Sod2 haploinsufficiency does not accelerate aging of telomere dysfunctional mice

Abstract

Telomere shortening represents a causal factor of cellular senescence. At the same time, several lines of evidence indicate a pivotal role of oxidative DNA damage for the aging process in vivo. A causal connection between the two observations was suggested by experiments showing accelerated telomere shorting under conditions of oxidative stress in cultured cells, but has never been studied in vivo. We therefore have analysed whether an increase in mitochondrial derived oxidative stress in response to heterozygous deletion of superoxide dismutase (Sod2(+/-)) would exacerbate aging phenotypes in telomere dysfunctional (mTerc(-/-)) mice. Heterozygous deletion of Sod2 resulted in reduced SOD2 protein levels and increased oxidative stress in aging telomere dysfunctional mice, but this did not lead to an increase in basal levels of oxidative nuclear DNA damage, an accumulation of nuclear DNA breaks, or an increased rate of telomere shortening in the mice. Moreover, heterozygous deletion of Sod2 did not accelerate the depletion of stem cells and the impairment in organ maintenance in aging mTerc(-/-) mice. In agreement with these observations, Sod2 haploinsufficiency did not lead to a further reduction in lifespan of mTerc(-/-) mice. Together, these results indicate that a decrease in SOD2-dependent antioxidant defence does not exacerbate aging in the context of telomere dysfunction.

Keywords: DNA damage; SOD2; aging; free radicals; oxidative stress; stem cells; superoxide; telomere shortening.

Figure 1.

Western blots showing SOD2 levels in liver (A), brain (B) and bone marrow (C) of 12 to 18 months old mice. Lower panels show representative western blots and upper panels show quantification of normalized SOD2 levels to actin controls from n=4 mice per group (1 to 2 repeat experiments per sample). Data is shown in arbitrary units ± SEM. (D) Basal ROS levels in muscle fibers stained with DHE. Signal quantification of G3 mTerc^-/-Sod2^+/- (n=235), G3 mTerc^-/-(n=211) mTerc⁺, Sod2^+/-(n=270 ) and mTerc⁺, Sod2^+/+ (n=203) nuclei from 5 mice per genotype. Data is shown as mean fluorescence intensity ± SEM. (E) Antioxidant capacity of LSK cells. DCFDA loaded bone marrow cells were incubated with 50 uM of antimycinA and DCFDA fluorescence was monitored in Lin^-Sca⁺cKit⁺ populations by FACS analysis. Data is shown in arbitrary units ± SEM of n=4 mice per group. (F) Antioxidant capacity of myeloid cells. Mitosox loaded bone marrow cells were incubated with 20 uM antimycinA and mitosox intensity monitored in myeloid population by FACS analysis. "Y" axis denotes arbitrary units for fluorescence intensity of n=5 to 6 mice per group.

Figure 2.

(A) Mitochondrial respiration of muscle fibers. 10 to 25 mg of permeabilized bundles were analyzed by high resolution respirometry. Results are expressed as oxygen consumption per mg of muscle (± SEM) normalized to citrate synthase activity of n=5 to 6 mice per group. State III respiration is shown after addition of malate, octanoyl-carnitine, ADP, glutamate, succinate and cytochrome c. After state III respiration determination, uncoupled respiration was determined with addition of FCCP to the respiring fibers. Rotenone and antimycin A were used to inhibit respiration at complex I and III respectively. (B) Oxidative modifications (fpg sites) in DNA from bone marrow cells of 12 to 17 month old mice. Data from n=4 to 9 mice per group is shown as number of lesions per 10⁶ bp ± SEM. (C) Representative pictures of gH2AX staining in intestinal crypts of aged mice and bar graphs (D) showing percentage of positive cells per crypt and number of foci per cell ± SEM of n=4 to 6 mice per group. 200 crypt cells were analyzed per mouse. (E) Telomere length analysis by qFISH in liver sections of n= 4 to 5 mice per group aged 12 to 18 months old. n=237 G3 mTerc^-/-, Sod2^+/-), n=234 (G3 mTerc^-/-, Sod2^+/+); n=242 (mTerc⁺, Sod2^+/-) and n=211 (mTerc⁺, Sod2^+/+) nuclei were analyzed for telomere fluorescence intensity (TFI). The black line indicates the mean TFI value of each genotype and the dotted line the threshold of critically short telomeres (TFI<3500).

Figure 3.

(A) Number of erythrocytes per ul of peripheral blood ± SEM in 12 to 18 months old mice. (B) Percentage of mature B cells defined as IgD⁺ IgM⁺ B220⁺ CD43^- cells in total bone marrow cells of 12 to 18 months old mice. n=21 (G3 mTerc^-/-, Sod2^+/-), n=17 (G3 mTerc^-/-, Sod2^+/+); n=9 (mTerc⁺, Sod2^+/-) and n=12 (mTerc⁺, Sod2^+/+) mice per group were analyzed by FACS. (C) Percentage of long term hematopoietic stem cells defined as Lin^- Sca⁺ cKit⁺ CD34^-/low cells in total bone marrow cells of 12 to 18 months old mice. n=9 to 20 mice per group were analyzed by FACS. (D) Competitive transplantation of total bone marrow of Ly5.2 test donor cells against Ly5.1 competitor cells. 8(10)⁵ cells of test donor cells were transplanted along with 4(10)⁵ competitor cells into 1 to 3 young lethally irradiated recipients per donor. Four different donors were used per group. White blood cell chimerism was verified at 1, 2 and 5 months after transplantation by FACS analysis. Data is shown as percentage of donor derived chimerism ± SEM (E) Representative pictures displaying the large intestine atrophy in telomere dysfunctional mice wildtype and heterozygous for Sod2. (F) Bar graph depicting the average number of intestinal crypts per visual field at a magnification of 40X of whole mounts from n=8 (G3 mTerc^-/-, Sod2^+/-), n=7 (G3 mTerc^-/-, Sod2^+/+); n=4 (mTerc⁺, Sod2^+/-) and n=4 (mTerc⁺, Sod2^+/+) mice per group.

Figure 4.

(A) Kaplan Meyer survival curves for G3 mTerc^-/-, Sod2^+/- (n=58); G3 mTerc^-/-, Sod2^+/+ (n=38); mTerc^-/-, Sod2^+/- (n=31) and mTerc⁺, Sod2^+/+ (n=34). (B) Survival curves for females G3 mTerc^-/-, Sod2^+/- (n=22); G3 mTerc^-/-, Sod2^+/+ (n=14); mTerc^-/-, Sod2^+/- (n=16) and mTerc⁺, Sod2^+/+ (n=19). (C) Survival curves for males G3 mTerc^-/-, Sod2^+/- (n=36); G3 mTerc^-/-, Sod2^+/+ (n=24); mTerc^-/-, Sod2^+/- (n=15) and mTerc⁺, Sod2^+/+ (n=15). Dot blots showing body weight of male (D) and female (E) mice throughout lifespan in the aging cohorts. Third order polynomial regression is shown as trendline. All mice were weighed monthly until death.