Date Published: February 3, 2017
Publisher: Public Library of Science
Author(s): Shinichi Someya, Gregory C. Kujoth, Mi-Jung Kim, Timothy A. Hacker, Marc Vermulst, Richard Weindruch, Tomas A. Prolla, Tadafumi Kato.
Mitochondrial DNA (mtDNA) mutations are thought to have a causative role in age-related pathologies. We have shown previously that mitochondrial mutator mice (PolgD257A/D257A), harboring a proofreading-deficient version of the mtDNA polymerase gamma (POLG), accumulate mtDNA mutations in multiple tissues and display several features of accelerated aging. Calorie restriction (CR) is known to delay the onset of age-related diseases and to extend the lifespan of a variety of species, including rodents. In the current study we investigated the effects of CR on the lifespan and healthspan of mitochondrial mutator mice. Long-term CR did not increase the median or maximum lifespan of PolgD257A/D257A mice. Furthermore, CR did not reduce mtDNA deletions in the heart and muscle, accelerated sarcopenia, testicular atrophy, nor improve the alterations in cardiac parameters that are present in aged mitochondrial mutator mice. Therefore, our findings suggest that accumulation of mtDNA mutations may interfere with the beneficial action of CR in aging retardation.
Calorie restriction (CR) is the most robust non-genetic intervention to consistently extend lifespan in a variety of species . CR also reduces risks for a variety of age-associated diseases, including diabetes, sarcopenia, cancer, cardiovascular diseases, and hearing loss in rodents and humans [1–3]. Furthermore, CR reduces the levels of reactive oxygen species (ROS) and associated oxidative damage, and mtDNA deletions in multiple tissues [1–2, 4–5]. Consistent with these reports, long-lived GH (growth hormone)-deficient mice display CR-like anti-aging effects, including increased expression of antioxidant enzymes and stress response genes in muscle cells and fibroblasts , reduced body size and extended median lifespan . The mitochondrial theory of aging postulates that ROS generated inside the mitochondria damage key mitochondrial components such as mitochondrial DNA (mtDNA), resulting in mitochondrial dysfunction . This in turn leads to energy depletion in multiple tissues and eventually to aging symptoms. Accordingly, CR is thought to slow the rate of aging or to reduce risks for many age-associated diseases through the protection of mitochondrial macromolecules, including mtDNA [1, 5, 8–10]. In the current study, we investigated the effects of CR on the healthspan and lifespan of mice that express a proofreading-deficient version of mtDNA polymerase gamma (PolgD257A/D257A) and display elevated mtDNA mutation frequencies throughout their tissues and accelerated aging phenotypes [11–14].
We and others have previously shown that D257A/D257A mice display a range of premature aging phenotypes beginning at ~9 months of age, including body weight loss, loss of bone mass, and reduced lifespan [11–12]. To investigate the effects of CR on the healthspan and/or lifespan of mitochondrial mutator mice, we reduced the calorie intake of wild-type (+/+), Polg+/D257A (+/D257A), and PolgD257A/D257A (D257A/D257A) mice in the C57BL/6 background to 75% (a 25% CR) of that fed to control diet (CD) mice starting in early adulthood (2 months of age), and this diet regimen was maintained until the animals reached middle-age (13–16 months of age) or spontaneous death of the animals occurred. CD mice were fed a control diet (Teklad, Madison, WI) and received 84 kcal/week of the diet, while CR mice were fed a restricted diet (Teklad, Madison, WI) of 63 kcal/week. The restricted diet was enriched in protein, vitamins, and minerals to avoid malnutrition. This is a well-established CR protocol that extends lifespan in inbred mouse strains, including the C57BL/6 strain . We first confirmed that this CR diet regimen resulted in a significant reduction of body weight in middle-aged +/+ males and females (Fig 1). CR also resulted in a significant reduction of body weight in middle-aged +/D257A males, but not in middle-aged +/D257A females. Although middle-aged D257A/D257A mice weighed less than their +/+ or +/D257A counterparts, CR did not further reduce the body weight of D257A/D257A males or females. We note that the specific cause of reduced weight in D257A/D257A mice is unknown, although it is likely to be a combination of reduced adipose tissue and muscle mass in these animals, as well as degeneration of other organs. These phenotypes become progressively worse with age . Thus, if CR reduced accelerated aging in D257A/D257A mice, we speculate that the weight of POLG mice should be more similar to that of +/+ animals at middle age.
Point mutations and deletions in mtDNA accumulate with age in a variety of tissues , and a lifelong accumulation of such mutations in postmitotic tissues that demand high energy is thought to result in mitochondrial energy depletion, causing tissue dysfunction and eventually age-related pathologies. In agreement with this hypothesis, there are over 80 pathogenic mutations in the human POLG gene, and some of these are associated with Alper’s syndrome, PEO (progressive external ophthalmoplegia), or ataxia, and cause a variety of symptoms, including ophthalmoplegia, cataracts, hearing loss, progressive muscle weakness, parkinsonism, and cardiac dysfunction [22–23]. Mitochondrial mutator mice display a 10-90-fold increase in mtDNA deletions  and a >100-fold increase in point mutations in mtDNA in the heart and brain . These mice display mitochondrial diseases and aging symptoms such as motor function decline, cardiac dysfunction and muscle mass loss, and hearing loss [20, 21, 23]. We have previously proposed that phenotypes of D257A/D257A mice are due to loss of somatic stem cells, since tissues displaying early onset phenotypes have high cellular turnover . Recent studies have confirmed that D257A/D257A mice have a profound somatic stem cell defect [24–25]. Thus, CR failed to extend the healthspan or lifespan of D257A/D257A mice likely because the dietary intervention failed to rescue somatic stem cell loss or dysfunction in the context of damaged mitochondria in D257A/D257A mice.