Abstract:

Aging is characterized by a progressive deterioration in various physiological functions and metabolic processes.  In both humans and animals, the aging process in several post-mitotic tissues (e.g., heart, skeletal muscle, and brain) has been associated with a decrease in the total number of viable cells.  It has been proposed that this reduction in total cell number may lead to a gradual decline in function in the heart, skeletal muscle, and brain.  In addition to necrosis, where cells die due to accident and lethal injury, apoptosis may be a major factor contributing to the loss of post-mitotic cells during normal aging.  Apoptosis describes a process of programmed cell death, a type of cell death involved in cellular development, which is distinct from necrosis.  Senescent aging probably involves both programmed changes in gene expression and “wear and tear” mechanisms, such as oxygen radicals, mitochondrial DNA damage, and the formation of glycooxidation products.  Programmed changes in gene expression and “wear and tear” mechanisms are likely to be interlinked, with both contributing to the aging process and apoptosis.  Although evidence exists that the incidence of apoptosis increases in post-mitotic cells with age, there are few studies to date that have attempted to identify the molecular mechanisms underlying this cell loss. Recent work suggests that mitochondria may play a key role in regulating apoptosis, with chronic life-long radical production by mitochondria leading to increase in oxidative stress.  Oxidative stress is one of many apoptotic cell death-inducing signals, which can trigger mitochondria to release caspase-activating proteins such as cytochrome c.  In our studies, we examined the role that mitochondria play in influencing apoptosis in the heart, brain and skeletal muscle.  If the mechanisms underlying age-associated cell loss can be identified, it could help explain the loss of function with age and may lead to specific therapeutic interventions (specific diet and exercise) that could attenuate this cell loss and improve the quality of life during old age.  

Radicals in Normal Aging and Disease:

There is both direct and indirect evidence which implicates oxidative damage in cells during aging; however, it is not clear as to the exact source and pathways that inflict the oxidative damage. Furthermore, it is of interst to study the antioxidant defenses, such as glutathione (GSH), vitamin E, vitamin C, uric acid and antioxidant enzymes with oxidative stress and with age.  

Research has shown that the levels of oxidized proteins increase with age, and certain unknown oxidation pathways may be partly responsible for protein oxidation. There are several factors that may influence the accumulation of oxidized proteins. For example, species-specific differences in metabolic rate may exist, which could influence the formation of oxidized macromolecules. Also, less efficient removal of oxidized proteins through proteolytic cleavage may promote the accumulation of oxidized protein with aging. Furthermore, several proteolytic enzymes responsible for degrading oxidized proteins may decline with age in tissues. In addition, defenses either enzymatic or non-enzymatic may decline with age in specific tissues. Very little is known about the complex biochemistry of the accumulation and removal of oxidized amino acids in vivo. However, it is reasonable to predict that the accumulation of oxidized proteins is dependent upon the balance between pro-oxidant, antioxidant, and proteolytic activities.

Abstract on Protein Oxidation and Caloric Restriction:

The decline in cellular function observed with age may be partially due to the accumulation of oxidatively damaged proteins.  Oxidized proteins may accrue within the cell during aging as a result of increased oxidant production, decreased degradation of oxidized proteins, or a combination of both.  The 20S proteasome is responsible for degrading the majority of oxidized proteins within the cell and thereby preventing the accumulation of potentially toxic protein aggregates.  Lifelong caloric restriction (CR), the only experimental intervention that has consistently been shown to slow the rate of aging and increase mean and maximum lifespan, reduces the rate of mitochondrial oxidant production and the accumulation of oxidized proteins and prevents some of the age-associated decline in 20S proteasome activity.  However, few studies have investigated whether short-term CR produces similar beneficial effects.  We determined whether 8 weeks of CR would impact mitochondrial oxidant production, antioxidant enzyme activity, and proteasome activity.  To test this, we isolated heart mitochondria and cytosol from ad libitum fed (AL, n=9) and caloric restricted (CR, n=9) six-month old, male Fischer 344 rats.  Mitochondrial hydrogen peroxide production was significantly reduced in the CR animals.  Short-term CR also caused a significant reduction in mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities but there were no differences in cytosolic SOD and GPX activities.  The reduction in mitochondrial antioxidant enzyme activities may be a result of diminished oxidant production since increased oxidant production serves as a signal to upregulate antioxidant enzyme activity.  Although the chymotrypsin-like and trypsin-like proteasome activities were not different between groups, the peptidylglutamyl-peptide hydrolase activity was significantly elevated in the CR animals.  These results indicate that several of the anti-aging effects of CR are evident after only 8 weeks in young, healthy animals.

Some of the questions our laboratory is interested in are the following:

1) What are the major sources of free radical production (reactive oxygen species or reactive nitrogen species) when we age? Oxidative damage, particularly to proteins, has been widely postulated to be a major causative factor in the loss of functional capacity during senescence. It is hypothesized that as we age the defense mechanisms preventing oxidation decline, and accelerated oxidative damage may, therefore, trigger the deterioration in physiological function. Many lines of evidence implicate, at least in part, oxidative damage to proteins, lipids and nucleic acids as an important component of the aging process. The main source of oxidant formation is believed to be generated by the mitochondria, which could, therefore,play an important role in the aging process. The mitochondria's mainfunction is energy production. However, during oxidative phosphorylation, highly reactive oxygen radicals are generated and subsequently attack cellular components such as respiratory chain proteins and mitochondrial DNA. It has been estimated that the release of reactive intermediate accounts for 2-5% of the oxygen consumed during respiration. Mutations in mitochondrial DNA can lead to the production of less functional respiratory chain proteins, resulting in an increased free radical production and possibly more mitochondrial DNA mutations. This phenomenon is often referred to as the mitochondrial theory of aging, which may lead to random accumulation of mitochondrial DNA mutations. This could ultimately reduce energy output and contribute to the common signs of normal aging. We, therefore, closely investigate the oxidants generated by the mitochondria, energy production, and the antioxidant defenses in the mitochondria.

2) Do different organs protect themselves sufficiently against reactive oxygen and reactive nitrogen species by adapting intracellular antioxidant defenses? Most research shows that antioxidant defenses are not dramatically impaired as we age. However, certain tissues like brain and liver show slight declines in antioxidant defences with age wich may explain some of the increase in oxidative damage in these tissues.

3) What happens to proteins modified by free radical? Are the modified amino acids removed effectively from proteins? Or do they accumulate as we age? Very little research has been conducted in this area. Therefore, our laboratory is very interested in elucidating some of the mechanisms for the removal of oxidized unnatural amino acids. We have data to support that oxidized amino acids may be damaged by free radicals, recognized, and then removed from proteins. In this recent study, we demonstrate that the level of protein oxidation dramatically increases in mitochondria isolated from animals subjected to an acute bout of exercise. This provides the first direct evidence that protein oxidation takes place in the mitochondria of exercised animals, and it strongly supports the hypothesis that exercise is a physiologically relevant source of oxidative stress. This study also shows that the increase in mitochondrial protein oxidation was transient. Remarkably, loss of damaged proteins from the mitochondria was associated with the excretion of oxidized amino acids in the urine of the animals. This provides the first convincing evidence in support of the notion that oxidized proteins are selectively targeted for proteolytic breakdown in vivo, and it raises the exciting possibility that oxidized amino acids in the urine will serve as a non-invasive marker for oxidative stress.

4) Can antioxidant therapy prevent oxidative stress? There is also evidence that antioxidant therapy can protect against the development of several degenerative diseases, such as atherosclerosis, cancer, by reducing oxidative stress. However, there is no evidence that antioxidants can prolong maximum life span in rodents or humans.  In general, the nature and the source of the reactive radical species that may contribute to oxidative stress and protein oxidation is only partially understood, because most of the methods currently used to investigate oxidative reactions are non-specific, and the reactive radical intermediates are short-lived and difficult to detect directly. Better understanding the antioxidant mechanisms in vivo may help to find combinations of antioxidants to prolong life. 

Mitochondria and apoptosis research:

Mitochondiral oxidative stress with aging may be liked to apoptosis.  Apoptosis is a highly regulated form of cell death characterized by specific morphological, biochemical, and molecular events.   However, its role during aging, particularly in post mitotic tissues such as the brain, heart and skeletal muscle has not been studied in depth.  Apoptosis appear to increase in post-mitotic tissues with age and it may be a major contributing factor to the observed loss in tissue function with age.  The mechanisms by which apoptosis are induced with advancing age and adaptations that may protect against apoptosis remain to be identified.  Oxidants and calcium initiate a sequence of events that may play a key role in the activation of the mitochondrial- and the endoplasmic reticulum-mediated pathways of cell death.  Wether increased levels of mitochondrial superoxide radical, hydrogen peroxide, peroxynitrite, and/or calcium cause the activation and adaptive potential of the mitochondrial-mediated pathway and endoplasmic reticulum -mediated pathway is unknown.  Moreover, the adaptations of major regulatory proteins upon the activation of the apoptotic signal transduction pathways during normal aging are unknown.  Caloric restriction - an intervention that reduces oxidant production, improves calcium handling, reduces cell loss and extends maximum life span - could be emplyed to further study the anti-apoptotic adaptations.  In addition, skeletal and heart muscle function with age could relate to apoptosis and apoptosis may be attenuated by caloric restriction.  Furhter research would allow us to understand the mechanisms of apoptosis in vivo with normal aging, and caloric restriction.

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