A new role for oxidative stress in aging: The accelerated aging phenotype in Sod1-/- mice is correlated to increased cellular senescence

Abstract

In contrast to other mouse models that are deficient in antioxidant enzymes, mice null for Cu/Zn-superoxide dismutase (Sod1^-/- mice) show a major decrease in lifespan and several accelerated aging phenotypes. The goal of this study was to determine if cell senescence might be a contributing factor in the accelerated aging phenotype observed in the Sod1^-/- mice. We focused on kidney because it is a tissue that has been shown to a significant increase in senescent cells with age. The Sod1^-/- mice are characterized by high levels of DNA oxidation in the kidney, which is attenuated by DR. The kidney of the Sod1^-/- mice also have higher levels of double strand DNA breaks than wild type (WT) mice. Expression (mRNA and protein) of p16 and p21, two of the markers of cellular senescence, which increased with age, are increased significantly in the kidney of Sod1^-/- mice as is β-gal staining cells. In addition, the senescence associated secretory phenotype was also increased significantly in the kidney of Sod1^-/- mice compared to WT mice as measured by the expression of transcripts for IL-6 and IL-1β. Dietary restriction of the Sod1^-/- mice attenuated the increase in DNA damage, cellular senescence, and expression of IL-6 and IL-1β. Interestingly, the Sod1^-/- mice showed higher levels of circulating cytokines than WT mice, suggesting that the accelerated aging phenotype shown by the Sod1^-/- mice could result from increased inflammation arising from an accelerated accumulation of senescent cells. Based on our data with Sod1^-/- mice, we propose that various bouts of increased oxidative stress over the lifespan of an animal leads to the accumulation of senescent cells. The accumulation of senescent cells in turn leads to increased inflammation, which plays a major role in the loss of function and increased pathology that are hallmark features of aging.

Keywords: Aging; Cellular senescence; DNA damage; Dietary restriction; Inflammation; Oxidative stress; Superoxide dismutase.

Graphical abstract

Fig. 1

Cellular senescence is increased in kidney of Sod1^−/− mice. (A) Transcript levels of p16INK4a and p21 in kidney measured by qRT-PCR and normalized to GAPDH. (B) The level of p16INK4a and p21 protein in kidney as measured by Western blot (top panel). Quantification of p16INK4a and p21 normalized to β-tubulin is shown in the bottom panel. (C) Images of SA β-Gal positive staining cells in kidney is shown in the left panel (arrow points to β-Gal positive cells). Percentage of SA β-Gal positive cells is quantified and graphically represented in the right panel. Four groups of mice were studied: young (4–6 month-old) WT (YWT, turquoise bar); old (24 month-old) mice (OWT, blue bar); young (4–6 month-old) Sod1^−/− mice (YKO, red bar); young (6-month-old) Sod1^−/− mice on DR (YKODR, yellow bar). The data are the mean±SEM of 4 mice per group and were statistically analyzed by one-way ANOVA followed by student T-test. The asterisk (*) indicates a significance (P<0.05) difference between either young WT mice or young Sod1^−/− mice on DR and old WT mice or young Sod1^−/− mice on DR. There were no significant differences between the old WT and young Sod1^−/− mice or the young WT and young DR young Sod1^−/− mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

DNA damage is increased in the kidney of Sod1^−/− mice. (A) DNA oxidative damage (ratio of 8-oxo-dG to dG). (B) Kidney sections immunostained for γH2AX, a marker for DNA double strand breaks. The arrows point to γH2AX positive nuclei. (C) γH2AX nuclei were quantified, and data shown as mean percentage of nuclei positively stained for γH2AX. The following mice were studied: young (4–6 month-old) WT (turquoise bar); young (4–6 month-old) Sod1^−/− mice (red bar); young (6-month-old) Sod1^−/− mice on DR (yellow bar). The DNA oxidative damage data are the mean±SEM of 4 mice per group and were statistically analyzed by one-way ANOVA followed by student T-test. The asterisk (*) indicates that the values for the Sod1^−/− mice are significantly different (P<0.05) from the WT mice and DR Sod1^−/− mice. The DSB data are the mean±SEM of 4 mice per group and were statistically analyzed by unpaired two-tailed T-test. The asterisk (*) indicates that the values for the Sod1^−/− mice are significantly different (P<0.05) from the WT mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Expression of inflammatory cytokines are elevated in Sod1^−/− mice. (A) The levels IL6, IL-1β, and IL-8 mRNA in kidney was measured by qRT-PCR and normalized to GAPDH. (B) NFkB p65 (pS536) levels in kidney were measured using a Simple Step ELISA kit from Abcam and expressed as the ratio of NF-κB p65 pS536/Total NF-κB p65. (C) Quantification of IL-6 in plasma by ELISA. Four groups of mice were studied: young (4–6 month-old) (turquoise bar) and old (24 month-old) mice (blue bar) WT mice and young (4–6 month-old) Sod1^−/− mice fed ad libitum (red bar) or young (6-month-old) Sod1^−/− mice on DR (yellow bar). The cytokines data are the mean±SEM of 4 mice per group and were statistically analyzed by one-way ANOVA followed by student T-test. The asterisks indicate statistical significance (*P<0.05 and **P<0.01) between either young WT mice or young DR Sod1^−/− mice and old wild type mice or young Sod1^−/− mice. There were no significant differences between the old WT and young Sod1^−/− mice or the young WT and young DR Sod1^−/− mice. The NF-κB p65 (pS536) data are the mean±SEM of 4 mice per group and were statistically analyzed by unpaired two-tailed T-test. The asterisk (*) indicates that the values for the Sod1^−/− mice are significantly different (P<0.01) from the WT mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)