Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging

Abstract

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1^-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1^-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1^-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1^-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1^-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1^-/∆ and aged WT mice. Chronic treatment of Ercc1^-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

Keywords: Aging; Cellular senescence; Endogenous DNA damage; Free radicals; Genotoxic stress; Oxidative lesions; Reactive oxygen species.

Graphical abstract

Fig. 1

DNA repair deficient Ercc1^-/Δ mice accumulate oxidative damage and senescent cells faster than WT mice. (a) Immunoblot detection of ERCC1 and XPF in fractionated liver lysates from two WT mice. COXIV was used as a loading control for the mitochondria (Mito) and PCNA was used for the nuclear (Nuc) fraction. (b) Levels of 8,5’-cyclopurine-2’-deoxynucleosides in DNA isolated from murine kidney. Graphed are the mean and SD from n = 3 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 calculated by two-way ANOVA. Data are derived from Wang et al. . (c) Staining for SA-β-gal activity on kidney and liver from Ercc1^-/Δ mice and aged WT mice compared to adult WT mice. Images were captured at 20X magnification. (d) Representative images of p16-luciferase signal in age-matched WT and a DNA repair-deficient mouse. (e) Total body luciferase activity in p16^luc/luc;Ercc1^-/Δ (blue) and p16^luc/luc (red) mice with increasing age. Dots represent individual animals. Black bars indicate the mean ± standard deviation. p values were calculated using a two-way ANOVA. ***p < 0.001, ****p < 0.0001. * over the blue dots indicate significant differences between the WT and Ercc1^-/Δ mice. * over the black bars indicate a significant difference between Ercc1^-/Δ of different age groups. (f) qPCR detection of p16^Ink4a expression in liver (n = 6–12), kidney (n = 4–6) and spleen (n = 7–10) of Ercc1^-/∆ mice (blue), age-matched WT mice (red) and old WT mice (green). Values represent the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 determined by one-way ANOVA with Tukey's test.

Fig. 2

Increased oxidative stress in tissues of progeroid Ercc1^-/Δ mice and old WT mice. (a) Levels of four cyclopurine lesions (R-cdG, S-cdG, R-cdA, S-cdA) measured in liver tissues of 4-month-old WT, Xpa^-/- and Ercc1^-/Δ mice (n = 3 per genotype) by LC-MS/MS/MS. (b) Detection of endogenous superoxide production by quantifying 2-OH-E⁺ by HPLC/electrochemical analysis in DHE-treated kidney (n = 3–7) and liver (n = 6–9 animals per genotype/age). (c) Representative images from immuno-spin trapping of endogenous, biomolecular free radicals with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The level of oxidant stress was determined by immunodetection of DMPO-adducted biomolecules in renal and liver sections. DMPO staining is illustrated in red, actin in green to illustrate tissue architecture and DAPI in blue to highlight cell nuclei. (d) Lipid peroxidation as measured by quantitation of 4-hydroxynonenal protein adducts via ELISA (n = 3–4 mice per group). For all panels, values represent the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 determined by one-way ANOVA with Tukey's test.

Fig. 3

Increased production of ROS in progeroid Ercc1^-/Δ mice and old WT mice. Xanthine oxidase (XO) activity was measured in the (a) livers (n = 7–9 per group) and (b) serum of Ercc1^−/Δ and WT mice (n = 3–4 per group) at multiple ages. (c) NADPH oxidase activity was measured in the livers of 4-month-old Ercc1^−/Δ and WT mice as well as 24-month-old aged WT mice (n = 6–13 per group). (d) Measurement of mitochondrial respiration using a Seahorse Bioscience XF Analyzer on mitochondria isolated from liver tissues of 2 month-old Ercc1^−/Δ and WT mice (n = 4 per group). Values represent the mean ± SD, *p < 0.05, **p < 0.01 as determined by one-way ANOVA with Tukey's test or unpaired two-tailed Student’s t-test.

Fig. 4

Reduced antioxidant capacity in progeroid Ercc1^-/Δ mice and old WT mice. (a) Liver metabolite string analysis of 3-month-old WT (n = 6) versus Ercc1^−/Δ mice (n = 7). Metabolites shaded in blue are significantly more abundant in the mutant animals. (b) Unbiased differential proteomic analysis of liver from 5 to 8-month-old adult WT, 25–30-month-old aged WT and 4-month-old progeroid Ercc1^−/Δ mice (n = 4–8) revealed a significant decrease in the abundance of antioxidant proteins. (c) Catalase activity in the liver of Ercc1^−/Δ and WT mice at various ages (n = 3–5 per group). (d) Cytosolic and (e) mitochondrial superoxide dismutase (SOD) activity in the liver of Ercc1^−/Δ and WT mice at various ages (n = 3 per group). (f) The ratio of oxidized glutathione to reduced glutathione (GSH/GSSG) in livers from Ercc1^−/Δ and WT mice of various ages (n = 3–14 per group). Values represent the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 determined by one-way ANOVA with Tukey’s test.

Fig. 5

A mitochondrial-targeted radical scavenger suppresses endogenous DNA damage, senescence and aging. (a) Schematic diagram of the treatment regimen with XJB-5–131. Littermate pairs of mutant mice were administered either vehicle (sunflower seed oil) or 2 mg/kg XJB-5–131 three times per week for 15 weeks, i.p., starting at five weeks of age. (b) Oxidative DNA damage in the liver of Ercc1^-/Δ mice treated with XJB-5–131 or vehicle only (n = 3 per group) was measured by LC-MS/MS/MS detection of cyclopurine adducts (R-cdG, S-cdG, R-cdA, S-cdA) in genomic DNA. Tissues were collected from 20-week-old animals at the end of the study. (c) Representative images of SA-β-gal staining of liver sections from vehicle- or XJB-treated mice. (d) Total body luciferase activity was measured in p16^luc/+;Ercc1^-/Δ mice treated with 8 mg/kg XJB-5–131, i.p., 3X per week for 4.5 weeks and plotted relative to the signal in Ercc1^-/Δ mice treated with vehicle only. Dots represent individual animals. Graphed is the mean ± SD. *p < 0.05 determined by an unpaired two-tailed Student’s t-test. (e) Representative images of 20-week-old Ercc1^-/Δ mice (siblings) treated with XJB-5–131 or vehicle only. The vehicle treated mouse shows greater ataxia (splayed-foot gait) and hind-limb wasting than the treated animal. (f) Representative images of H&E stained sections of liver, kidney and pancreas. XJB-5–131 had less necrosis and ballooning degeneration of hepatocytes in liver, fewer hyaline casts in the renal tubules and more islets in the pancreas, compared to mice administered vehicle only. (g) Immuno-stain detection of glial fibrillary acidic protein (GFAP, a marker of neurodegeneration) in cerebellar sections of XJB-treated mice compared to vehicle only. Nuclei were counter-stained with hematoxylin. (h) MicroCT analysis of the vertebral column to detect osteoporotic changes in bone.