Relationship between Cognitive Dysfunction and Age-Related Variability in Oxidative Markers in Isolated Mitochondria of Alzheimer's Disease Transgenic Mouse Brains

Abstract

Many neurodegenerative disorders, including Alzheimer's disease (AD), are strongly associated with the accumulation of oxidative damage. Transgenic animal models are commonly used to elucidate the pathogenic mechanism of AD. Beta amyloid (Aβ) and tau hyperphosphorylation are very famous hallmarks of AD and well-studied, but the relationship between mitochondrial dysfunction and the onset and progression of AD requires further elucidation. In this study we used transgenic mice (the strain name is 5xFAD) at three different ages (3, 6, and 20 months old) as an AD model. Cognitive impairment in AD mice occurred in an age-dependent manner. Aβ1-40 expression significantly increased in an age-dependent manner in all brain regions with or without AD, and Aβ1-42 expression in the hippocampus increased at a young age. In a Western blot analysis using isolated mitochondria from three brain regions (cerebral cortex, cerebellum, and hippocampus), NMNAT-3 expression in the hippocampi of aged AD mice was significantly lower than that of young AD mice. SOD-2 expression in the hippocampi of AD mice was lower than for the age-matched controls. However, 3-NT expression in the hippocampi of AD mice was higher than for the age-matched controls. NQO-1 expression in the cerebral cortex of AD mice was higher than for the age-matched controls at every age that we examined. However, hippocampal NQO-1 expression in 6-month-old AD mice was significantly lower than in 3-month-old AD mice. These results indicate that oxidative stress in the hippocampi of AD mice is high compared to other brain regions and may induce mitochondrial dysfunction via oxidative damage. Protection of mitochondria from oxidative damage may be important to maintain cognitive function.

Keywords: Alzheimer’s disease; cognitive impairment; mitochondria; oxidative stress; reactive oxygen species.

Figure 1

Differences in cognitive function between control and Alzheimer’s disease (AD) transgenic mice depending on age. The time to goal (escape latency) in the Morris water maze test is shown in panel (A). The swimming trajectory and swimming speed are shown in panels (B,C). The ratio of staying time in the platform quadrant is shown in panel (D). Three different ages of AD mice (3 M, 6 M, 20 M) were used (3 M AD, n = 10; 6 M AD, n = 10; 20 M AD, n = 5). Age-matched C57BL/6 mice were used as a control group (3 M control, n = 15; 6 M control, n = 15; 20 M control, n = 10). * p < 0.05, vs. the age-matched control group. The data are shown as means ± SE. Statistical analyses of goal time were performed using two-way analysis of variance. Statistical analysis of goal time among all group was performed using the two-way analysis of variance. Statistical analyses of the goal time of each day, swimming speed, and the ratio of staying time in the platform quadrant were performed using the Tukey–Kramer method.

Figure 2

Time to fall in the rota-rod test. Three different ages of AD mice (3 M AD, n = 8; 6 M AD, n = 8; 20 M AD, n = 8) and age-matched C57BL/6 mice (3 M control, n = 8; 6 M control, n = 8; 20 M control, n = 5) were used. * p < 0.05 *** p < 0.001 vs. 3 M control. The data are shown as means ± SE. Comparisons were performed using the Tukey–Kramer method.

Figure 3

Changes in the levels of Aβ protein according to Western blot and immunohistochemical analyses. Western blotting experiments were performed using three different ages (3, 6, and 20 months) and three different brain regions (cerebral cortex, Cortex; cerebellum, Cer; hippocampus, Hip). Total protein expression in all bands was calculated for Aβ1-40 analysis (A). For Aβ1-42 analysis, the high-molecular-weight band (amyloid fibrils) was used for calculation (B). The ratio of each protein band intensity to Ponceau S intensity is shown in panels (A) and (B), with the ratios of each brain region of 3 M control samples set to 1. Black columns show 3 M, 6 M, and 20 M AD transgenic mice (3 M AD, n = 9; 6 M AD, n = 8; 20 M AD, n = 5), and white columns show age-matched C57BL/6 mice (3 M control, n = 10; 6 M control, n = 10; 20 M control, n = 10). Asterisks show significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The data are shown as means ± SE. Comparisons were performed using the Tukey–Kramer method; 6 M AD and control mice were used.

Figure 4

Western blotting analysis of the levels of tau and phospho6-tau expression in the brains of three different ages of AD mice. All experiments were performed using three different brain regions (cerebral cortex, Cortex; cerebellum, Cre; hippocampus, Hip). Western blotting images are shown in panel (A). Tau protein expression was calculated and shown in panel (B). Phospho-tau protein expression was calculated using two different band sizes (52 (C) and 75 kDa (D)). The ratio of each phospho-tau band intensity to Ponceau S intensity is shown, with the ratios of each brain region of 3 M control samples set to 1. Black columns show 3 M, 6 M, and 20 M AD mice (3 M AD, n = 10; 6 M AD, n = 10; 20 M AD, n = 5), and white columns show age-matched C57BL/6 mice (3 M control, n = 10; 6 M control, n = 10; 20 M control, n = 5). Asterisks show significant differences (* p < 0.05, ** p < 0.01). The data are shown as means ± SE. Comparisons were performed using the Tukey–Kramer method.

Figure 5

Western blotting analysis of the levels of neurotrophic-factor-related proteins in the brains of three different ages of AD transgenic mice. All experiments were performed using three different brain regions (cerebral cortex, Cortex; cerebellum, Cre; hippocampus, Hip). The ratio of each protein band intensity to Ponceau S intensity is shown, with ratios of each brain regions of 3 M control samples set to 1. Black columns show 3 M, 6 M, and 20 M AD mice (3 M AD, n = 8; 6 M AD, n = 8; 20 M AD, n = 4), and white columns show age-matched C57BL/6 mice (3 M control, n = 8; 6 M control, n = 8; 20 M control, n = 8). Asterisks show significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001). The data are shown as means ± SE. Comparisons were performed using the Tukey–Kramer method.

Figure 6

Western blotting analysis of the levels of each protein in isolated mitochondria of brains of three different ages of AD-transgenic mice. All experiments were performed using three different brain regions (cerebral cortex, Cortex; cerebellum, Cer; hippocampus, Hip). The mitochondrial isolation method is shown in panel (A). Western blotting images are shown in panel (B). The number of each sample is shown in panel (C). The ratios of each protein band intensity to COX-IV intensity are shown, with ratios of each brain region in 3 M control samples set to 1 (D). Black columns show 3 M, 6 M, and 20 M AD mice. White columns show age-matched control mice. Asterisks show significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The data are shown as means ± SE. Comparisons were performed using the Tukey–Kramer method.