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. Author manuscript; available in PMC: 2016 May 4.
Published in final edited form as: Mech Ageing Dev. 2016 Feb 1;154:1–8. doi: 10.1016/j.mad.2016.01.005

Liver specific expression of Cu/ZnSOD extends the lifespan of Sod1 null mice

Yiqiang Zhang a, Yuhong Liu b, Michael Walsh b, Alex Bokov c, Yuji Ikeno d,e, Young C Jang f, Viviana I Perez g, Holly Van Remmen h,i, Arlan Richardson i,j,*
PMCID: PMC4855307  NIHMSID: NIHMS778670  PMID: 26839948

Abstract

Genetic ablation of CuZn-superoxide dismutase (Sod1) in mice (Sod1−/− mice) leads to shortened lifespan with a dramatic increase in hepatocellular carcinoma and accelerated aging phenotypes, including early onset sarcopenia. To study the tissue specific effects of oxidative stress in the Sod1−/− mice, we generated mice that only express the human SOD1 gene specifically in the liver of Sod1−/− mice (Sod1−/−/hSOD1alb mice). Expression of hSOD1 in the liver of Sod1−/− mice improved liver function, reduced oxidative damage in liver, and partially restored the expression of several genes involved in tumorigenesis, which are abnormally expressed in the livers of the Sod1−/− mice. However, liver specific expression of hSOD1 did not prevent the loss of body weight and muscle mass and alterations in the structure of neuromuscular junctions. The expression of hSOD1 in the liver of Sod1−/− mice significantly improved the lifespan of Sod1−/− mice; however, the lifespan of the Sod1−/−/hSOD1alb mice was still significantly shorter than wild type mice.

Keywords: CuZnSOD, Oxidative stress, Lifespan, Liver-specific transgenic mice

1. Introduction

Oxidative stress induced by reactive oxygen species (ROS) is implicated in a variety of pathological conditions and diseases including ischemia–reperfusion injury, degenerative diseases, diabetes, and cancer (Barnham et al., 2004). It is also the focal point of the free radical theory of aging proposed by Harman (Harman, 1956). ROS are generated in aerobic organisms by several processes and several defense mechanisms exist to minimize the damage incurred by high levels of ROS. One of the major antioxidant enzymes is CuZn-superoxide dismutase (CuZnSOD), which is found primarily in the cytosol; however, small amounts of CuZnSOD are also found in inter-membrane space of mitochondria.

Mice with a null mutation in the Sod1 gene have been generated and characterized (Reaume et al., 1996; Huang et al., 1997). The Sod1−/− mice have significantly shortened lifespan (30% reduction) compared to wild type mice and many aging phenotypes are accelerated, e.g., early onset of muscle atrophy, loss of hearing and hair, and thinning of skin, cataract formation (Elchuri et al., 2005; Muller et al., 2006; Ohlemiller et al., 1999; McFadden et al., 1999a; Keithley et al., 2005). However, the Sod1−/− mice also developed enlarged liver at as early as 3 months of age with many of the mice developing hepatocellular carcinoma (HCC) (Elchuri et al., 2005). The development of HCC is correlated with an increase in DNA mutations and an up-regulation of several oncogenic genes in liver (Elchuri et al., 2005; Elchuri et al., 2007; Han et al., 2008).

The mechanism(s) by which the deletion of Cu/ZnSOD shortens lifespan and leads to accelerated aging phenotypes is poorly understood. Because Elchuri et al. (2005) initially reported that 70–80% of the Sod1−/− mice have hepatocellular carcinoma when they die while hepatocellular carcinoma is rarely observed in the strain of wild type mice studied, it has been argued that the shortened lifespan of the Sod1−/− mice may be related to liver cancer and as opposed to acceleration in aging arising from the increase in ROS and oxidative stress. However, our group recently showed that only 30% of the Sod1−/− mice in our aging colony developed hepatocellular carcinoma even though the survival of all of the Sod1−/− mice was reduced 30% compared to wild type mice. In addition, the Sod1−/− mice still show an early onset of many phenotypes found in old mice (Zhang et al., 2013a). To gain a better insight into the effect of the null Sod1 mutation on aging, we have generated mice that express CuZnSOD selectively in liver by crossing liver specific hSOD1 transgenic mice (hSOD1alb) to Sod1−/− mice producing Sod1−/−/hSOD1alb mice.

2. Materials and methods

2.1. Generation of transgenic mice

The cDNA of human SOD1 gene was subcloned from a hSOD-1 construct (kindly provided by Dr. Ting Ting Huang from Stanford University) into the plasmid pBSAlb/αFet (kindly provided by Dr. Guntrar Shut). The transgenic mice expressing liver specific hSOD1 gene (hSOD1alb) were generated by the Transgenic Animal Core facility at the University of Michigan. Transgenic mice were genotyped using isolated tail DNA by polymerase chain reaction (PCR) with the following specific primers: 5′-ATGAAATGCGAGGTAAGTATGG-3′ and 5′-ACATTGCCCAAGTCTCCAAC-3′. Genomic DNA was extracted from mouse tail with one-step lysis buffer (Viagen Biotech, Inc., TX) plus protease K at 55°C overnight followed by inactivation at 85°C for 1 hour. The amplification conditions are: 1 cycle of 95°C 5 minutes, 35 cycles of 95°C for 30 seconds + 54°C for 30 seconds + 72°C for 45 seconds, 1 cycle of 72°C for 5 minutes.

The hSOD1alb mice were crossed to Sod1−/− mice generating Sod1+/−/hSOD1alb mice, which were subsequently bred to each other to obtain the following three genotypes used in this study: wild type, Sod1−/−, and Sod1−/−/hSOD1alb mice. Genotyping of these mice were performed using two set of primers for PCR amplification: one for hSOD1 transgene as described above, the other for the knockout of mouse Sod1 as described previously (Huang et al., 1997).

2.2. Animals and lifespan study

All mice were fed ad libitum a standard NIH-31 chow and maintained in micro-isolator cages on a 12-h dark/light cycle. For tissue collection, animals were sacrificed by CO2 inhalation followed by cervical dislocation, and the tissues were immediately excised and placed on ice or liquid nitrogen. All procedures involving the mice were approved by the Subcommittee for Animal Studies at the Audie L. Murphy VA Medical Center and the University of Texas Health Science Center at San Antonio.

For the lifespan experiments, wild type, Sod1−/−, Sod1−/−/hSOD1alb mice were housed four animals per cage starting at 2 months of age. Mice were assigned to survival groups at 4–8 months of age and were allowed to live out their lifespan, i.e., there was no censoring of mice when measuring survival. Lifespans for mice were determined by recording the age of spontaneous death. The mean, median, 10% (the mean lifespan of longest-lived 10% animals), and maximum (the age of death for the longest-lived mouse in the cohort) lifespans were calculated from the survival data for each genotype.

2.3. Western blot analysis of protein expression

Tissues were homogenized with ice-cold RIPA buffer (50 mM Tris pH7.6, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, and 0.5% sodium deoxycholate) supplemented with cocktails of protease and phosphatase inhibitors (Roche Life Science). Protein concentration was determined with Pierce™ BCA Protein Assay kit. Equal amount of total protein was separated on SDS-PAGE gel and transferred onto nitrocellulose membrane. The following specific antibodies were used to detect the target proteins: cyclin D1, Met (hepatocyte growth factor), Stat3 (signal transducer and activator of transcription 3), pStat3, MIF (macrophage migration inhibitory factor) (Cell Signaling), β-tubulin (Sigma-Aldrich, MO), and CuZnSOD (Stressgen Biotechnologies Corporation, Canada).

2.4. CuZnSOD activity assay

The enzymatic activity of CuZnSOD was measured by nativegel analysis as previously described (Van Remmen et al., 1999). Briefly, liver tissues were homogenized in 25 mM Tris buffer with 0.1% Triton-X 100. After centrifugation at 10,000 × g for 10 min at 4°C, protein of the supernatant was quantified using the BCA method following standard protocol. Equal amount of protein was separated on native gel in cold room and CuZnSOD activity was determined by the inhibition of the xanthine plus xanthine oxidase mediated-reduction of cytochrome c.

2.5. Isolation of Skeletal Muscle Mitochondria and measures of mitochondria function

Mitochondria from skeletal muscle were isolated as previously described by Muller et al. (Muller et al., 2007). Briefly, the muscles were excised and incubated with 3 mg of nagarse/g tissue for 5 minutes in Chappell–Perry buffer (100 mM KCl, 50 mM Tris–HCl, 5 mM MgCl2, 1 mM EDTA, and 1 mM ATP). The muscles were homogenized in pre-chilled Chappell–Perry buffer using a Potter–Elvehjem glass homogenizer with a teflon pestle. The homogenate was diluted with Chappell–Perry buffer and centrifuged at 600 × g for 10 min. The resulting supernatant was centrifuged at 14,000 × g for 10 minutes. The mitochondrial pellets were suspended in modified Chappell–Perry buffer (100 mM KCl, 50 mM Tris (pH 7.5), 1 mM MgCl2, 0.2 mM EDTA, and 0.2 mM ATP), and were centrifuged at 7000 × g for 10 min. Mitochondrial pellets were then resuspended in a half volume of modified Chappell–Perry buffer and centrifuged at 3500 × g for 10 minutes.

The assay of mitochondrial generated ROS is based on the detection of H2O2 in the medium using the Amplex Red fluorescent dye (Mohanty et al., 1997). The Amplex Red reagent reacts with H2O2 with a 1:1 stoichiometry producing highly fluorescent resorufin, in the presence of horseradish peroxidase. Briefly, Amplex Red reagent (1 µM) and horseradish peroxidase (5 U/2 ml) were added as previously described by Muller et al. (Muller et al., 2007). All the assays were performed at 37°C and fluorescence was followed at an excitation wavelength of 545 nm and an emission wavelength of 590 nm using a Fluoroskan Ascent Type 374 multiwell plate reader. The slope of the increase in fluorescence was converted to the rate of H2O2 production with the use of a H2O2 standard curve.

Mitochondrial respiration was measured by oxygen consumption using a Clark electrode system (Hansatech Instruments Ltd., Norfolk, UK), as previously described by Jang et al. (2010). The respiratory buffer consisted of 125 mM KCl, 10 mM HEPES, 5 mM MgCl2, and 2 mM K2HPO4, pH 7.44, with 0.3% BSA. Respiration rates were measured using substrates that enter the electron transport chain selectively at the following specific complexes: for complex I, glutamate (1.7 mM) and malate (1.7 mM); for complex II, succinate (2.5 mM) with an NADH dehydrogenase inhibitor (5 mg/ml rotenone); and for complex III, duroquinol (500 mM). State 3 respiration was determined by the addition of ADP (375 nM final concentration) to the above reactions. State 4 respiration was defined as oxygen consumption in the presence of adequate substrate but without added ADP. The respiratory control ratio (RCR) was calculated as the ratio of state 3 to state 4 respiration rates. The activity of cytochrome oxidase, which represents the activity of complex IV, was measured with a Clark electrode (24) using 500 mg of mitochondrial protein in the following buffer: 50 mM potassium phosphate (pH 7.4), 40 mM cytochrome c, 12.5 mM ascorbate, 0.63 mM N,N,N9,N9 tetra-methyl-p-phenylenediamine, and 0.03% Triton X-100.

2.6. Measurement of lipid peroxidation

Lipid peroxidation measured as the levels of F2-isoprostanes was performed as previously described (Ward et al., 2005). The tissues (200 mg) were homogenized, and whole lipid was extracted with chloroform–heptane. The levels of F2-isoprostanes were determined with gas chromatography–mass spectrometry (GC–MS) as initially described by Morrow and Roberts (1999) and currently used in our laboratory.

2.7. Neuromuscular junction staining

Immunofluorescence images of NMJs were prepared as previously described by Jang et al. (2010, 2012). Briefly, gastrocnemius muscle was fixed for 1 h at room temperature (4% paraformaldehyde in PBS), followed by teasing into bundles of muscle fibers and incubation with 100 mM glycine. After removal of the overlying connective tissue, muscles were permeabilized and blocked for 1 h in PBS containing 2% bovine serum albumin, 4% normal goat serum, and 0.5% Triton X-100. To label axons, acetylcholine receptors (AChR), and muscle fibers, muscles were incubated with rabbit polyclonal antibodies against neurofilament (Chemicon, Temecula, CA) and synaptophysin (Zymed, San Francisco, CA), followed by incubation with fluorescein-conjugated species specific secondary antibodies, Alexa594-conjugated α-Bungarotoxin, and Alexa647-conjugated phalloidin. To quantify the number and density of AChRs, muscles were stained only with Alexa594 α-BBungarotoxin followed by fixation with 1% formaldehyde in PBS and flat-mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA). Images were captured using a Nikon Eclipse TE2000-U (Melville, NY) or Zeiss (Oberkochen, Germany) LSM 510 confocal microscope.

2.8. Statistical analysis

All data are presented as mean ± SEM, except the survival data which is explained in the following paragraph. Statistical evaluation between samples or treatments was conducted by one- or two-way ANOVA or student t-test. Difference was considered statistically significant with p < 0.05.

The survival data were analyzed with the help of software developed by Therneau (Therneau and Grambsch, 2000; Team, 2012). The Cox proportional hazard model was fitted to the data with the Sod1−/− mice as the reference group being compared to the wild type and Sod1−/−/hSOD1alb mice. This contrast was chosen because it was the most direct way to test the hypothesis that the Sod1−/− mice have impaired survival and expressing Cu/ZnSOD in the liver significantly reverses this impairment. Sex was also included as a covariate or a stratifying variable. Restricted means and standard errors were also calculated from Kaplan-Meier curves and are reported. Estimates for the ages of median and 90% mortality were also obtained for each group from the Kaplan-Meier curves where the sample size was sufficient. For the purposes of survival analysis, the following genotypes were grouped as wild type: Sod1+/+ or Sod1+/− mice as well as Sod1+/+ or Sod1+/− mice carrying the hSOD1-transgene.

3. Results

3.1. Generation of Sod1−/− mice that express hSOD1 specifically in the liver

Liver-specific hSOD1 transgenic mice were generated as described in the Methods section using the human cDNA for SOD1 fused to the albumin promoter. As can be seen in Fig. 1A and B, the expression of hSOD1 gene was detected only in the liver of the transgenic mice as measured by hSOD1 mRNA and Cu/ZnSOD protein levels. No hSOD1 expression was detected in skeletal muscle, heart, kidney and brain. The liver specific male hSOD1 transgenic mice were bred with female Sod1+/− mice to generate Sod1−/− mice that are null for Sod1 but express hSOD1 specifically in the liver, i.e., Sod1−/−/hSOD1alb mice. As shown in the Western blots in Fig. 1C, Sod1−/−/hSOD1alb mice expressed the human CuZnSOD in the liver at a level approximately 20% of the level found in wild type mice. The human CuZnSOD protein expressed in the mouse liver was enzymatically active as measured by native gel activity assay (Fig. 1D), and the activity of CuZnSOD was also approximately 20% of the CuZnSOD found in wild type mice.

Fig. 1.

Fig. 1

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The generation of liver specific transgenic mice crossed to Sod1−/− mice. Panel A: The expression of hSOD1 transcripts in various tissues of the hSOD1-transgenic (TG) and wild type (WT) mice are shown. Panel B: Western blots are shown for the levels of Cu/ZnSOD in tissues of hSOD1-transgenic (TG) and wild type (WT) mice. The human CuZnSOD protein runs slower than mouse CuZnSOD protein on SDS-PAGE gel electrophoresis (e.g., 23 vs.19 kDa). Panel C: Western blots are shown for the levels of hSOD1 and mSod1 in the liver and muscle of Sod1−/− (KO) and wild type (WT) mice (1 and 2 represents two different mice) compared to Sod1−/−/hSOD1alb (ALB) that are hemizygous (+/−) and homozygous (+/ +) for the hSOD1-transgene. Panel D: CuZnSOD enzymatic activity is compared in the liver of Sod1−/− (KO) and Sod1−/−/hSOD1alb (ALB) mice at two different levels of protein added to the gels. Abbreviations for tissues: K—kidney, M—skeletal muscle, L—liver, H—heart, B—whole brain.

One of the major phenotypes observed in Sod1−/− mice is a dramatic increase in oxidative damage in all tissues (Elchuri et al., 2005; Muller et al., 2006). Therefore, we first determined if re-introducing hSOD1 into the liver of Sod1−/− mice could significantly reduce oxidative damage by measuring the levels of F2-isoprostanes, which are a marker of lipid oxidation (Roberts and Morrow, 2002). As shown in Fig. 2, the F2-isoprostane levels were ~2.5-fold higher in the Sod1−/− mice compared to the wild type mice, and the levels of F2-isoprostanes were significantly reduced (40%) in the livers of the Sod1−/−/hSOD1alb mice. However, the Sod1−/−/hSOD1alb mice still showed a significantly higher level of oxidative damage than the wild type mice, which is not unexpected because the level of Sod1 expression in the Sod1−/−/hSOD1alb mice was only ~20% of wild type. The data in Fig. 2 also show that while the levels of F2-isoprostanes were significantly increased in brain, heart, and skeletal muscle of the Sod1−/− mice, the levels of F2-isoprostanes were not reduced in these tissues of Sod1−/−/hSOD1alb mice. The data in Fig. 2 showing that the level of oxidative damageis only reduced in the liver of Sod1−/−/hSOD1alb mice confirms the tissue specific expression of Sod1 in the Sod1−/−/hSOD1alb mice.

Fig. 2.

Fig. 2

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Effect of liver-specific expression of hSOD1 on oxidative damage. The levels F2-isoprostanes in liver, skeletal muscle, brain, and heart from wild type (white bar), Sod1−/−/hSOD1alb (black bar) and Sod1−/− (gray bar) mice are shown for 12- to 14-month-old mice (n = 6 mice per group). The data are expressed as the mean ± SEM and the statistical significance shown as follows: icance: **p < 0.01 between wild type and Sod1−/− mice; *p < 0.05 between Sod1−/− or Sod1−/−/hSOD1alb mice and wild type mice. There is no significant difference between Sod1−/− and Sod1−/−/hSOD1alb mice in all the tissues, except the liver.

3.2. Liver specific expression of hSOD1 reduces liver damage and improves liver function in Sod1−/− mice

First, we determined whether liver-specific expression of hSOD1 improved the physiological parameters of the liver previously reported to be altered in Sod1−/− mice, including body weight and liver size (Muller et al., 2006). As shown in Fig. 3A, the liver-specific expression of hSOD1 in Sod1−/− mice did not significantly increase the body mass of Sod1−/− mice to that of wild type mice. The supplementary data show that this lower body weight in the Sod1−/− and Sod1−/−/hSOD1alb mice was maintained throughout the lifespan of the mice. However, the liver-specific expression of hSOD1 attenuated the liver hypertrophy seen in the Sod1−/− mice. The liver weight of the Sod1−/−/hSOD1alb mice was comparable to the wild type mice, which was 30% smaller than that of Sod1−/− mice.

Fig. 3.

Fig. 3

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The effect of liver specific expression of hSOD1 on liver of Sod1−/− mice. These data were obtained from 12- to 14-month-old Sod1−/− (gray bar), wild type (white bar), and Sod1−/−/hSOD1alb (black bar) mice. Panel A: Body weight and liver wet weight (n = 14, 9, and 13 for wild type, Sod1−/− and Sod1−/−/hSOD1alb mice, respectively). The data shown combine both sexes; however, the differences observed are significant for both males and females. Panel B: Plasma levels of free fatty acids (FFA) and lactic acid (n = 4 mice per group). Panel C: Liver mitochondrial function (n = 4 mice per group). The data are expressed as the mean ± SEM and the statistical significance shown as follows:*p < 0.05 and **p < 0.01 indicates statistical significance between Sod1−/− mice and wild type or Sod1−/−/hSOD1alb mice.

Because liver damage or disease can lead to increased plasma free fatty acids and lactic acid (Kalhan et al., 2011; Ritz and Heidland, 1977). we measured the plasma levels of free fatty acids and lactic acid in the livers of the mice (Fig. 3B). Plasma levels of free fatty acids and lactic acid were significantly higher in Sod1−/− mice compared to wild type mice but was restored to wild type levels in the Sod1−/−/hSOD1alb mice, demonstrating an improvement of liver function in Sod1−/−/hSOD1alb mice that is comparable to wild type mice.

Our group has previously shown that skeletal muscle mitochondria isolated from Sod1−/− mice show dramatic alterations in mitochondria function, e.g., mitochondria from Sod1−/− mice generate more ROS and produced less ATP with reduced membrane potential (Muller et al., 2007; Jang et al., 2010). As shown in Fig. 3C, the function of liver mitochondria isolated from Sod1−/−/hSOD1alb mice is comparable to that of wild type mice; no significant differences were observed in ATP production, oxygen consumption, membrane potential, and ROS generation.

To further characterize the liver of the Sod1−/−/hSOD1 mice, we measured the expression of several genes that have been previously shown altered in the liver of Sod1−/− mice that could play a role in promoting tumorigenesis or tumor growth (Elchuri et al., 2005, 2007; Han et al., 2008). We focused on Met and cyclin D1 because they have been associated with liver tumorigenesis/progression (Goyal et al., 2013; Matsuda et al., 2013). As shown in Fig. 4, the levels of cyclin D1 were increased significantly in the liver of adult (8–10 months) Sod1−/− mice and the levels of cyclin D1 were significantly reduced in the Sod1−/−/hSOD1alb mice to levels that are comparable to wild type mice. The difference in the expression level of cyclin D1 diminished in old animals (20–24 months). The levels of Met on the other hand were similar in the three groups of mice in adult mice; however, Met levels were significantly increased in the old Sod1−/− mice compared to the Sod1−/−/hSOD1alb or wild type mice. We also measured the phosphorylation of Stat3, an indicator of the activation of Stat3 signaling, which regulates a number of pathways important in tumorigenesis. The phosphorylation of Stat3 was increased significantly in the liver of adult and old Sod1−/− mice. The introduction of hSOD1 attenuated the increase in Stat3 phosphorylation in the liver of Sod1−/− mice. Thus, the reduction in liver weight, circulating fatty acids and lactate, and reduction in the expression of several genes upregulated in hepatocarcinoma, demonstrate that overexpressing hSOD1 in the livers of mice null for Sod1 is able to rescue the liver dysfunction observed in the Sod1−/− mice.

Fig. 4.

Fig. 4

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Expression of genes in liver associated with tumorigenesis. The levels of Met and cyclin D1 and the phosphorylation of Stat3 (pStat3/Stat3 ratio) were measured in liver tissue isolated from of 8- to 10- or 20- to 24-month-old wild type (white bars), Sod1−/−/hSOD1alb (gray bars), and Sod1−/− (black bars) mice with n = 6 mice for each group. The data are expressed as the mean ± SEM and the symbols denote statistical significance at p < 0.05: between Sod1−/− and wild type or Sod1−/−/hSOD1alb mice (*); between Sod1−/− mice and wild type mice (&); between Sod1−/− mice and Sod1−/−/hSOD1alb mice (#).

3.3. Effect of liver-specific expression of hSOD1 on skeletal muscle in Sod1−/− mice

Because the accelerated age-related loss of muscle mass is one of the most dramatic phenotypes observed in the Sod1−/− mice (Muller et al., 2006), we determined whether the liver-specific reintroduction of hSOD1 would rescue the muscle loss in Sod1−/− mice. It has been argued that the accelerated muscle loss may be caused by liver cancer, a condition known as cachexia (Argiles et al., 2014). However, indirect evidence from our group suggested that the sarcopenia phenotype in the Sod1−/− mice was not due to cachexia (Muller et al., 2006). As shown in Fig. 5A, the muscle mass of the hind leg of Sod1−/−/hSOD1alb mice was significantly smaller than wild type control mice and similar to the Sod1−/− mice for the Sod1−/−/hSOD1alb mice. The same was true for the mass of specific muscle types, e.g., quadriceps, gastrocnemius, and tibias anterior (Fig. 5A). Correlated with the failure to prevent muscle loss, liver-specific expression of hSOD1 did not reduce the level of oxidative damage in skeletal muscle, e.g., the level of lipid oxidation was similar to that found in the muscle of Sod1−/− mice (Fig. 2). In addition, we found that the alterations in the function of mitochondria isolated from gastrocnemius muscle of Sod1−/−/hSOD1alb mice were similar to that of Sod1−/− mice (Fig. 5B), i.e. mitochondria produced more ROS, generated less ATP, and consumed less oxygen than age-matched wild type littermates.

Fig. 5.

Fig. 5

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Effect of liver specific expression of hSOD1 on skeletal muscle. Panel A: Wet weight of different types of skeletal muscle from wild type (n = 10, white bars), Sod1−/− (n = 6, black bars), and Sod1−/−/hSOD1alb mice, (n = 8, gray bars). Panel B: Skeletal muscle mitochondrial function as measured by ROS generation, oxygen consumption, and ATP generation (n = 4 mice for each group). Panel C: Structure of neuromuscular junctions visualized through fluorescence immunostaining and confocal microscopy for wild type (WT), Sod1−/− (KO), and Sod1−/−/hSOD1alb (ALB) mice. The data are expressed as the mean ± SEM and the asterisk denotes statistical significance at (*) p < 0.05 and (**) p < 0.001 either between wild type mice and Sod1−/− mice or between wild type and Sod1−/−/hSOD1alb mice.

Our group has also shown that one of the characteristics of the sarcopenia phenotype observed in the Sod1−/− mice is the disaggregation of neuromuscular junctions (Jang et al., 2010). As shown in Fig. 5C, the neuromuscular junctions in Sod1−/−/hSOD1alb mice exhibited similar structural abnormalities as those found in the Sod1−/− mice, further indicating that the muscle loss in Sod1−/− mice is independent of liver dysfunction. These data indicate that the accelerated sarcopenia phenotype observed in Sod1−/− mice is largely due to oxidative damage in the muscle and neuronal tissue and is not due to liver dysfunction/cancer.

3.4. Liver specific hSOD1 expression extends the lifespan of Sod1−/− mice

Because previous studies have shown that the Sod1−/− mice have a significantly shortened lifespan (Elchuri et al., 2005; Zhang et al., 2013a; Jang et al., 2012), we compared the lifespan of the Sod1−/−/hSOD1alb mice to Sod1−/− and wild type mice to determine the role of liver damage in the shortened lifespan of the Sod1−/− mice. Both male and female Sod1−/−/hSOD1alb mice lived longer than Sod1−/− mice. However, the lifespan was only partially restored by the liver specific expression of Cu/ZnSOD because the Sod1−/−/hSOD1alb mice lived significantly shorter than wild type mice. Fig. 6 shows the survival curves and data (Table 1) we obtained from the limited number of mice we studied. Combining data from both sexes, mean survival was lower for the Sod1−/− mice than for wild type or Sod1−/−/hSOD1alb mice. In females, the age at median and 90% mortality followed the same pattern. In males the sample size of the Sod1−/−/hSOD1alb mice was insufficient to obtain valid median and 90% mortality ages; however, the male Sod1−/−/hSOD1alb attained median and 90% mortality at earlier ages than the control wild type males. The same pattern as observed with the females. These age differences are strictly qualitative because the sample size was not sufficient to obtain bootstrap confidence intervals that are required for hypothesis testing.

Fig. 6.

Fig. 6

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Survival of Sod1−/−/hSOD1alb mice compared to Sod1−/− and wild type mice. The Kaplan–Meier survival curves are shown for male and female mice combined. The curves represent Sod1−/−/hSOD1alb (Alb-red line, n = 31), Sod1−/− (KO-blue line, n = 25), and wild type (WT-green line, n = 34) mice. Censored events are represented by the symbol ‘X’. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1.

Survival characteristics of Sod1−/− (KO), wild type (WT) and Sod1−/−/hSod1Alb (Alb) mice.

Sex M F M F M F
Genotype KO KO WT WT Alb Alb
Records 13 12 16 18 8 23
Censored 3 3 6 7 3 7
Events 10 9 10 11 5 16
armean survival (days) 705 769 851 853 831 825
aSEM for rmean (days) 46 28 47 53 65 32
Lower Bound 50% mortality 527 780 789 820 – 813
Estimated 50% mortality 730 797 922 980 – 848
Upper Bound 50% mortality – – – – – –
Lower Bound 90% mortality – 830 981 1042 – 901
Estimated 90% mortality 914 876 1026 1095 – 988
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a

Restricted mean.

We further analyzed the mortality rate of the three groups of mice using the method developed by Therneau as described in the method section and shown in Table 2. After adjusting for sex, the mortality risk of the wild type mice was 28% that of the Sod1−/− mice (p = 0.0004) and the mortality risk of the Sod1−/−/hSOD1alb mice was 49% that of the Sod1−/− mice (p = 0.0174). Thus, the mortality risk of Sod1−/− mice was 3.7-fold higher than that of wild type mice and 2.3-fold higher than that of Sod1−/−/hSOD1alb mice. Analysis of deviance was performed to determine whether it was necessary to also include an interaction term in this model, and it was found not to be necessary (p = 0.65). Similar hazard ratios were obtained if the data were stratified by sex or if the male and female animals were treated as one group. In all cases, the Sod1−/− mice had significantly greater mortality risk than either the wild type or Sod1−/−/hSOD1alb mice, indicating that the survival of Sod1−/−/hSOD1Alb mice was significantly improved over Sod1−/− mice.

Table 2.

Mortality risk analysis of survival.

β Hazard ratio SEM Z P
Stratified by sex
Control vs KO −1.3200 0.2671 0.3758 −3.5128 0.0004
ALB vs KO −0.8382 0.4325 0.3523 −2.3791 0.0174
Adjusting for sex
Control vs KO −1.2815 0.2776 0.3681 −3.4818 0.0005
ALB vs KO −0.7130 0.4902 0.3411 −2.0901 0.0366
F vs M −0.1959 0.8221 0.2721 −0.7201 0.4715
Pooling M and F
Control vs KO −1.2979 0.2731 0.3694 −3.5134 0.0004
ALB vs KO −0.7588 0.4683 0.3349 −2.2657 0.0235
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4. Discussion

The Sod1−/− mouse is of interest to the aging community because it is the only mouse model with a loss of function in a gene coding for an antioxidant protein that is not embryonically/prenatally lethal and has a shortened lifespan with accelerated aging phenotypes (Muller et al., 2006; Ohlemiller et al., 1999; Olofsson et al., 2005; McFadden et al., 1999b). For example, mice lacking Sod2 (MnSOD) die within days after birth (Li et al., 1995), mice lacking Gpx4 (glutathione peroxidase 4) are embryonically lethal (Ran et al., 2003) and mice lacking either Gpx1 or MsrA (methionine sulfoxide reductase A) show no change in lifespan and no major phenotype (Zhang et al., 2009; Salmon et al., 2009). The Sod1−/− mouse is also a valuable model to study the role of oxidative stress/damage on physiological processes because the level of oxidative stress/damage in tissues of the Sod1−/− mouse is higher than any other viable mouse model with a reduction in an antioxidant enzyme (Perez et al., 2009). In addition, the ability to alter the expression of Sod1 in specific tissues makes the Sod1−/− mouse valuable for studying the biological importance of oxidative stress in specific tissues or cell types. Using genetic approaches, one can study how increased expression of Cu/ZnSOD in a tissue alters the phenotypes observed in the Sod1−/− mouse or one can determine the effect of knocking out the Sod1 gene in a specific tissue or cell type. In other words, the Sod1−/− mouse allows investigators to determine how changes in oxidative stress/damage in a specific tissue affect the global physiological status of the mouse. For example, our group has studied the effect of neuronal and muscle specific expression of Sod1 on the accelerated sarcopenia phenotype that is a hallmark of the Sod1−/− mouse. Sakellariou et al. (2014) showed that expressing the hSOD1 transgene in neuronal tissue of Sod1−/− mice prevented the loss of muscle mass and neuromuscular junction disaggregation (Sakellariou et al., 2014). On the other hand, knocking out the Sod1 gene in muscle (Zhang et al., 2013b) or neurons (Sataranatarajan et al., 2015) alone did not recapitulate the accelerated sarcopenia phenotype observed in Sod1−/− mice. These data demonstrate the importance of the interplay of between motor neurons and muscle in the mechanism underlying the loss of muscle arising from increased oxidative stress (Sataranatarajan et al., 2015).

In this study, we were interested in determining what role increase liver damage/pathology played in the shortened lifespan of the Sod1−/− mice. Hepatocellular carcinoma is rare in the strain of mice used in these studies (Elchuri et al., 2005; Muller et al., 2006), and it is often argued that the decrease in lifespan, and even some of the accelerated aging phenotypes, are secondary to the dramatic increase in hepatocellular cancer. In this study, we first generated transgenic mice that expressed Cu/ZnSOD specific in liver using the albumin promoter fused to the hSOD1 cDNA, and then generated mice that expressed Cu/ZnSOD specifically in the liver of Sod1−/− mice by crossing the liver specific hSOD1 transgenic mice to Sod1−/− mice. The Sod1−/−/hSOD1alb mice displayed a reduction in oxidative damage in liver, improved liver function, and reduced markers of hepatocellular carcinoma. The liver-specific expression of hSOD1 increased significantly the lifespan of Sod1−/− mice; however, the lifespan of the Sod1−/−/hSOD1alb mice was still significantly lower than wild type mice, and the Sod1−/−/hSOD1alb mice still showed the accelerated aging phenotype of sarcopenia, e.g., the early onset of muscle atrophy, mitochondrial dysfunction, and the disaggregation of neuromuscular junctions. Thus, while expressing SOD1 in liver can partially rescue lifespan of Sod1−/− mice, these mice still show the accelerated aging phenotype in muscle. Thus, the Sod1−/−/hSOD1alb mice allows one to study the effect of increased oxidative stress on aging without the confound of the dramatic increase in liver pathology in the Sod1−/− mice.

Supplementary Material

Appendix A

Acknowledgments

This study was funded as part of a program project grant from the National Institute on Aging (AG-020591) and VA Merit Grants to HVR and AR.

Footnotes

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2016.01.005.

Contributor Information

Yuhong Liu, Email: LiuY2@uthscsa.edu.

Michael Walsh, Email: walshme125@gmail.com.

Alex Bokov, Email: BOKOV@uthscsa.edu.

Young C. Jang, Email: young.jang@gatech.edu.

Viviana I. Perez, Email: Viviana.Perez@oregonstate.edu.

Holly Van Remmen, Email: Holly-VanRemmen@omrf.org.

Arlan Richardson, Email: arlan-richardson@ouhsc.edu.

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Supplementary Materials

Appendix A
NIHMS778670-supplement-Appendix_A.pdf (78.5KB, pdf)

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