ReviewMitochondrial DNA alterations and reduced mitochondrial function in aging
Introduction
Mitochondria are unique organelles because they contain their own DNA. Multiple copies of the mitochondrial genome are present in each mitochondrion. The mammalian mitochondrial genome is composed of ∼16.5 kilobases of circular, double-stranded DNA encoding for 2 ribosomal RNAs, 22 transfer RNAs, and 13 protein subunits of the electron transport chain (Anderson et al., 1981, Bibb et al., 1981), all of which are essential for proper mitochondrial function. The majority of mitochondrial proteins are encoded by the nuclear genome and transported to mitochondria. Mitochondrial DNA (mtDNA) is organized into protein-DNA complexes called nucleoids within the mitochondrial matrix (Gilkerson, 2009). Although mtDNA is packaged into nucleoids, which provide more protection to the genome than was originally thought, it remains in close proximity to the electron transport chain (located in the inner mitochondrial membrane) which is the main source of reactive oxygen species (ROS) within the cell.
Mitochondria generate chemical energy (ATP), which is required to fuel many thermodynamically unfavorable processes within cells (e.g., ion transport against electrochemical gradients, protein synthesis, ubiquitin-dependent protein degradation, and contractility). Mitochondrial oxidative phosphorylation generates ATP through a set of coupled reactions where macronutrients are oxidized, oxygen is reduced to water, and ADP is phosphorylated to ATP. First, carbon substrates enter the tricarboxylic acid cycle either through acetyl CoA or anaplerotic reactions. These substrates are then oxidized to generate reducing equivalents in the form of NADH and FADH2, which provide electron flow though respiratory chain complexes I (NADH dehydrogenase) and II (succinate dehydrogenase), respectively. Electron flow from complexes I and II converges on complex III (ubiquinone-cytochrome c reductase), along with electrons shuttled in from electron transferring flavoproteins (beta oxidation), through the mobile electron carrier coenzyme Q. A second mobile electron carrier (cytochrome c) transfers electrons on to complex IV (cytochrome c oxidase) where they are finally transferred to oxygen, yielding water. A proton gradient across the inner mitochondrial membrane is generated as a result of electron transport through complexes I, III, and IV. Complex V (ATP synthase) phosphorylates ADP to ATP by harnessing the potential energy of this gradient.
During the process of ATP production by oxidative phosphorylation, ROS are also generated at complex I and complex III. Electron leak at these complexes can reduce oxygen to form the superoxide radical, •O2−. ROS are highly reactive molecules that can damage nucleic acids, lipids, and proteins. Because of the close proximity of mtDNA to the electron transport chain it is thought that mtDNA is highly susceptible to oxidative damage by ROS. The mitochondrial theory of aging proposes that ROS-induced oxidative damage to mitochondria and its DNA is a major contributor to aging (Harman, 1972, Linnane et al., 1989). Key to this theory is the idea that a vicious cycle of mitochondrial damage resulting in dysfunction drives the aging process. Briefly, ROS generated during oxidative phosphorylation may damage mtDNA, lipids, and proteins. Mutations that may occur within the mtDNA as a result of unrepaired oxidative damage may lead to the production of dysfunctional proteins. Dysfunctional electron transport chain proteins may produce more ROS which would continue the damaging cycle.
Accumulating evidence suggests that the ROS production rate is largely dependent on the mitochondrial membrane potential (Δψ) (Balaban et al., 2005). Mitochondrial redox potential is high when membrane potential is high, allowing for greater potential for backflow of electrons through complexes I and III. In contrast, state 3 respiration minimizes this effect by increasing the forward flux of electrons along the cytochromes. The mitochondrial membrane potential is also influenced by uncoupling proteins that are expressed in tissues such as adipose tissue and skeletal muscle. The uncoupling of mitochondrial oxygen consumption from ATP production by uncoupling proteins (UCP-1) is an important mechanism of thermogenesis in brown adipose tissue in animals. Mitochondrial uncoupling and thermogenesis also occur in skeletal muscle, which, by virtue of its mass, plays a key role in regulating energy expenditure and insulin-stimulated glucose uptake. Proteins with similar structural homology to UCP-1 are expressed in skeletal muscle (UCP-2,3) (Boss et al., 1997, Fleury et al., 1997). Although the role of UCP-3 as an uncoupler is questionable (Cadenas et al., 1999, Dulloo et al., 2001), this protein has been determined to be important in fatty acid (FA) metabolism by shuttling FA anions out of the mitochondrial matrix. FAs may participate in “flip-flop acidification” whereby neutral FAs, but not FA anions, can rapidly flip-flop across the inner mitochondrial membrane. An acid-base equilibrium is then achieved via protonation of FA anions on the cytosolic side of the membrane and deprotonation of neutral FAs on the matrix side of the membrane. This “flip-flop acidification” would lead to dissipation of the mitochondrial proton gradient and uncoupling of oxidative phosphorylation (Jezek and Garlid, 1998). FA anion carriers such as UCP-3 transport these deprotonated FAs back to the cytosolic side of the membrane. By exporting FA anions from the mitochondrial matrix, UCP-3s are believed to minimize peroxidation of lipids that would occur in close proximity to the superoxide-generating electron transport chain. Furthermore, although UCP-3s are not believed to act as proton channels such as UCP-1, they are key components in the process of FA cycling, which dissipates a portion of the proton gradient (Jezek and Garlid, 1998).
Mitochondrial structure is maintained by the balanced processes of fusion and fission. Mitochondrial fission, mediated by mitofusins, is believed to be an initial step in maintaining the functionality of the mitochondrial reticulum through separation and subsequent autophagy of dysfunctional regions of the organelle (Legros et al., 2002, Chan, 2006). During mitochondrial fusion, the contents of the mitochondrial matrix are exchanged between organelles. This process is regulated by dynamin-related protein Drp1 and the mitochondrial fission protein hFis1 (Lopez-Lluch et al., 2008). While some believe that fusion causes mutated mtDNA to diffuse between organelles and become evenly distributed about the cell, there is evidence to suggest that mitochondrial nucleoids are bound to regions of the inner membrane, which restricts their diffusion (Miyakawa et al., 1987). Nevertheless, it is possible that defects in fusion and fission could contribute to declines in mitochondrial function with old age because mtDNA deletions and mutations may not be effectively diluted by fusion or eliminated by fission and autophagy. The role of mitochondrial remodeling in aging has recently been demonstrated by depletion of hFis by RNA interference, which induces a senescent phenotype (Lee et al., 2007). Furthermore, the activity of Lon protease, which degrades oxidized mitochondrial proteins, decreases with age (Lee et al., 1999). The resulting accumulation of oxidized, dysfunctional proteins could further increase oxidative stress and oxidative damage to proteins and DNA with aging.
Section snippets
Oxidative damage
Consequences of oxidative damage to mtDNA include point mutations, deletions, and strand breaks. Exposure to ROS can result in a number of oxidative modifications to mtDNA including the lesions 8-oxoguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5-formamidopyrimidine (FapyA), and thymine glycol. The most commonly studied of these oxidative lesions is 8-oxoguanine. 8-oxoguanine can be removed from mtDNA through the process of base excision repair by the mitochondrial
Mitochondrial DNA abundance in aging
A decline in mtDNA abundance could contribute to the age-related decline in mitochondrial function. Reduced mtDNA template availability could negatively impact the levels of mitochondrial gene transcripts resulting in reduced levels of mitochondrial proteins. An age-related decline in mtDNA abundance has been demonstrated by some investigators. Barazzoni et al. found that mtDNA copy number was reduced by 25% in gastrocnemius, 40% in soleus, and 50% in liver of old rats (27 months) compared to
Changes in mitochondrial function with aging
Although there is some question concerning the physiological impact of many reported age-related changes to mtDNA, it remains a distinct possibility that even small changes to mtDNA integrity could exert trickle-down effects, ultimately altering the overall function of mitochondria. Damage, deletions, or mutations to mtDNA are likely to influence downstream processes such as transcription, translation, enzyme activity, mitochondrial ATP synthesis, and overall organ function.
Threshold effects
The previous paragraphs have reviewed much of the evidence linking aging with changes downstream from the mitochondrial genome. A lingering question is: to what extent can the documented changes in transcript levels, translational rates, protein expression, enzyme activity, and mitochondrial function be related back to mtDNA mutations and deletions that occur with aging? The answer to this is not straightforward, but much insight can be gleaned from a large body of literature supporting the
Summary
The exact mechanism responsible for the decline in mitochondrial function with aging remains to be determined. A proposed model is presented in Fig. 4. Oxidative damage increases with age and can result in alterations of the mitochondrial genome including point mutations, deletions, and DNA strand breaks. The extent of mutations and deletions within a tissue increase with aging, however, the overall mutational load typically remains low. Recent evidence that oxidatively damaged mtDNA is
Acknowledgements
Support was provided by NIH grants RO1-AG09531, UL1-RR024150, T32-DK07352 (S.L. Hebert), and the David Murdock-Dole Professorship (K.S. Nair).
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