Rajindar S. Sohal - US grants

DESCRIPTION (Adapted from the applicant's Abstract): The long-term goal of this study is to understand the role of oxidative stress in the aging process. Mitochondria have been widely hypothesized to play a key role in the causation of oxidative stress and aging in animals. This hypothesis has been based on the findings that mitochondria are the main intracellular producers of superoxide anion radical (O2-) and H2O2, which are the progenitors of a variety of other reactive oxygen species (ROS). Furthermore, the rates of mitochondrial O2- and H2O2 generation as well as amounts of mitochondrial oxidative damage increase with age, whereas oxidative phosphorylation capacity declines during aging. It has been shown that oxidative damage to mitochondria can cause both the age-related increase in O2- and H2O2 generation and the loss in respiratory capacity. The hypothesis that attenuation of mitochondrial oxidative damage will retard the progression of age-related deleterious alterations and extend the life span of the organism, will be tested in Drosophila melanogaster using transgenic approaches. Three different experimental strategies will be used to decrease the level of mitochondrial oxidative damage. The first will be to simultaneously overexpress Mn-SOD and ectopic catalase in the mitochondrial matrix; some lines of flies will additionally overexpress CuZn-SOD and catalase in the cytosol. Such overexpression of antioxidant enzymes should decrease the rates of mitochondrial O2- and H2O2 generation and lower the overall level of oxidative stress in cells. The second strategy will be to use regulatable gene promoters to overexpress SOD and catalase in order to control the timing and tissue-specificity of antioxidant gene overexpression. This approach may uncover beneficial effects of reduced oxidant production, which are masked by counterbalancing harmful effects of antioxidant elevation during sensitive stages of development. The third strategy will be to overexpress DNA glycosylase within the mitochondrial matrix to enhance DNA oxidative damage repair capacity. This latter approach is expected to result in an actual reversal of damage, whereas all previous strategies have concentrated on slowing the rate at which it accumulates. The effects of the different gene overexpression on age-related changes in mitochondria, and on the biochemical and physiological patterns of aging of the flies, including life spans, will be determined. The significance of this study is that it will provide a direct test of a basic tenet of the oxidative stress hypothesis of aging, namely that mitochondrial oxidative damage plays a key role in the aging process.

The general goal of the prospective studies is to understand the causes of the aging process in animals. Endogenously-generated oxygen free radicals have been widely postulated to play a causal role in the aging process; however, few studies have been conducted to directly test the validity of this hypothesis. The purpose of the proposed experiments is to directly test the main prediction of this hypothesis: that a decrease in the level of oxidative stress should slow down the aging process. Extra copies of genes that play key roles in the antioxidative system will be introduced into the Drosophila melanogaster genome and the impact of overexpression on age-related biochemical and physiological changes will be assessed. Effects of the overexpression of the Mn superoxide dismutase, glutathione reductase, glutamylcysteine synthetase and glucose-6-phosphate dehydrogenase will be studied; we have previously examined the effects of Cu-Zn superoxide dismutase and catalase. Because the overexpression of single antioxidative genes may create a biochemical imbalance in an inter- related defense mechanism, a major effort will be made to construct transgenic strains with balanced antioxidative defenses. Results of this study should critically test the veracity of the free radical hypothesis of aging by indicating if the major defenses against reactive oxygen species play a causal role in the aging process of Drosophila. Knowledge gained from this study should help in developing strategies for the manipulation of the aging process in mammalian systems.

biomedical equipment resource; biomedical equipment purchase;

The long-term goal of this investigation is to elucidate the mechanisms by which molecular oxidative damage causes losses in cellular functions during the aging process. In the current view, age-related attenuation in physiological functions is due to the accumulation of randomly- inflicted molecular oxidative damage. The proposed study will test an alternate hypothesis that "senescence-associated losses in cellular functions are due to the accrual of oxidative damage to specific proteins." The validity of this hypothesis will be tested in different tissues of the mouse by determining whether an age-associated increase in oxidative damage and a loss in catalytic activity involve only a limited number of proteins and whether the amount of such damage and loss of protein function are related to the life expectancy of the animals. Life expectancy of the mice will be experimentally varied by caloric restriction. Protein oxidation will be detected by the presence of carbonyl groups. Specific aims are: (1) Immunochemically detect carbonylated proteins in mitochondrial, microsomal and cytosolic fractions of various tissues in aged (24-month old) mice. (2) Quantify the age-associated increase in carbonylation of specific proteins in ad libitum-fed (AL) and calorically restricted (CR) mice. Select the proteins showing attenuation of carbonylation by CR (referred to here as 'CR-sensitive carbonylated proteins'.) (3) Purify and identify by microsequencing the 'CR-sensitive carbonylated proteins'. (4) Determine if carbonylated proteins lose catalytic activity during aging. (5) Elucidate mechanisms of carbonylation and loss of catalytic activity by determining the source of carbonylation and its effect on catalytic activity of the proteins. Results of this study should provide significant new information by indicating how oxidative molecular damage to specific proteins is mechanistically linked to senescence-associated losses in cellular functions. Results will also provide a test of the validity of oxidative stress hypothesis of aging.

The long-term goal of the proposed research is to understand the mechanisms by which oxidative stress causes senescence-associated losses in cellular functions. Hypothesis: The specific hypothesis to be tested is that "accrual of oxidative damage to specific proteins is responsible for the senescence-associated losses in cellular functions." The main idea to be scrutinized is that oxidative damage to specific proteins determines both the nature and the rate of progression of deleterious functional alterations occurring during the aging process. Specific aims: Studies will be conducted on mitochondrial and cytosolic proteins in the flight muscles of Drosophila melanogaster, as the flying ability of the flies gradually declines during aging. Specific proteins exhibiting an age-related increase in oxidative damage, indicated by carbonylation, and loss in catalytic activity will be identified. Causal association between oxidative damage to protein targets and the aging process will be tested in transgenic and mutant flies, which, respectively, overexpress and underexpress the genes encoding the proteins susceptible to oxidative damage. In addition, comparison of oxidative damage will be made between transgenic or genetically selected long-lived and control flies. Significance: Results of this study should provide important new knowledge about: (1) Mechanisms linking oxidative stress to the losses in physiological functions during aging and thus provide a further critical test of the validity of oxidative stress hypothesis of aging. (2) Identify targets of protein oxidative damage during aging and the consequent metabolic failures. Novelty: The idea that protein oxidative damage is a selective and not a random phenomenon is new and challenges the current concepts. The experimental approach will employ new methodology for the quantification of carbonylation of specific proteins as well as unique genetic strategies to test cause-and-effect relationships between the oxidation of specific proteins and the rate of the aging process.

The main purpose of this study is to determine whether the long-term administration of co-enzyme Q (CoQ) , or ubiquinone, retards or accelerates the aging process in mice and to elucidate the underlying mechanisms. CoQ is present in the hydrophobic interior of virtually all the cellular membranes and has versatile functions. It is best known as a component of the electron transport system in the inner mitochondrial membrane, where, in concert with alpha-tocopherol, it also acts as an antioxidant, inhibiting oxidative damage. Paradoxically, auto-oxidation of CoQ is also the main source of mitochondrial superoxide anion radical and H2O2 generation CoQ is being widely consumed by humans as a dietary supplement, even though the effects of long-term intake of CoQ are virtually unknown. Whether the long-term intake of CoQ leads to an attenuation or an exacerbation of oxidative stress has not been determine. The present study will elucidate the nature of biochemical and behavioral perturbations following the administration of exogenous CoQ10 to mice. The specific hypothesis to be validated or refuted is that "CoQ administration will decrease the level of oxidative stress, improve and preserve mitochondrial respiratory functions, enhance motor and cognitive performance, and extend life span of mice." Specific Aims include determination of the effects of CoQ administration on: (1) the longevity of mice; (2) age-associated changes in mitochondrial respiratory functions and oxidative damage; (3) perturbations of homeostasis among the main anti-oxidative defenses; (4) age-related accrual of oxidative damage to lipids, proteins and DNA in tissue homogenates; and (5) age-associated decline in cognitive and motor abilities of the mice. Results of this study will provide an understanding of the mechanisms by which CoQ intake may have a beneficial or deleterious effect on the aging process in mice.