroxidation products malondialdehyde MDA and 4-hydroxynonenal HNE in two different APP transgenic mouse models at 3–4 and 12–15 months of age. No changes in any parameter were observed in brains from PDGF-APP695SDL mice, which have low levels of Aβ and no plaque load. In contrast, Thy1-APP751SL mice show high Aβ accumulation with aging and plaques from an age of 6 months. In brains of these mice, HNE levels were increased at 3 months (female transgenic mice) and at 12 months (both gender), that is, before and after plaque deposition, and the activity of Cu/Zn-SOD was reduced. Interestingly, β-amyloidogenic cleavage of APP was increased in female Thy1-APP751SL mice, which also showed increased HNE levels with simultaneously reduced Cu/Zn-SOD activity earlier than male Thy1-APP751SL mice. Our results demonstrate that impaired Cu/Zn-SOD activity contributes to oxidative damage in Thy1-APP751SL transgenic mice, and these findings are closely linked to increased β-amyloidogenic cleavage of APP.
Keywords: Aging; Alzheimer's disease; Amyloid beta; Amyloid precursor protein APP; Gender; Lipid peroxidation; Transgenic mouse; Oxidative stress; Superoxide dismutase; Transgenic
Abbreviations: AD, Alzheimer's disease; Aβ, amyloid beta; APP, amyloid precursor protein; FAD, familial Alzheimer's disease; GPx, glutathione peroxidase; GR, glutathione-disulfide reductase; HNE, 4-hydroxynonenal; MDA, malondialdehyde; PS1, presenilin 1; PS2, presenilin 2; sAPP, soluble APP; SOD, superoxide dismutase
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Fig. 1. (a) Levels of Tris-soluble Aβ accumulate in aged Thy1-APP751SL transgenic mice. Brain homogenates were blotted after Tris-buffered saline extraction and probed for APP, sAPP alpha, C99, and Aβ. Expression of full-length APP does not change with age, but there is a small rise in C99 peptide levels with aging and profound accumulation of Aβ levels. sAPP alpha is represented by the weakly stained bands below the APP band. Lanes 1 and 2: 3 months; lanes 3 and 4: 7 months; lanes 5 and 6: 14-month-old Thy1-APP751SL transgenic mice; each lane representing one individual animal. (b) Comparison of SDS-soluble brain homogenates from 3-month-old Thy1-APP751SL mice (lanes 1–4) and 13- to 15-month-old PDGF-APP695SDL mice (lanes 5 and 6); each lane representing one individual animal. Lower panel: actin loading control band. The different amino acid forms of full-length APP are detected at different retentions according to the number of amino acids expressed: the 751 amino acid form expressed in Thy1-APP751SL mice (lanes 1–4) is detected at higher retention than the 695 amino acid form in PDGF-APP695SDL mice (lanes 5 and 6). Aβ peptide as well as high levels of C99 fragment can be already detected in 3-month-old Thy1-APP751SL mice, whereas Aβ is undetectable and the intensity of the C99 band is much lower in 13- to 15-month-old PDGF-APP695SDL mice. Note the difference in C99 levels and Aβ peptide between female (lanes 1 and 3) and male (lanes 2 and 4) Thy1-APP751SL transgenic mice.
Fig. 2. Comparison of Aβ levels between male and female Thy1-APP751SL transgenic mice. (a) Aβ bands in Western blots from SDS-soluble brain extracts from female (lanes 1, 3, 4, and 6) and male (lanes 2, 5, and 7) 3-month-old Thy1-APP751SL transgenic mice; each lane representing one individual animal. (b) Densitometric evaluation of SDS-soluble Aβ bands. Intensity of Aβ bands is 32% higher in female compared to age-matched male Thy1-APP751SL transgenic mice. **P < 0.01 Student's t test. (c) Comparison of soluble Aβ1–40 levels between 3- and 12-month-old male and female Thy1-APP751SL transgenic mice analyzed by ELISA. Levels of soluble Aβ1–40 are much higher in aged animals (right axis) compared to young animals (left axis). In both age groups, female transgenic mice have higher levels of soluble Aβ1–40 than male mice. Two-way ANOVA: P < 0.05 effect of gender, P < 0.001 effect of aging, interaction P = 0.05. *P < 0.05 Student's t test, 12-month-old female vs. male.
Fig. 3. Levels of lipid peroxidation products in young (3 months) and aged (12 months) non-transgenic and Thy1-APP751SL mice (a). HNE levels. Two-way ANOVA revealed a significant effect of transgene (P < 0.01). Post hoc t test: *P < 0.05, 12-month-old Thy1-APP751SL vs. 12-month-old non-transgenic mice; P = 0.07 young vs. aged non-transgenic mice. (b). MDA levels. Two-way ANOVA revealed a significant effect of age (P < 0.0001). Post hoc t test: P < 0.01, young vs. aged non-transgenic mice.
Fig. 4. Activity of Cu/Zn-SOD in young (3 months) and aged (12 months) non-transgenic and Thy1-APP751SL mice. Two-way ANOVA revealed a significant effect of transgene (P < 0.01) and age (P < 0.05) with no significant interaction between the two parameters. Post hoc t test: *P < 0.05, 12-month-old Thy1-APP751SL vs. 12-month-old non-transgenic mice; P < 0.05, 3-month-old vs. 12-month-old non-transgenic mice.
Fig. 5. Levels of lipid peroxidation in female and male mice aged 3 and 12 months. (a) HNE levels. Two-way ANOVA revealed a significant effect of transgene (P < 0.01) in 12-month-old mice and a significant gender effect in both age groups (P < 0.01 in 3 months old, P < 0.05 in 12-month-old mice). Post hoc t test: *P < 0.05, 3-month-old female non-transgenic vs. female Thy1-APP751SL transgenic mice; +P < 0.05, 12-month-old male non-transgenic vs. male Thy1-APP751SL transgenic mice. (b) MDA levels. Two-way ANOVA revealed a significant effect of gender in both age groups (P < 0.01 in 3 months old, P < 0.05 in 12-month-old mice). Post hoc t test: *P < 0.05, 3-month-old female non-transgenic vs. female Thy1-APP751SL transgenic mice.
Fig. 6. Cu/Zn-SOD activity in female and male mice aged 3 and 12 months. Two-way ANOVA revealed a significant effect of transgene (P < 0.05) in 12-month-old mice. Post hoc t test: *P < 0.05, 3-month-old female non-transgenic vs. female Thy1-APP751SL transgenic mice.
Table 1.
Levels of lipid peroxidation products in non-transgenic littermate control animals and PGDF-APP695SDL mice at 3–4 and 13–15 months
(+) post hoc t test P = 0.06 vs. 3-month-old non-transgenic mice.
* Post hoc
t test
P < 0.05 vs. 3 months old non-transgenic mice.
Table 2.
Activities of antioxidant enzymes in non-transgenic littermate control animals and PGDF-APP695SDL mice at 3–4 and 13–15 months
* Post hoc
t test
P < 0.0001 vs. Cu/Zn-SOD activity of 3- to 4-month-old non-transgenic mice.
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