In this subchapter, we consider three major classes of cellular and molecular processes that may be proximate causes of organismic aging: (1) damage caused by oxidative stress and other factors, (2) inadequate repair of damage, and (3) dysregulation of cell number. No one of these is the underlying mechanism of aging. The basic mechanism of aging is likely to be the long-term imbalance between damage and repair. During growth and development, the genetic program not only creates a complex structure, but also repairs damaged molecules that arise in the process. Following development is a brief adult period when damage and repair are in balance, and then begins a long-term imbalance in favor of damage.
The factors underlying the imbalance vary among species and among individuals within species, as a result of both genetic and environmental variability. For example, oxidative stress is one of many damaging processes that underlie aging, but an individual's genome and environment determine the extent to which it is an important causal factor.
Oxidative stress and related processes that damage macromolecules may have a causal role in aging
Raymond Pearl in 1928 proposed that organisms have a finite amount of a “vital principle,” which they deplete at a rate proportional to the rate of energy expenditure. Although this once-dominant “rate of living” theory of aging has now been discarded, some of its concepts helped to spawn the oxidative stress theory of aging. N62-5
Free-Radical Theory of Aging
Contributed by Edward Masoro
Also contributing to the development of the oxidative stress theory of aging was the free-radical theory of aging. In 1954, Denham Harman published an article setting forth the free-radical theory of aging. Free radicals are highly reactive chemical entities containing unpaired outer orbital electrons. Harman proposed that free radicals are generated in living organisms from both endogenous and exogenous sources. He theorized that these highly reactive entities damage biologically important molecules, resulting in aging.
Reactive Oxygen Species
As illustrated in Figure 62-3A, reactive oxygen species (ROS) include molecules such as hydrogen peroxide (H2O2), neutral free radicals such as the hydroxyl radical (.OH), and anionic radicals such as the superoxide anion radical (). Free radicals have an unpaired electron in the outer orbital, shown in red in Figure 62-3A. These free radicals are extremely unstable because they react with a target molecule to capture an electron, so that they become a stable molecule with only paired electrons in the outer shell. However, the target molecule left behind becomes a free radical, which initiates a chain reaction that continues until two free radicals meet to create a product with a covalent bond. ROS—particularly .OH, which is the most reactive of them all—have the potential to damage important biological molecules, such as proteins, lipids, and DNA. However, ROS also play important physiological roles in the oxidation of iodide anions by thyroid peroxidase in the formation of thyroid hormone (see pp. 1006–1010), as well as in the destruction of certain bacteria by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase in phagocytic cells. N62-6 Finally, the highly reactive signaling molecule nitric oxide (see p. 66) is a free radical (see Fig. 62-3A). N62-7
FIGURE 62-3 Reactive oxygen species.
Physiological Roles of ROS
For more information about ROS and their physiological roles, see the following Web page, particularly the discussion under the heading “ROS Are Essential”: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/ROS.html (accessed February 2015).
For more information on the chemistry and physiology of nitric oxide (NO), visit the following websites:
1. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/NO.html (accessed February 2015)
2. http://herkules.oulu.fi/isbn9514268512/html/i231674.html (accessed February 2015)
Quantitatively, the most important source of ROS is the mitochondrial electron transport chain (see p. 118). Complex I and complex III of the electron transport chain generate as byproducts (see Fig. 62-3B). The enzyme superoxide dismutase (SOD) converts to hydrogen peroxide, which in turn can yield the highly reactive .OH.
Only a small fraction of the oxygen used in aerobic metabolism (<1%) generates ROS. However, even that amount would be lethal in the absence of protective mechanisms. Fortunately, organisms have two potent antioxidant defenses. The major defense is enzymatic, specifically SODs, catalase, and glutathione peroxidase (Fig. 62-4). In addition, low-molecular-weight antioxidants, such as vitamins C and E, play a minor role in the defense against the metabolically produced radicals.
FIGURE 62-4 Enzymatic defenses against ROS. SODs eliminate the superoxide radical but generate hydrogen peroxide, which, as shown in Figure 62-3B, can yield the highly reactive hydroxyl radical via the Fenton reaction. The hydrogen peroxide is eliminated by catalase or glutathione peroxidase, which yield relatively nonreactive products: water, molecular oxygen, and oxidized glutathione.
Because these antioxidant defense mechanisms are not fully protective, the dominant concept of the oxidative stress theory is that an imbalance between the production and removal of ROS by antioxidant defenses is the major cause of aging. Nevertheless, recent studies using genetically engineered mice—with either deficient or overexpressed antioxidant enzymes—do not support this theory.
Glycation and Glycoxidation
Glycation refers to nonenzymatic reactions between the carbonyl groups of reducing sugars (e.g., glucose) and the amino groups of macromolecules (e.g., proteins, DNA) to form advanced glycation end products (AGEs). Figure 62-5 shows an interaction of open-chain D-glucose with a lysine residue on a protein, yielding a Schiff base and water. The Schiff base undergoes an intramolecular rearrangement to form an open-chain Amadori compound that undergoes the Amadori rearrangement to form a ring structure called an Amadori product. In cooking, Amadori products undergo a series of further reactions to produce polymers and copolymers called melanoidins, which give a brown color to cooked food. N62-8 In humans, the Amadori product can undergo a series of intramolecular and intermolecular rearrangements that include oxidation—glycoxidation—to form AGE molecules. For example, the Amadori product in Figure 62-5 can either form carboxymethyllysine or react with an arginine residue on the same or a different protein to form a cross-link called pentosidine.
FIGURE 62-5 Examples of glycation, glycoxidation, and the formation of AGEs. R1 and R2 refer to two different proteins or two different domains of the same protein.
Contributed by Emile Boulpaep, Walter Boron
In the Maillard reaction, the amino group of an amino acid reacts with the carbonyl group of a sugar. As shown in the left side of Figure 62-5, the products are water plus an N-substituted glycosylamine (i.e., an open-chained Amadori compound). This reaction, discovered by the physiologist Louis Camille Maillard (see http://en.wikipedia.org/wiki/Louis_Camille_Maillard) in the 1910s is responsible for the brown color of toast and the color of seared meat. For more information about the reaction, visit the following websites:
The formation of AGEs is especially important for long-lived proteins and appears to play a role in the long-term complications of diabetes. The similarity between the aging phenotype and that of the diabetic patient led Anthony Cerami to propose the glycation hypothesis of aging. Although glucose is not the only reducing sugar involved in glycation, it is an important one. Thus, the level of glycemia is a major factor in glycation, and periods of hyperglycemia are probably the reason glycation—including the glycation of hemoglobin (see Box 29-1)—is enhanced in patients with diabetes. Proteins containing AGEs exhibit altered structural and functional properties. For example, AGE formation in lens proteins of the eye probably contributes to age-associated opacification. Moreover, with advancing age, the increased stiffness of collagen in connective tissues (e.g., blood vessels; see pp. 458–459) may also, in part, be due to AGE-mediated collagen cross-links. AGE-induced DNA damage may lead to alterations in genomic function.
Because mitochondria are the major source of ROS, they are also likely to be a major target of oxidative damage. Damage to mitochondrial DNA (mtDNA) increases greatly with age because, unlike genomic DNA, mtDNA is not protected by histones (see pp. 75–76). According to the mitochondrial theory of aging, the damage to mtDNA reduces the ability of the mitochondria to generate ATP, and this decreased production of ATP results in the loss of cell function and hence aging.
Damage to genomic and mitochondrial DNA can occur as the result of radiation and other environmental agents, such as toxic chemicals. In recent years, oxidative stress has been recognized as a major source of DNA damage. Cells can repair much of the damage to DNA, and the level of damage is in a steady state between damaging and repair processes. According to the DNA damage theory of aging, accumulated DNA damage interferes with DNA replication and transcription, thereby impairing the ability of cells to function and causing aging. Moreover, this loss of function increases as the steady-state level of DNA damage increases with advancing age. However, it is not clear that DNA damage and mutations in somatic cells are sufficient to cause the organismic functional deterioration that characterizes the aging phenotype.
Inadequacy of repair processes may contribute to the aging phenotype
Many biogerontologists believe that even more important than damage per se is the progressive age-associated loss in the ability to repair such damage. N62-9
Age-Associated Inadequacy of Repair Processes
Contributed by Edward Masoro
Those who believe in the concept of primary and secondary aging would feel that, because repair processes are an intrinsic component of an organism, their inadequate functioning is a component of primary aging. Some distinguished biogerontologists do not regard the concept of primary and secondary aging as useful. On the other hand, other distinguished biogerontologists, including some in the medical area, still subscribe to the concept.
As noted above, the steady-state level of damaged DNA depends on the balance between damage and repair processes. The DNA repair theory of aging proposes that DNA repair declines with advancing age, which causes a rise in the steady-state level of damaged DNA and thereby compromises the integrity of the genome. Because DNA repair is a complex process, it is difficult to measure in vivo. Moreover, not only the rate but also the accuracy of the repair processes could contribute to aging. Particularly in stem cells, unrepaired or inappropriately repaired DNA may play a major role in aging.
In addition to oxidative stress and nonenzymatic glycation, a host of other processes—including deamidation, racemization, and isomerization—may lead to deterioration of proteins, resulting in changes in the secondary and tertiary structures as well as aggregation and fragmentation. Protecting the organism from an excessive accumulation of altered proteins are proteolytic degradation and replacement—protein turnover.
As noted beginning on page 33, cells have mechanisms for monitoring and maintaining the quality of their proteome. A major role of these mechanisms is the maintenance of the appropriate conformation of the proteins in the face of factors tending to unfold and misfold them, thereby abolishing function and often converting the protein into a toxic agent. An age-associated decrease in chaperone proteins—involved in protein folding and refolding—may be a factor in the accumulation of protein oligomers and aggregates. Small oligomers, not mature amyloid fibers, are believed to be the most toxic species in some age-associated diseases, such as Alzheimer disease. The ubiquitin/proteasome system (see pp. 33–34) degrades many of the proteins not refolded by the chaperones. The catalytic activity of the proteasome may decrease with age.
The rate of total-body protein turnover in humans decreases with age. Thus, the average lifetime of most but not all protein species increases with age. Long-lived proteins in the extracellular matrix, particularly collagen and elastin, undergo age-associated changes such as oxidation, glycation, and cross-linking. These changes, for cells embedded in the matrix, probably alter proliferation, migration, and the response to extracellular signals.
Damaged proteins that are not either refolded or degraded by the ubiquitin/proteasome system may be broken down by lysosomal proteases. In addition to proteases, lysosomes contain enzymes that can break down lipids, carbohydrates, and nucleic acids. Degradation of cellular components by the lysosomes is referred to as autophagy, of which there are three types.
Macroautophagy involves the sequestering of proteins and other cytosolic components inside double-membrane vesicles called autophagosomes, the outer membrane of which fuses with a lysosome. The lysosomal enzymes degrade the cytosolic cargo.
In microautophagy, the lysosome membrane engulfs single damaged cytosolic molecules, exposing them to the lysosomal enzymes.
In chaperone-mediated autophagy, a complex of a damaged cytosolic protein and a chaperone traffics and then binds to a receptor on the lysosomal membrane. Lysosomal machinery then unfolds the protein and translocates it into the lysosome.
With increasing age, lysosomes in most tissues undergo marked changes, including an expansion of the lysosomal compartment, changes in lysosomal enzyme activities, and the accumulation of undegraded lysosomal products in the form of lipofuscin. A loss of lysosomal function adversely affects all three types of autophagy. In addition, other age-associated defects may be specific to macroautophagy and to chaperone-mediated autophagy.
Dysfunction of the homeostasis of cell number may be a major factor in aging
For most cell types, the total number of cells remains nearly constant over much of adult life. An imbalance in favor of cell division results in hyperplasia (see p. 990), such as occurs in the prostates of elderly men, or in neoplasia (i.e., formation of new, abnormal cells), a disease process that also increases in frequency with age. An imbalance in favor of cell removal results in a reduction of cell number, such as occurs with age in some skeletal muscles. Of course, in cell types that are truly postmitotic in adult life, any age-associated loss of cells results in a decrea se in number.
Limitations in Cell Division
In 1961, Leonard Hayflick and Paul Moorhead reported that human fibroblasts in culture could divide only a limited number of times, a phenomenon known as the Hayflick limit that also applies to many other somatic cell types in culture. Although Hayflick hypothesized that this limited cell proliferation in culture is a “test tube” model of aging, more recent findings indicate that the in vitro cell-culture system falls short as a valid model of organismic aging. Nevertheless, intensive study of the Hayflick limit led to consideration of the role of telomeres in aging.
Telomeres are elements at the ends of linear chromosomes and are composed of repeated specific DNA sequences and associated proteins. In the late 1980s, Calvin Harley found that the telomeres of human cells in culture shorten with each mitotic division. When the telomeres shorten to a critical length, the cell can no longer divide—a probable basis of the Hayflick limit. Such cells also exhibit other functional changes.
Are the telomere findings in culture systems relevant to organismic senescence? A reduction in telomere length could play a role in cell types that exhibit an age-associated decrease in cell number. Clearly, a reduction in telomere length cannot be a factor in the aging of cells that are truly postmitotic during adult life. Telomeres do not shorten in the germline, which, unlike somatic cells, contains significant levels of telomerase, an enzyme that catalyzes the extension of telomere length. Cancer cells are also rich in telomerase. For their work on telomeres and telomerase, Elizabeth Blackburn, Carol Greider, and Jack Szostak shared the 2009 Nobel Prize in Physiology or Medicine. N62-10
Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak
For more information about Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak and the work that led to their Nobel Prize, visit http://www.nobelprize.org/nobel_prizes/medicine/laureates/2009/ (accessed July 2015).
Although not associated with a major dysregulation of cell number, aging may impair the burst in proliferation necessary to meet certain challenges. For example, with increasing age, the immune system is less effective in protecting the organism from infection. An important factor may be that an antigenic challenge may be less effective in triggering a proliferation of T lymphocytes, perhaps because of an age-associated decrease in the length of T-lymphocyte telomeres.
Necrosis, apoptosis, and necroptosis are major processes by which the body loses cells. Necrosis, a cellular response to severe trauma, is manifested by uncontrolled breakdown of cellular structure, cell lysis, and an inflammatory response. The morphological characteristics of necrosis are cell swelling and loss of membrane integrity.
Apoptosis is a form of programmed cell death (see p. 22). N62-11 It plays a key role in organogenesis and tissue renewal, and it also occurs in response to relatively mild damage. Apoptosis requires ATP, is gene driven, and is characterized by preservation of organelles, maintenance of membrane integrity, absence of inflammation, cell shrinkage, and fragmentation of the cell into multiple membrane-enclosed apoptotic bodies. Macrophages or neighboring cells remove the apoptotic bodies by phagocytosis.
For a summary of apoptosis, consult http://herkules.oulu.fi/isbn9514266676/html/i267388.html (accessed February 2015).
Three interacting pathways N62-12 lead to apoptosis (Fig. 62-6). First, in the extrinsic pathway, extracellular signals bind to cell-surface receptors—death receptors (DRs)—of the tumor necrosis factor receptor (TNFR) family, which are examples of receptors that act at least in part via regulated proteolysis (see p. 48). The DRs include Fas, TNFR1, DR3, DR4, and DR5, all of which have a cytosolic death domain. In the case of the receptor Fas, the homotrimeric ligand called FasL binds to three Fas molecules, which results in the formation of a trimer of ligand-receptor complexes. This clustering allows the death domains on Fas to bind to death domains of an intracellular adapter protein called FADD (Fas-associated death domain). Death-effector domains of FADD recruit several copies of procaspase-8, the aggregation of which leads to the autoproteolytic cleavage of procaspase-8 and thereby releases active caspase-8. This initiator caspase is a member of the caspase family of proteolytic enzymes. N62-13 Relatively small numbers of caspase-8 molecules, by proteolytic cleavage, activate much larger numbers of executioner caspases, including caspases 3 and 7, as well as another initiator caspase called caspase-9. This amplified cascade results in the proteolysis of numerous cytosolic and nuclear proteins, leading to apoptosis. One caspase target is a gelsolin, which, when cleaved, severs actin filaments and leads to a loss of normal cell shape. Another caspase target is inhibitor of caspase-activated deoxyribonuclease (ICAD), which normally binds to and thereby inactivates caspase-activated deoxyribonuclease (CAD). Cleavage of ICAD releases active CAD, which translocates to the nucleus and severs chromosomal DNA. N62-14
FIGURE 62-6 Apoptotic signaling pathways. Bax, Bcl-2–associated X protein (proapoptotic protein); Bcl-2, B-cell lymphoma 2 (antiapoptotic protein); Cyt c, cytochrome c.
Contributed by Emile Boulpaep, Walter Boron
For an example of interacting pathways in a connected diagram, see Figure 25-1C.
Contributed by Edward Masoro
Caspases are proteases with a cysteine (Cys) residue at their active site. They cleave a protein substrate at an aspartate (Asp) residue. The Cys and Asp give rise to the name caspase. They exist as inactive precursors called procaspases or zymogen caspases. Only after cleavage of the procaspase does the enzyme become active.
ICAD and CAD
Contributed by Emile Boulpaep, Walter Boron
CAD (caspase-activated DNase) is also known as DNA fragmentation factor 40 (DFF40) because it has a mass of 40 kDa. It is encoded by the DFFB gene.
ICAD (inhibitor of caspase-activated DNase) is also known as DNA fragmentation factor 45 (DFF45) because it has a mass of 45 kDa. It is encoded by the DFFA gene.
Wikipedia. s.v. DFFA. [Last modified May 5] http://en.wikipedia.org/wiki/Inhibitor_of_caspase-activated_DNase; 2015 [Accessed July 15, 2015].
Wikipedia. s.v. DFFB. [Last modified May 5] http://en.wikipedia.org/wiki/DFFB; 2015 [Accessed July 15, 2015].
The second pathway involves damage to the mitochondria (see Fig. 62-6), triggered by such agents as ROS, increases in [Ca2+] in the mitochondrial matrix, and caspases. The result is the opening of a large pore in the mitochondrial inner membrane—the mitochondrial permeability transition pore (MPTP)—followed by mitochondrial swelling, rupture of the outer mitochondrial membrane, and release of cytochrome c into the cytosol. There, apoptotic protease-activating factor (Apaf-1) complexes with the cytochrome c and ATP, forming a wheel-like structure that contains seven of each molecule—an apoptosome. This structure recruits seven procaspase-9 molecules, which results in the formation of active caspase-9 and thus commits the cell to apoptosis.
The third pathway is triggered by damage to nuclear DNA (see Fig. 62-6). The tumor-suppressor p53, a nuclear protein, recognizes certain base-pair mismatches. In cases of modest DNA damage, p53 increases the transcription of p21, which in turn halts the cell cycle. In cases of more severe DNA damage, p53 upregulates its own transcription. In addition, p53 increases the transcription of the proapoptotic protein Bax and decreases the transcription of the antiapoptotic protein Bcl-2. The increase in the Bax/Bcl-2 ratio appears to activate MPTP, thereby precipitating the events leading to apoptosis described in the second pathway. Finally, p53 also upregulates Fas, which reinforces the first pathway.
Dysregulation of apoptosis promotes aging. Failure of apoptosis to remove damaged cells could result in abnormal function or increase the risk of cancer. Excess apoptosis would unnecessarily decrease cell number.
Necroptosis is a second important mode of regulated cell death. Like apoptosis, necroptosis involves a highly orchestrated sequence of intracellular signaling reactions. Notably, necroptosis is activated in response to many of the same microbial or environmental stresses that trigger apoptosis. In cells in which the stress or infectious stimuli suppress initiator caspases (e.g., caspase-8; see pp. 1241–1242) or executioner caspases (e.g., caspase-3; see pp. 1241–1242), the cells activate the necroptotic cascade as an alternative to apoptosis. Indeed, some microbial pathogens express potent inhibitors of host cell caspases, which allows the pathogens to establish protected replicative niches within infected host cells. Engagement of the necroptosis pathway facilitates death of the infected host cells with consequent release of the internalized pathogens into extracellular compartments, where multiple innate immune mechanisms more readily destroy the pathogens. These mechanisms include complement-mediated cytolysis and neutrophil-mediated production of reactive oxygen and nitrogen species (see pp. 1238–1239). Recently identified pronecroptotic gene products include the RIP1 and RIP3 protein kinases and the mixed-lineage kinase-like (MLKL) protein, which appears to function as an endogenous porin (see p. 109) when recruited to the plasma membrane. Ischemia-reperfusion injury of organs such as the brain, kidney, and liver may trigger necroptosis and thus cell death.