As the presently reluctance to associate mitochondrial dysfunction with aging through ROS and oxidative damage are largely based on the notion that these phenomena were apparently not involved in aging in mtDNA mutator mice [ 19 , 24 , 25 ], and as our present data indicate the opposite to be the case, our observations may also be of significance for discussions of the nature of aging and the possibility to ameliorate the aging process therapeutically.

Experimentally, an alternative avenue to examine the possible involvement of ROS in the development of aging characteristics would be to examine the ability of mitochondrially targeted antioxidants to ameliorate the health problems associated with experimentally induced aging. In this paper, we find that the mitochondrially targeted antioxidant 10-(6’-plastoquinonyl)decyltri-phenylphosphonium cation (SkQ1) [ 21 ] substantially counteracts the acquisition of aging characteristics in the mtDNA mutator mice. We also find that parameters for oxidative damage not earlier examined (cardiolipin depletion and accumulation of hydroxynonenal protein adducts) are diminished by SkQ1 treatment. These data and other antioxidant data (mitochondrially targeted catalase [ 22 ] and N-acetyl-L-cysteine treatment [ 23 ]) clearly indicate that ROS production and oxidative damage are substantial factors in the development of aging characteristics in the mtDNA mutator mice.

However, the significance of ROS for the aging process has been doubted [ 10 , 11 ], particularly based on observations in the mtDNA mutator mice [ 12 – 14 ]. These mice accumulate errors in their mtDNA and demonstrate subsequent alterations in their respiratory chain composition [ 15 ]. They also demonstrate an early occurrence of characteristics normally associated with aging, and they die at a young age. However, there has been no convincing evidence that oxidative damage causes these problems; rather an absence of oxidative damage has been reported [ 13 , 16 – 19 ], but see [ 20 ].

As a cause for the decreasing health status that accompanies aging, mitochondrial deterioration has been repeatedly suggested [ 1 – 4 ]. Particularly, it has been discussed that an accumulation of errors in mitochondrial DNA (mtDNA) replication would lead to mitochondrial dysfunction, including increased production of reactive oxygen species (ROS) that may both further deteriorate the mitochondria and affect the function of the rest of the cell [ 5 – 9 ].

Results

In order to examine the significance of possible enhanced ROS production for the development of early aging characteristics in the mtDNA mutator mice, we have here followed such mice that were treated or not with SkQ1. In the following, we first describe phenotypic manifestations of the SkQ1 treatment and we then examine the effect of such a treatment on markers of oxidative damage. We also examine the effect of SkQ1 treatment on total lifespan in mtDNA mutator mice.

SkQ1 treatment attenuated the manifestation of kyphosis and alopecia in mtDNA mutator mice Already at an age of less than 230-250 days, mtDNA mutator mice show features normally associated with aging, as exemplified with the ≈250 days old mtDNA mutator mouse in Fig. 1A which confirms observations originally published by [12,13]. Strikingly, treatment of these mice with the SkQ1, not from birth but only from an age of ≈100 days, markedly diminished this phenotype, resulting in mice presenting a mouse much more similar to wild-type mice of this age (Fig. 1A). These dramatic improvements were quantitatively analyzed in the ensuing graphs. Figure 1. Effect of SkQ1 treatment on appearance, kyphosis and alopecia in mtDNA mutator mice. (A) Pictures of mtDNA mutator mice: a non-treated (Mut) mouse and an SkQ1-treated (Mut+SkQ1) littermate female mouse, 248 and 256 days of age respectively. (B) Scores of kyphosis manifestation (arbitrary units) in mtDNA mutator mice. Each bar represents the mean ± S.E. score from mice of both genders: 21 Mut (10 females + 11 males) and 22 Mut+SkQ1 (13 f + 11 m) in ages 213-268 days; 11 Mut (5 f + 6 m) and 13 Mut+SkQ1 (6 f + 7 m) in ages 268–290 days; in ages 290–356 days, the number of mice decreased with age according to survival; the lowest number of mice in a group was 4 (2 f + 2 m). Day number under each bar indicates the center day of 11 days of mouse age. (C) X-ray picture of a non-treated mouse and an SkQ1-treated littermate female mtDNA mutator mouse (290 ± 4 days old). White circle arc indicates curvature angle measurements. Note that the angle is calculated from the extended line of the lower spine. (D) Angle of thoracic-lumbar curvature determined as in C. Each bar represents the mean ± S.E. from 10–11 mice of both genders in each group; Mut, 287 ± 7 days old, 5 f + 6 m; Mut + SkQ1, 284 ± 6 days old, 6 f + 5 m. (E) Bone minerality of spine cord area from scapula to lumbar. Each bar represents the mean ± S.E. from 6–8 female mice in each group. Mut, 290 ± 11 days; Mut + SkQ1, 287 ± 9 days. (F) Scores of alopecia manifestation in mtDNA mutator mice. The mice are the same as those in (B). * in B-F indicates a statistically significant difference between non-treated and SkQ1-treated mtDNA mutator mice (p < 0.05).

One feature of the aging process is the occurrence of (hyper)kyphosis, i.e. (over)curvature of the spine [12,13]. The severity of this malfunction has been manually scored in Fig. 1B every 11th day (as exemplified in Supp. Table S1). Whereas hyperkyphosis developed gradually but consistently in the non-treated mtDNA mutator mice, the occurrence was low and fairly stable in the SkQ1-treated age-matched mice, even though the SkQ1-treated mice lived longer (see below) and thus could be followed to a higher age. Hyperkyphosis can be quantitated through X-ray analysis. Fig. 1C illustrates such a measurement, performed on mice of equal age. The mean values of these measurements are compiled in Fig. 1D. As seen, there was a significant effect of SkQ1 in diminishing the degree of kyphosis. Measurement of bone minerality of the spinal cord from scapula to lumbar revealed a higher bone mineral content of this area in the SkQ1-treated mtDNA mutator mice (Fig. 1E). Non-treated mtDNA mutator mice showed severe alopecia (hair loss) [12,13]. This pathology was much less evident in mice treated with SkQ1 (Fig. 1F). It may especially be noted that these cohorts of mice were single-caged. Thus, the alopecia and the loss of whiskers were mouse-autonomous effects. Earlier studies have kept mice in larger groups where mouse-mouse interaction (particularly between males) may substantially affect the outcome, leading to exaggerated fur loss (barbering). – As fur protects against heat loss [26] and as the mice were living at 22 °C, i.e. below their thermoneutral zone, the implication is that the SkQ1-induced amelioration of the fur status is not only a general indication of less rapid advancement of aging features but also that it can diminish the heat loss and thus the risk for hypothermia [27].

SkQ1 treatment prevented loss of fat and improved the estrus state in mtDNA mutator mice The SkQ1-treated mtDNA mutator mice showed no decrease with age in body weight and body fat content. Both these parameters significantly decreased in the non-treated mice (Fig. 2A, B). Lean body mass was kept at stable levels longer than fat mass, but from an age of ≈270 days, it decreased in the non-treated mtDNA mutator mice (Fig. 2C). SkQ1 treatment prevented this loss of lean body mass (Fig. 2C). Acute body weight loss is an important criterion for the running assessment of mouse health (Supp. Table S1). As seen on Fig. 2D, this parameter was much improved in the SkQ1-treated mice. Figure 2. Effects of SkQ1 treatment on body energy stores and intake in mtDNA mutator mice. (A) Body weight, (B) body fat content and (C) lean body mass as a function of age (± 7 days). The points are means ± S.E. In these “paired death” experiments, 8 female mice were in each group until age 238 days; after this, the number of mice decreased with time, depending on survival, with 4 mice in each group at the final point. (D) Scoring of acute body weight decrease in mtDNA mutator mice. The mice are the same as in Fig. 1B. (E) Skin morphology. The skin samples were from the back region of non-treated and SkQ1-treated littermate female mice of the same age (290 ± 4 days). Bar is 100 µm. The subdermal fat region is indicated by arrow. (F) Food intake. The mice are the same as in Fig. 2A. (G) Dietary, fecal and metabolic energy in mtDNA mutator at age 250 ± 7 days. Metabolic energy values were obtained by subtraction of fecal energy from dietary energy consumed. * in A-D and F indicates a statistically significant difference between non-treated and SkQ-treated mtDNA mutator mice (p < 0.05, n = 8 mice in each group). (H) Water intake in mtDNA mutator mice treated or not with SkQ1. The points were obtained by combining remaining water from all mice in each group (8 female mtDNA mutator mice) and subtracting this from the total amount of water supplied (values per mouse).

In mtDNA mutator mice of 290 days, there were nearly no subdermal lipid stores left whereas the stores were well maintained in the SkQ1-treated mice (Fig. 2E). During the time when the weight curves for the non-treated and SkQ1-treated mice diverged, the food intake and metabolic energy consumption in the SkQ1-treated animals was slightly larger than that of the non-treated mtDNA mutator mice (Fig. 2F and G). This was probably secondary to the generally better status of the SkQ1-treated mice. Addition of SkQ1 to the drinking water did not change the water intake (Fig. 2H). Through leptin signaling [28], body lipid stores may affect ovulation in the mice. Accordingly, non-treated mtDNA mutator mice exhibited a successively impaired estrus cycle with irregularity and loss of estrus (Fig. 3). The SkQ1 treatment increased the number of estruses and ameliorated the irregularity of the estrus cycle (Fig. 3). Figure 3. Effects of SkQ1 treatment on estrus cycle in mtDNA mutator mice. (A) Examples of graphs of estrus cycles in non-treated and SkQ-treated mtDNA mutator female mice. Examination of estrus cycle was done by light microscopy of vaginal smears. Estrus was characterized by the presence of large cornified cells with degenerated nuclei and was indicated as estrus stage 2 on the graphic presentation. Diestrus was identified by presence of leucocytes and mucous and was indicated as 0 on graphs. Proestrus and metestrus were characterized by the presence of nucleated epithelial cells or leucocytes together with cornified cells and were indicated on the graphs as intermediate stages 0.5 – 1.5. (B) Number of estruses during 12 days measured as a function of age (± 7 days). The points are means ± S.E. of 7-8 female mice for each group. * indicates a statistically significant difference between non-treated and SkQ1-treated mice (p < 0.05).



Positive effects of SkQ1 treatment could be attributed to its antioxidant properties The data collected above demonstrate that treatment of mtDNA mutator mice with SkQ1 significantly delays the aging characteristics observed in the mtDNA mutator mice. As SkQ1 was originally developed as a mitochondrially targeted antioxidant [21], we examined whether the treatment of mtDNA mutator mice with SkQ1 would affect ROS-related parameters that may be linked to SkQ1’s beneficial effects. In the typical lipid peroxidation process, ROS will interact with omega-6 and omega-3 fatty acids (mainly linoleic acid and arachidonic acid) in the membrane phospholipids, leading to the release of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) that may form adducts with proteins. We examined whether these parameters were affected by the SkQ1 treatment. We followed the formation of MDA in kidney lysates (Fig. 4A). The rate of formation was markedly lower in the lysates from the SkQ1-treated mtDNA mutator mice. We made similar observations in lysates from liver and brain (Fig. 4B). These experiments thus indicated that at least in vitro, tissues obtained from SkQ1-treated mice were less prone to lipid peroxidation than tissues from non-treated mice. Figure 4. MDA formation and content of 4-HNE adducts in tissues from mtDNA mutator mice non-treated and treated with SkQ1. (A) MDA formation in kidney lysate exposed to normal atmospheric oxygen concentration and pressure at 37 °C. (B) Levels of MDA formed in liver, brain and kidney lysates after 40 min of exposure of tissue to normal atmospheric oxygen concentration and pressure. (C) Representative immunoblot analysis of 4-hydroxynonenal (4-HNE) adducts in kidney (left panel), liver (middle panel) and gastrocnemius skeletal muscle (right panel) tissue lysate from non-treated (M) and SkQ1-treated (S) mtDNA mutator mice (15 µg protein/lane). Numbers 1, 2, 3 indicate age-and gender-matched samples. Validation of the assay is presented in Fig. S1A. Loading control was performed by Ponceau Red staining (shown for kidney in Fig. S1C). (D) Profile of 4-HNE-adducts in kidney lysate. (E) Relative 4-HNE-adducts in kidney, liver, skeletal muscle (SKM) and brain tissue lysates. Tissue samples were collected in parallel from non-treated and SkQ1-treated mice (268–300 days old, both genders) in the paired death experimental setup. In B and E, for each pair, the mean level of 4-HNE-adducts in the non-treated mouse was set to 100% (indicated as dashed line) and the amount in the paired (age- and gender-matched) SkQ1-treated mouse was expressed relative to this. The means ± S.E. for 4-7 mice are shown. * and ** in A, B and E indicate statistical significance between SkQ1-treated and non-treated mice (p < 0.05 and p < 0.01, respectively).

To examine whether similar differences occurred in vivo, we estimated the endogenous level of lipid peroxidation by analysis of the in vivo formed 4-HNE adducts to cellular proteins. A lower content of 4-HNE-adducts was observed in kidney samples from SkQ1-treated mice as compared with the level in kidneys of non-treated mice (Figs. 4C - E). Also in liver and skeletal muscle, 4-HNE adducts were significantly (although less markedly) lower in SkQ1-treated mice (Fig. 4C - E); in brain, no difference was observed (Fig. 4E). These findings are in line with our data that the in vivo administrated SkQ1 accumulates in brain in much smaller amount than in kidney, liver and skeletal muscle (A. Andreev-Andreevsky et al., in preparation). As one of the main sources of the released MDA and 4-HNE are the polyunsaturated fatty acids of the mitochondrial membrane phospholipids [29], we examined the effect of SkQ1 on the phospholipid composition of mitochondria from different tissues. This experiment is exemplified for skeletal muscle and liver in Table 1. As seen in Table 1, neither the mutation nor the treatment with SkQ1 had any marked effect on the content of most phospholipid classes. However, one phospholipid class, cardiolipin, was relatively decreased in the mtDNA mutator mice as compared to wild-type mice. This effect was revealed in both skeletal muscle and liver (Table 1). Further, the SkQ1-treatment restored the cardiolipin amount in the mtDNA mutator mice to wild-type levels in the mtDNA mutator mice (Table 1). Table 1. Phospholipids and fatty acyl composition in mitochondria from wild-type mice [SkQ1-treated (WT+SkQ1) or non-treated (WT)] and mtDNA mutator mice [SkQ1-treated (Mut+SkQ1) or non-treated (Mut)]. Mitochondria

from tissue Phospholipid and their

fatty acyls (FA) WT WT+SkQ1 Mut Mut+SkQ1 Skeletal muscle Phosphatidylcholine 51.0±3.6 50.0±2.8 52.0±4.2 52.0±3.1 Phosphatidylethanolamine 19±2 18±1 22±2 19±2 Cardiolipin 17±1 20±2 13±1# 18±1* Phosphatidylinositol 5.0±0.7 4.0±0.6 5.0±0.6 4.0±0.4 Phosphatidylserine 5.0±1.1 5.0±0.9 4.0±0.8 4.0±0.7 Phosphatidic acid 3.0±0.6 3.0±0.2 4.0±0.4 3.0±0.3 Saturated FA 20±1 20±2 31±2# 23±1* Monounsaturated FA 22±2 20±1 21±1 22±0 Polyunsaturated FA n-3 22±1 24±1 24±1 22±1 Polyunsaturated FA n-6 37±3 37±1 24±1# 33±1* Liver Phosphatidylcholine 45.5±4.2 44.2±2.6 47±3 44.5±3.8 Phosphatidylethanolamine 34±2 32±2 35±2 33±3 Cardiolipin 15±2 20±2* 12±1 18±1* Phosphatidylinositol 5.0±1.1 3.0±1.0 5.0±0.7 4.0±0.9 Phosphatidylserine 0.5±0.2 0.8±0.2 1.0±0.5 0.5±0.1 Saturated FA 19±1 21±1 29±2# 22±2* Monounsaturated FA 24±1 22±1 22±1 23±1 Polyunsaturated FA n-3 21±1 23±2 26±2 24±1 Polyunsaturated FA n-6 36±2 35±2 23±2# 32±1* The values are expressed in mol % and represent the means ± S.E. of 6 independent mitochondrial preparations isolated in parallel from 4 groups of mice, 252–259 days old. * indicates statistical difference between SkQ1-treated and non-treated mice (p < 0.05). # indicates statistical difference between wild-type and mtDNA mutator mice (p < 0.05). We further analyzed the fatty acyl composition of the total phospholipids of the mitochondrial membranes. The content of polyunsaturated n-6 fatty acids (PUFA n-6) in mtDNA mutator mice was markedly decreased to only 2/3rd of wild-type levels both in skeletal muscle and liver mitochondria of mtDNA mutator mice (Table 1). The lowering of polyunsaturated fatty acids level was compensated for by saturated fatty acids. The SkQ1 treatment of the mtDNA mutator mice fully prevented this remodeling, such that wild-type levels were retained. As cardiolipin generally contains 4 linoleyl (18:2, n-6) moieties per molecule, it is likely that the decrease in cardiolipin amount (by ≈5 mol %) is largely responsible for the loss of n-6 PUFAs (by ≈10 mol %), and that the retention of the n-6 PUFAs by SkQ1 treatment is due to the preservation of cardiolipin in the SkQ1-treated mtDNA mutator mice.

SkQ1 normalized mitochondrial ultrastructure in liver and heart Cardiolipin is believed to be important for mitochondrial structure and function [32]. We therefore examined whether the large changes in cardiolipin amount were associated with alterations in these parameters. Fig. 5 shows electron microscopic pictures of liver cells from wild-type mice, mtDNA mutator mice and SkQ1-treated mtDNA mutator mice. The mitochondria in mtDNA mutator mice seemed larger than those in wild-type mice. Moreover, some of the mitochondria in the mtDNA mutator mice contained elongated electron-dense inclusions composed of series of membranes packed in a myelin-like manner. The SkQ1 treatment prevented both these changes. Figure 5. Liver mitochondrial structure. Electron micrographs of liver from wild-type mice (left panel), mtDNA mutator mice (middle panel) and mtDNA mutator mice treated with SkQ1 (right panel). Animals were 245 – 252 days old. Insertion in the middle panel, lower micrograph: intramitochondrial myelin-like structure at higher magnification. Similar findings were observed in 4-5 other mice of each group.

Safdar et al. [33] observed similar mitochondrial alterations in skeletal and heart muscles of mtDNA mutator mice. We confirmed this observation in heart muscle. Also in this tissue, SkQ1 prevented disorganization of the mitochondrial ultrastructure (Fig. 6). Moreover, the number of mitochondria per µm2 of heart muscle was increased by SkQ1 from 100 ± 5% to 128 ± 6% (P < 0.05) in the wild-type mice and from 69 ± 3% to 125 ± 6% (P < 0.001) in the mtDNA mutator mice. SkQ1 did not influence the area occupied by the inner mitochondrial membrane in the wild-type mice but prevented 42% (P < 0.01) decrease in this area observed in the mtDNA mutator mice. Figure 6. Heart mitochondrial structure. Electron micrographs of heart from wild-type mice (left panel), mtDNA mutator mice (middle panel) and mtDNA mutator mice treated with SkQ1 (right panel). Animals were 245 – 252 days old. Similar findings were observed in 4-5 other mice of each group.



SkQ1 improved the function of isolated mitochondria In the mtDNA mutator mice, there is a significant decline of ADP- and FCCP-inducible respiration of liver and heart mitochondria, as compared to wild-type mitochondria [15]. To examine whether SkQ1 treatment would affect the development of this mitochondrial deterioration, we analyzed the oxygen consumption of skeletal muscle mitochondria isolated from SkQ1-treated and non-treated mtDNA mutator mice. Fig. 7A exemplifies the bioenergetic kinetics observed in such experiments. As summarized in Fig. 7B, ADP-stimulated respiration was higher in mitochondria from SkQ1-treated mtDNA mutator mice than in mitochondria from mtDNA mutator mice. There was no observable effect on state 4 respiration (i.e. after oligomycin) but there was a tendency to a higher maximal respiratory capacity (i.e. after addition of the artificial uncoupler FCCP). Thus, both ultrastructural and bioenergetic features of mitochondria from mtDNA mutator mice were ameliorated by SkQ1 treatment. Figure 7. Effect of SkQ1 treatment on function of isolated mitochondria. (A) Example of an oxygen consumption trace in mitochondria isolated from skeletal muscle of mtDNA mutator mouse. Additions were 0.25 mg skeletal muscle mitochondria (SKM), 5 mM pyruvate (Pyr), 250 µM ADP, 2 µg /ml oligomycin (Olig) and 1.4 µM FCCP. (B) Rates of oxygen consumption in mitochondria isolated from skeletal muscle of SkQ1-treated and non-treated mtDNA mutator mice. Analysis was performed as shown in 7A. (C) Amplex Red fluorescence in intact mitochondria isolated from skeletal muscle of mtDNA mutator mouse. Additions were 0.25 mg skeletal muscle mitochondria (SKM), 5 mM pyruvate (Pyr), 5mM succinate (Succ). Malate (3 mM) was present in the medium. The analyses were performed in the same mitochondrial preparations as in (A) in parallel with the oxygen consumption measurements. (D) Rates of hydrogen peroxide production in mitochondria isolated from skeletal muscle of wild type mice (WT), SkQ1 non-treated (Mut) and treated (Mut + SkQ1) mtDNA mutator mice. Analysis was performed as shown in C. P+M indicates the presence of complex I substrates (pyruvate + malate) and P+M+S indicates the presence of three substrates (pyruvate + malate + succinate). In A and D, the values represent the means ± S.E. of 6 independent mitochondrial preparations isolated in parallel from treated and non-treated groups of mice of an age of 252 – 259 days. * in A-D indicates statistical difference between SkQ1-treated and non-treated mice (p < 0.05).



Higher mitochondrial ROS production in SkQ1-treated mice reflects an improved respiratory chain capacity Hydrogen peroxide production resulting from oxidation of a complex I substrates (pyruvate+malate) or of mixed complex-I and complex-II substrates (pyruvate +malate+succinate) was measured in skeletal muscle mitochondria from non-treated and SkQ1-treated mtDNA mutator mice, as exemplified in Figs. 7C and D. Hydrogen peroxide production supported by complex I substrates alone was not affected by SkO1-treatment (Fig. 7D). However, on mixed substrates hydrogen peroxide production rate in isolated mitochondria from SkQ1-treated mice was higher than in mitochondria from non-treated mice (Fig. 7D). This effect of SkQ1 may be the result of an improved respiratory electron transfer activity and/or more directly from a higher membrane potential in the mitochondria of SkQ1-treated mice. It is shown that the ROS production that results from reverse electron flow from exogenously provided succinate is augmented by a higher membrane potential [34–37]. As to the in vivo SkQ1 treatment, it does not lead to increased general oxidative damage (4-HNE adduct formation was actually decreased following SkQ1 treatment, see Fig. 4E).

SkQ1 treatment improves thermogenic capacity of brown adipose tissue Hypothermia is a characteristic feature of mtDNA mutator mice and develops markedly after about 220 days, as evaluated qualitatively in Fig. 8A. SkQ1-treated mtDNA mutator mice show almost no indication of hypothermia during this period. Additionally, whereas mtDNA mutator mice to a very high degree developed hypothermia towards the end of their life, the SkQ1-treated mtDNA mutator mice did not present with this problem even as they became moribund (Fig. 8A). Figure 8. Effect of SkQ1 treatment on thermogenesis and brown adipose tissue of mtDNA mutator mice. (A) Scoring of manifestations of hypothermia (based on body temperature, shivering and posture position) in SkQ1-treated and non-treated mtDNA mutator mice. Gender and number of mice in each group are as in 1B. (B) Wet weight of interscapular brown adipose tissue (BAT). (C) Western blots analyses of VDAC and UCP1 in BAT (tissue homogenate protein, 10 µg per lane). (D) Relative concentration of mitochondrial proteins. Western blots as in C were quantified. The mean level of UCP1 and VDAC in control mice was set to 100% and the levels of protein in BAT from SkQ1-treated mice expressed relative to this. (E) Total protein content per BAT depot. (F) Total content of UCP1 and VDAC per mouse. Content of each protein per µg homogenate protein estimated from Western blot analysis (in E) was multiplied with the total protein content of BAT (in C) from the same mouse. The values in B and D - F represent the means ± SE of 4-5 independent tissue preparations in each group, analyzed singly or in duplicate. * indicates statistical significance between SkQ1-treated and non-treated mice (p < 0.05).

A main source for sustained regulatory heat production in mammals is brown adipose tissue. There was a significant positive effect of SkQ1 treatment on interscapular brown adipose tissue mass (Fig. 8B). Analysis of mitochondrial proteins in brown adipose tissue homogenate revealed a higher level of mitochondrial voltage-dependent anion channel (VDAC) per mg brown adipose tissue total protein (Figs. 8C, D) in SkQ1-treated mtDNA mutator mice, indicating an increase in the number of mitochondria. More functionally, there was also an increase in the amount of the rate-limiting protein for thermogenesis, i.e. uncoupling protein (UCP1) (Fig. 8C, D). The total brown adipose tissue protein was also higher in SkQ1-treated mtDNA mutator mice (Fig. 8E). For thermogenesis on whole organism level, the total thermogenic capacity is more important than concentration of proteins. Therefore we estimated the total content of VDAC (≈ amount of mitochondria) and UCP1(≈ total thermogenic capacity) in the interscapular brown adipose tissue (Fig. 8F) by multiplying the specific content of each of these proteins (Fig. 8D) with the total amount of protein (Fig. 8E). The outcome was that the total mitochondrial content and particularly the total content of UCP1 was more than 50% higher in the SkQ1-treated mtDNA mutator mice than in the non-treated mice (Fig. 8F). These observations indicate that the lack of hypothermia in the SkQ1-treated mtDNA mutator mice can largely be ascribed to an enhanced (or preserved) capacity for heat production in their brown adipose tissue.

SkQ1 treatment delays development of mtDNA mutator mice immobility Oxygen consumption of mice at their living temperature 22 °C (Fig. 9A) reflects mouse general activity inclu-ding adaptive thermogenesis. The SkQ1-treated mtDNA mutator mice exhibited higher metabolism (oxygen consumption rates) above the resting rates (Fig. 9B and C), probably related to visibly higher mobility of animals (Fig. 9D), better retention of mitochondrial oxidative phosphorylation (Fig. 7B) and improved brown adipose tissue thermogenic capacity (Fig. 8F). Figure 9. Effect of SkQ1 treatment on mouse metabolism, motility and survival. (A) Representative trace of oxygen consumption (metabolism) of a mouse. Running oxygen consumption rate is indicated as black bold curve, the lowest (resting at 22 °C environmental temperature) oxygen consumption rate indicated as a dashed straight line and the oxygen consumed above the resting rate is indicated as grey area. Note that the oxygen consumption rate at 22 °C does not represent the basal metabolic rate, as the mice are examined below their thermoneutral temperature zone.(B) Metabolic rates in non-treated and SkQ-treated mice (≈ 200 days of age) at 22 °C ambient temperature measured as shown in A. (C) Metabolic activity of mtDNA mutator mice. Activity was determined as amount of oxygen consumed above resting metabolic rate during two hours in the metabolic chamber (as shown in 9A). In B and C, the values represent the means ± S.E. of 6 mice in each group. (D) Scoring of manifestations of immobility in SkQ1-treated and non-treated mtDNA mutator mice. (E) Survival curves of mtDNA mutant mice non-treated (thin line) or treated with SkQ1 (thick line), n= 38 (15 males and 23 females) in the non-treated group and n= 17 (6 males and 10 females) in SkQ1-treated group. No gender dependence on life-span was observed (not shown). Mice were single caged. Mean survival time for the non-treated group was 277 ± 6 days, for the SkQ1-treated 321 ± 10 days (+ 16%); median lifespan was 290 days for non-treated mice and 335 days (+ 16%) for SkQ1-treated mice. Comparison of survival curves with log-rank (Mantel-Cox) test yields p < 0.0001 and with Gehan-Breslow-Wilcoxon test yields p = 0.0012.



Significance of improved thermogenic capacity and physical activity on cause of death in SkQ1-treated mtDNA mutator mice In Table 2, we have collected descriptions of phenotypic features of mtDNA mutator mice treated and non-treated with SkQ1 at the point of euthanasia. It may be noted that in the non-treated mtDNA mutator mice, more than 50% of the mice displayed phenotypes related to body temperature regulation problems: low body temperatures, piloerection, shivering. In the SkQ1-treated mice, these features were not at all prominent. Thus, the SkQ1 treatment retained mitochondrial bioenergetic capacity, both in muscle and in brown adipose tissue, thereby eliminating a major cause of death. In the SkQ1-treated group, anemia and body weight decrease became the prominent features in the moribund mice. Table 2. Incidence of symptoms in mtDNA mutator mice at the point of euthanasia during the survival experiment. mtDNA mutator mice mtDNA mutator mice + SkQ1 Symptoms Number

(and %)

of mice in the group from total 28 Symptoms Number

(and %) of mice in the group from total 13 Keeping posture position; shivering 25 (89%) Severe paleness 9 (69%) Body temperature below 34 °C 23 (82%) Mouse hides, lies still but is startled when touched 8 (62%) Chronic body weight loss by 15% during several weeks 21 (75%) Chronic body weight loss by 15% during several weeks 8 (62%) Severe piloerection 20 (71%) Mouse is unable to stand up on back legs; dis-coordination, asymmetry of movement 5 (38%) Severe anus prolapse and bleeding 6 (21%) Keeping posture position; shivering 5 (38%) Soft feces, diarrhea 5 (18%) Severe dehydration 5 (38%) Acute body weight loss (2-3 g during 1 week) 5 (18%) Severe anus prolapse and bleeding 3 (23%) Severe paleness 4 (14%) Soft feces, diarrhea 3 (23%) Mouse is unable to stand up on back legs; dis-coordination, asymmetry of movement 3 (11%) Acute body weight loss (2-3 g during 1 week) 3 (23%) Severe dehydration 2 (7%) Severe piloerection 1 (8%) Mouse hides, lies still but is startled when touched 1 (4%) Body temperature below 34 °C 1 (8%) The heavy line separates the symptoms seen in >50% of all mice from those seen in <50%. Postmortem examination also revealed positive effects of SkQ1 treatment (Table 3). Particularly, the heart pathology (Fig. S2A) often observed in the mtDNA mutator mice [12,22] was much rarer (Table 3). Liver (Fig. S2B), as well as kidney (Fig. S2C), pathology was milder, and the hydrocephalus observed occasionally in the mtDNA mutator mice was not found in the SkQ1-treated mice (Table 3). In contrast, certain other pathological findings occurred irrespectively of SkQ1 treatment: cecum and colon displayed pathological changes in all animals in both groups, and particularly the spleen was also pathological in nearly all animals, irrespective of SkQ1 treatment or not (Table 3). The spleen pathology is probably related to the paleness observed to a high extent in the SkQ1-treated mice during their last days (Fig. S2D). It would simply seem that the SkQ1-treated mtDNA mutator mice lived so much longer than the non-treated mice that anemia had time to develop sufficiently to become life-threatening. Table 3. Histopathological findings at necropsy of mtDNA mutator mice untreated or treated with SkQ1. Organ Histopathology Mut

n = 11 Mut+SkQ1

n = 11 Heart 1) The myocardium exhibited moderate myocardial degeneration, characterized by irregularly shaped nuclei and rich cytoplasmic vacuolation in myocytes

2) Perivascular or interstitial fibrosis and Anitschkow cells 5

2 2

0 Brain Mild internal hydrocephalus 3 0 Kidney The renal cortex displayed occasional dilated tubules and increased cytoplasmic basophillia in the epithelial lining an occasional proximal tubule - characteristics of nephropathy:

mild

moderate 4

5 6

0 Liver 1) Chronic degenerative changes characterized by enlarged periportal hepatocytes having granular cytoplasm or large and small fatty vacuoles. Portal areas displayed moderate fibrosis and hyperplastic oval cells at sites:

1a) mild

1b) moderate

2) Degenerative changes with abnormal nuclear features (pseudo-inclusions) 1

5

3 2

0

0 3) The sinusoids exhibited scattered, small aggregates of hematopoietic cells or/and myelopoetic cells 5 5 Spleen 1) The red pulp displayed extensive hematopoetic activity

2) The white pulp displayed small lymphatic follicles 10

9 11

9 Cecum and the proximal colon Severe mucosal hyperplasia with chronic catarrhal inflammation. Epithelium degeneration, necrosis, apoptosis. Infiltration leukocytes predominately macrophages. Mucosal atrophy coexisted with sites of the mucosa thickened by hyperplasia, forming plague-like or irregular papillary projections lined by crowed, tall columnar, basophilic cells with numerous mitotic figures. 11 11 11 mice in each group were analyzed in the survival experimental setup.