Reversal of age-related learning deficits and brain oxidative stress in mice with superoxide dismutase/catalase mimetics

Abstract

Oxidative stress has been implicated in cognitive impairment in both old experimental animals and aged humans. This implication has led to the notion that antioxidant defense mechanisms in the brain are not sufficient to prevent age-related increase in oxidative damage and that dietary intake of a variety of antioxidants might be beneficial for preserving brain function. Here we report a dramatic loss of learning and memory function from 8 to 11 months of age in mice, associated with marked increases in several markers of brain oxidative stress. Chronic systemic administration of two synthetic catalytic scavengers of reactive oxygen species, Eukarion experimental compounds EUK-189 and EUK-207, from 8 to 11 months almost completely reversed cognitive deficits and increase in oxidative stress taking place during this time period in brain. In particular, increase in protein oxidation was completely prevented, whereas increase in lipid peroxidation was decreased by approximately 50%. In addition, we observed a significant negative correlation between contextual fear learning and levels of protein oxidation in brain. These results further support the role of reactive oxygen species in age-related learning impairment and suggest potential clinical applications for synthetic catalytic scavengers of reactive oxygen species.

Fig. 1.

Structures of EUK-189 and EUK-207.

Fig. 2.

Effects of chronic treatment with EUK-189 or EUK-207 on context and tone conditioning. Eight-month-old C57 mice were treated for 3 months with a low (1.5 mM) or a high (15 mM) concentration of EUK-189 or EUK-207 delivered i.p. continuously through Alzet minipumps. At the end of 3 months, mice were conditioned to context and tone as described in Materials and Methods. Results were calculated as percent time the mouse expressed freezing behavior during the 8-min observation period for context (A) and 6 min for tone (B). The data were then expressed as the percent of the corresponding values in vehicle control mice. Shown are means ± SEM of 16–18 mice. *, P < 0.01; †, P < 0.005 (Student's t test). Control 8-month-old mice were subjected to the same training and testing procedures, and the data were calculated as percent time the mouse expressed freezing behavior for the 8-min observation period for context (C) and 6 min for tone (D). Results are means ± SEM of 16–18 mice. Repeated-measures ANOVA indicated that the effect of age was highly significant (P < 0.001), as was the effect of EUK-207 (P < 0.01 vs. 11-month-old vehicle control). EUK-207 treatment completely reversed the aging effect on context, but only partially on tone.

Fig. 3.

Effects of chronic treatment with EUK-189 or EUK-207 on lipid peroxidation and protein oxidation in brain homogenates. At the end of the behavioral experiments, the mice were killed and their brains (minus cerebellum) were frozen. Levels of lipid peroxidation and protein oxidation (protein carbonyls) were determined as described in Materials and Methods. Results were expressed as percentage of vehicle control value (mean ± SEM, n = 12; A and B). *, P < 0.05; †, P < 0.01 (Student's t test). Levels of lipid peroxidation and protein carbonyls were also determined in brain homogenates from the 8-month-old control C57 mice. The levels of lipid peroxidation were expressed as nmol malondialdehyde equivalent per mg of protein (C) and the levels of protein oxidation as nmol carbonyl per mg of soluble extracted protein (D), and are means ± SEM of 12 mice [C: *, P < 0.05 as compared with either 8- or 11 month-old control mice; D: *, results not significantly different from 8-month-old control mice; †, P < 0.005 as compared with 11-month-old vehicle mice (Student's t test)].

Fig. 4.

Correlation between performance in the contextual fear-conditioning task and brain levels of protein carbonyls. Individual data for contextual fear conditioning and brain protein carbonyl contents for the 8-month-old control mice were plotted and exhibited a significant negative correlation (Pearson's correlation coefficient, γ =-0.76, P < 0.005). The levels of protein oxidation were expressed as nmol of carbonyl per mg of soluble extracted protein, and contextual fear conditioning was scored as percentage of freezing duration per min (mean ± SEM of 12 mice). Note that deleting the two outlying points still resulted in a significant correlation, γ = -0.74, P < 0.005.

Fig. 5.

EUK-207 treatment decreases levels of oxidized nucleic acids in hippocampus and amygdala. Eleven-month-old control mice (A, C, E, and G) and EUK-207 (1.5 mM)-treated mice (B, D, F, and H) were perfused with 4% paraformaldehyde. Brains were sectioned at 25 μm and sections were processed for immunohistochemistry by using a mAb to oxo8dG/oxo8G as described in Materials and Methods. Note the clear decrease in oxo8dG/oxo8G immunoreactivity in the pyramidal cells of CA3 of treated mice (B and D) compared with control mice (A and C), and decrease in diffuse staining in amygdala (F and H vs. E and G). High-magnification Insets in A, B, G, and H illustrate the cytoplasmic cellular localization of the oxidized nucleic acids. Similar results were observed in five mice.