Mitochondrial DNA Mutations in Aging

Konstantin Khrapko*; Doug Turnbull†    * Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA
† LLHW Centre for Ageing and Vitality, Newcastle University, Newcastle, United Kingdom

Abstract

The relationship of mitochondrial DNA mutations to aging is still debated. Most mtDNA mutations are recessive: there are multiple copies per cell and mutation needs to clonally expand to cause respiratory deficiency. Overall mtDNA mutant loads are low, so effects of mutations are limited to critical areas where mutations locally reach high fractions. This includes respiratory chain deficient zones in muscle fibers, respiratory-deficient crypts in colon, and massive expansions of deleted mtDNA in substantia nigra neurons. mtDNA “mutator” mouse with increased rate of mtDNA mutations is a useful model, although rates and distribution of mutations may significantly deviate from what is observed in human aging. Comparison of species with different longevity reveals intriguing longevity-related traits in mtDNA sequence, although their significance is yet to be evaluated. The impact of somatic mtDNA mutations rapidly increases with age, so their importance is expected to grow as human life expectancy increases.

Keywords

Aging

Mutations

Mitochondrial DNA

Clonal expansion

Evolution of aging

1 Introduction: The Different Faces of the Mitochondrial Theory of Aging

Mitochondrial involvement in aging was proposed over 30 years ago by Denham Harman, based on his original theory that aging is caused by the accumulation of damage resulting from reactive oxygen species (ROS).1 ROS are the inevitable by-products of normal cellular processes, most notably the process of oxidative phosphorylation, which is the primary function of the mitochondrion. Harman noted that as a major source of ROS, mitochondria should also be its major target.1 Hence, as the part of the cell most vulnerable to ROS, mitochondria could play the role of the “aging clock,” the limiting aging component of the system. Later, Linnane and others2 specifically implied accumulating mitochondrial DNA (mtDNA) mutations (as opposed to general damage, which usually means also chemical modifications of DNA and other macromolecules) as the main culprit in aging. The logic is that mitochondria are renewable organelles. Some of them grow, replicate their DNA, and divide. Other mitochondria are destroyed and replaced by newly divided ones. This means that all their damaged components are constantly replaced by newly synthesized ones. The only part that saves the record of past damage despite this turnover is the sequence of mtDNA. Indeed, mtDNA mutations once they arise are replicated and faithfully transmitted to the daughter mitochondria. This supports the hypothesis that accumulation of mtDNA mutations is one of the “primary” aging processes. The significance of mtDNA mutations for aging is also corroborated by a few other observations, of which most notable is the presence in old tissues, muscle, brain, and colon in particular, of cells that are deficient in mitochondrial function caused by mtDNA mutations (Fig. 2.1).

Of note, mitochondrial mutational hypothesis of aging should be distinguished from a few other mitochondrial hypotheses of aging, which do not explicitly refer to mtDNA mutations (genetic damage), but rather to chemical damage, such as oxidation, cross-links, and other covalent modification of macromolecules (including DNA). The various concepts are not mutually exclusive and all may very well have their share in the aging process. For example, “mitochondrial-lysosomal axis” theory of aging3 maintains that in aged cells, accumulating oxidated cellular waste (primarily damaged mitochondria) “chocks” cellular autophagy systems. This further hampers turnover of damaged mitochondria creating a self-accelerating loop that eventually cumulates in dysfunctional cells or cell death.

Another, more traditional “mitochondrial free radical theory of aging” (Chapter 1), postulates that aging is caused by accumulating oxidative damage, which does not necessarily involve a self-accelerating component. In this view, aging can be caused by a constant rate of oxidative damage. In this theory, while mitochondria are also considered a critical component, the main source and main target of oxidative damage, mtDNA mutations do not play a central role in this theory, they are just a part of damage.

Despite its attractiveness, the idea that mutations in mtDNA cause at least some aspects of aging remains controversial. Mitochondrial defects in aging tissues (Fig. 2.1) are yet to be convincingly related to age-related pathologies. To make the case of mtDNA mutations even more challenging, there are a few examples where artificially increased number of somatic mtDNA mutations in genetically engineered mice does not result in accelerated aging,4 implying that at least some types mtDNA mutations are not involved in aging. Furthermore, controversy is fueled by the difficulties in accurately measuring of the fraction of mutant molecules. Some estimates yield rather unimpressively low overall fractions of mtDNA mutations, which seem to challenge the idea that these mutations may be of any significance. High estimates by other methods are sometimes perceived as gross overestimates.78 To make things even more complicated, to estimate the functional impact of mtDNA mutations, one needs to know, in addition to overall mutant fraction, the detailed cell-to-cell distribution of mutations. This is because mutant mtDNA tend to affect cellular physiology only once they reach certain percentage within the mtDNA population of each individual cell, called the physiological threshold (Section 3). Here, we will review the evidence in support of the mtDNA mutational hypothesis of aging, which ranges from data on the detailed cell-by-cell distribution of mtDNA mutations to the inverse correlation of the number of repeats in mtDNA sequence and species’ longevity.

2 Mitochondria, mtDNA, and mtDNA Mutations

2.1 Mitochondrial biology and mtDNA

Mitochondria are subcellular organelles responsible for generation of ATP, the cell's universal energy carrier, in a process of oxidative phosphorylation. This process is performed by a set of five multisubunit enzyme complexes (I, II, III, IV, and V) called the respiratory chain (RC), which are located on the inner membrane of the mitochondrion (for illustration see Ref. 5). Depending on the metabolic requirement of the cell, the number of mitochondria can vary markedly. In the majority of cells, mitochondria appear as a thread-like network throughout the cell and are constantly undergoing fission and fusion, a process that seems to be very important in terms of physiology and turnover of mitochondria.6,7

Mitochondria carry their own genome, i.e., a small (about 16,500 base pairs in animals) circular DNA molecule, which is replicated by the mtDNA polymerase gamma. Animal mtDNA encodes 13 polypeptides, which are subunits of all but one (complex II) of the RC complexes, as well as mitochondrial 22 tRNA and 2 rRNA.8 All other subunits of the RC enzymes (~ 80 of them) are encoded in the nuclear genome,5 as are the rest of several hundred mitochondrial proteins that are located in mitochondria but are not directly involved in the RC. Of note, outside the animal kingdom, mtDNA types are rather diverse.9 MtDNA is maternally inherited in mammals since all the mitochondria from the sperm are destroyed on entry into the ovum. MtDNA is present in multiple copies in all cells. The number of copies of mtDNA varies markedly between cells, with tissues with high energy requirements, such as brain, heart, and skeletal muscle, harboring the largest numbers of mitochondrial genomes. MtDNA is believed to be organized into “nucleoids,” i.e., compact nucleoprotein particles containing from one to a few mtDNA molecules. The exact composition, structure, and function of nucleoids are under intense investigation.10

2.2 mtDNA mutations

The mitochondrial genome principally suffers from two types of mutations: point mutations and large genome rearrangements, of which most studied are deletions, i.e., loss of large portions of the genome (from a few hundred base pairs to almost the entire mitochondrial genome).

There are several possible sources of mtDNA mutations, which can be broadly classified into spontaneous errors and damage-induced mutations. Spontaneous polymerase errors result from inherent polymerase infidelity. For example, an incorrect nucleotide may be inserted opposite to a normal nucleotide of the DNA template to generate base substitution. DNA damage is the other source of mutations. In the simplest case, DNA polymerase may be prompted to insert...