Mitochondria

MITOCHONDRIA

Mitochondria are organelles found in the cytoplasm of all eukaryotic cells. They vary considerably in shape and size, but are all composed of four compartments: a smooth outer membrane, a convoluted inner membrane that forms recognizable structures called cristae, the intermembrane space, and the matrix. Mitochondria are the "powerhouses" of cells; their function is to convert energy found in nutrient molecules and store it in high-energy phosphate bonds in a molecule called adenosine triphosphate, which is the universal energy-yielding component necessary for the reactions that modulate many fundamental cellular processes. Mitochondrial ATP is produced through the process of oxidative phosphorylation, a process that uses molecular oxygen as the final electron acceptor.

The products of metabolism are carried from the cytoplasm into the mitochondrial matrix, where they go through the citric acid, or Krebs cycle. The Krebs cycle promotes the reduction of the catabolism-generated coenzymes NAD⁺ (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) to NADH and FADH₂, respectively, which are rich in electron energy. As these molecules are reoxidized, they supply electrons that are carried to final electron acceptor via an elaborate respiratory, or electron, transport chain. The electron transport system is a chain of electron acceptors located in the inner membrane of the mitochondria.

Hydrogens are passed down from NADH to the electron transport chain in a series of redox reactions, where they become dissociated from their electrons and are released as protons. The electrons entering the electron transport system have a relatively high energy content. As they are transferred from one acceptor molecule to the next, they lose much of their energy, some of which is used to pump the protons across the inner mitochondrial membrane. This sets up an electrochemical gradient across the inner mitochondrial membrane, which provides the energy for ATP synthesis. Therefore, the function of this chain is to permit the controlled release of free energy to drive the synthesis of ATP from ADP (adenosine diphosphate, formed from the breakdown of ATP) and inorganic phosphate. This oxidative phosphorylation process engages five respiratory-chain enzyme complexes located within the inner mitochondrial membrane. Four of these complexes—I (NADH dehydrogenase), II (succinate dehydrogenase), III (cytochrome-c reductase), IV (cytochrome-c oxidase)—catalyze the transport of electrons to molecular oxygen. Complex V (ATP synthase) uses the proton motive force to form ATP from ADP and inorganic phosphate. Oxygen is the final electron acceptor in the electron transport system, which is why organisms that respire aerobically require oxygen.

Mitochondria contain their own deoxyribonucleic acid (DNA). Each human cell contains several hundred mitochondria and thousands of copies of the mitochondrial genome (mtDNA). The human mtDNA molecule is a closed circular molecule and is 16,569 base pairs (bp) in length. Out of the thirty-seven mitochondrially encoded genes, thirteen encode polypeptides that are subunits of the respiratory chain enzyme complexes; twenty-two encode transfer RNA and two encode ribosomal RNA. The twenty-four genes that encode RNA are needed for mitochondrial protein synthesis.

Relationship of energy functions to cellular and organismic aging

Although oxygen plays an essential role as the terminal electron acceptor during respiration, oxygen and its metabolites are potentially cytotoxic (toxic to cells). During the course of normal oxidative phosphorylation, between 1 percent to 3 percent of all oxygen reduced by mitochondria escape from the electron transport chain into the mitochondrial inner membrane and are converted into reactive oxygen species (ROS) that have the ability to oxidize macromolecules. These oxidants, produced continuously as by-products of the anaerobic metabolic process, include superoxide (0-₂), hydrogen peroxide (H₂0₂), and hydroxyl radicals (HO-) and are a continuous threat to cellular macromolecules— ROS attacks result in molecular defects found in proteins, lipids, and DNA. However, the damage to the cells is balanced by the existence of cellular enzymatic defenses, which have evolved to battle reactive oxygen species. Unfortunately, these defenses are not perfect, and, cellular macromolecules can become damaged. The accumulation of damaged macromolecules is thought to contribute significantly to aging.

In 1956, Denham Harman first proposed that free radicals play a major role in the aging process by causing cumulative macromolecule damage. Harman subsequently extended his theory and proposed that mitochondria are the major players in aging, since mitochondria are the major targets of free radicals (Harman, 1981). The free-radical theory of aging has gained a lot of support. Many age-correlated genetic data implicate mitochondrial dysfunction in the process of aging. Because of their vulnerability, the mitochondrial DNA molecules are particularly affected. In contrast to nuclear DNA, which is assembled in nucleosomes and protected by histones and other proteins, mtDNA is "naked," facilitating direct ROS attacks. In addition, mtDNA is attached to the inner mitochondrial membrane, and is therefore accessible by the by-products of respiration and a primer target for damage by ROS. Yakes and Van Houten (1997) have shown that the mtDNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress.

Age-related mitochondrial bioenergetic defects have also been reported in the electron transport chain and oxidative phosphorylation. Defective electron transport chains increase the production of mitochondrial free radicals, which in turn cause a further decline in mitochondrial functions, leading ultimately to a decline of the ATP level. Since a sufficient supply of ATP is necessary for life, the accumulation of bioenergetically defective cells is a key factor in the process of aging. Furthermore, during aging the ROS-scavenging enzymes decline, which further increases both free radicals and oxidative stress within the mitochondria.

Three different kinds of studies have been used to show that mitochondrial respiratory functions decline with age. First, histochemical analysis of respiratory enzymes has revealed an age-correlated deficiency in cytochrome-c oxidase (complex IV of the respiratory chain). This was first shown by Josef Müller-Höcker (1989) using cardiomyocytes in the human heart. Cox-deficient cardiomyocytes (heart muscle cells) are regularly present in humans beginning in the sixth decade of life. The second type of study, derives from measurements of enzymatic activities of each respiratory chain complex, as shown first by Yen et al. (1989) and Trounce et al. (1989). The third kind of study involves monitoring the changes of the mitochondrial membrane potential. The development of mitochondrial fluorescent indicators and sophisticated fluorescence microscopy has enabled organellar events to be studied (Smiley et al., 1991). As explained above, the energy released during oxidation reactions in the mitochondrial respiratory chain is stored as an electrochemical gradient consisting of transmembrane electrical potential. Since maintenance of membrane potential is essential for ATP synthesis, the decline seen in mitochondrial membrane potential is a good indicator of mitochondria malfunction.

Potential role of DNA damage and DNA mutations

What happens to the mitochondrial genome as it gets older has now been extensively studied and documented. It is known that mtDNA mutations can compromise the mitochondria function in many ways: they can disrupt both transcription and the translation of encoded proteins; they can produce nonfunctional ribosomal RNA, (RNA), transfer RNA (+RNA) and proteins; and they can impair mtDNA replication. The mitochondrial genome has a great ability to mutate during the life span, producing a heterogeneous array of somatic mutations. The mutation rate for the mitochondrial genome is ten to twenty times larger than for nuclear DNA study by Khrapko et al. (1997) showed a several-hundred-fold higher rate of somatic mutations both in vivo and in vitro in human mtDNA than in nuclear DNA. This increased rate is due to both a high spontaneous mutation rate and the sensitivity of the mitochondrial genome to exogenous environmental mutagens. Five different types of mtDNA mutations have been shown to be age-associated: point mutations, deletions, additions, duplications and rearrangements. One problem that has been raised by some experts concerning studies of the age-related accumulation of specific mutations in human mtDNA is that although the level of a specific mtDNA mutation increases substantially with age, any of these age-associated mutations affects no more than 1 percent of the organelle mtDNA molecules. However, a large number of specific mutations are likely to occur at each of the 16,569 nucleotide positions within the mitochondrial genome during a lifetime, so that even if each mutation is found at a low level, the increasing accumulation of a large number of mutations will eventually reach a critical level, leading to nonfunctional mitochondria. Furthermore, the load of mtDNA mutations is usually underestimated, since most of the mutations are only detectable using the polymerase chain reaction (PCR). This technique, routinely used to estimate the relative proportions of age-associated mutant DNA may give biased results, as it is dependent on the choice of primers and PCR conditions selected by the individual conducting the study.

One of the most reported mtDNA mutations is the so-called common deletion. Initially identified by Cortopassi and Arnheim (1990), the accumulation of mtDNA molecules exhibiting a 4,977 base pairs deletion increases with age. This deletion occurs between two thirteen base pairs sequence repeats, removing almost five kilobase pairs of mtDNA that encodes six essential polypeptides of the respiratory chain as well as five tRNAs. This deletion was subsequently shown by many other investigators to increase with age in many different tissues. Many other age-associated mtDNA mutations have been identified Khrapko et al. (1999) used long PCR techniques in single cell cardiomyocyte from elderly patients to show that multiple mutations coexist in various tissues of aged individuals and that single mutations occur within individual cells. A large age-dependent accumulation of specific mutations in a critical control region for mtDNA replication has been shown in human fibroblasts (Michikawa et al., 1999).

The incidence of mutant mtDNA has been found to correlate with oxidative damage to mtDNA. Adachi et al. (1993) provided the first evidence that ROS is responsible for the occurrence of mtDNA deletions. A large number of DNA base modifications resulting from oxidative stress have been reported—the one that has been the most widely studied is oxidized nucleotide 8-OH-dG (8-hydroxy-deoxyguanosine). This specific product of oxidative damage to DNA has been shown to accumulate with age, and it correlates with an increase of mtDNA 7.4 kilobase pairs deletion (Mecocci et al., 1993).

As mitochondrial respiration and oxidative phosphorylation gradually uncouple from each other, the activity of the mitochondrial respiratory chain gradually declines. The immediate consequence of a decline of respiratory functions is a decline of ATP synthesis, which will further elevate ROS generation. As the production of ROS species in mitochondria increases, the oxidative damage is reflected by an increasing number of mtDNA mutations. Therefore, respiratory enzymes will incorporate the defective mtDNA-encoded subunits and show impaired respiratory function. This vicious circle operates in an age-dependent manner and plays an important role in aging. This scenario can be also amplified by exogenous factors—many types of mtDNA mutations occur more frequently in sun-exposed skin and mtDNA deletions in the human lung are significantly increased by cigarette smoking, suggesting that ROS resulting from environmental factors play a role in promoting mtDNA damage during aging. Although the mitochondrial free-radical theory of aging has gained prominence, it is important to remember that aging is a multifactorial biological process and that many other cellular components are involved.

Ultrastructural changes are also seen in the mitochondria of aged individuals. The mitochondria become larger and less numerous and they exhibit vacuolization, cristae rupture, and accumulations of occlusions.

Christiane Fauron

See also Cellular Aging; DNA Damage and Repair; Theories of Biological Aging: DNA Damage; Theories of Biological Aging: Error Catastrophe.

BIBLIOGRAPHY

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MODERNIZATION THEORY

See Status of older people: tribal societies; Theories, social