Age-related increases in oxidatively damaged proteins of mouse kidney mitochondrial electron transport chain complexes

Mitochondrial dysfunction generates reactive oxygen species (ROS) which damage essential macromolecules. Oxidative modification of proteins, DNA, and lipids has been implicated as a major causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain complexes I and III are the principal sites of ROS production, and oxidative modifications to the complex subunits inhibit their in vitro activity. Therefore, we hypothesize that mitochondrial complex subunits may be primary targets for oxidative damage by ROS which may impair normal complex activity by altering their structure/function leading to mitochondrial dysfunction associated with aging. This study of kidney mitochondria from young, middle-aged, and old mice reveals that there are functional decreases in complexes I, II, IV, and V between aged compared to young kidney mitochondria and these functional declines directly correlate with increased oxidative modification to particular complex subunits. We postulate that the electron leakage from complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function seen in aged mouse kidney. In conclusion, increasing mitochondrial dysfunction may play a key role in the age-associated decline in tissue function.     

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Mitochondrially generated ROS increase with age and are a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS, which may impair normal complex activity. This study of heart mitochondria from young, middle-aged, and old mice reveals that there is an age-related decline in complex I and V activity that correlates with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of the aged mouse heart.     

Age-related alterations in oxidatively damaged proteins of mouse skeletal muscle mitochondrial electron transport chain complexes

Age-associated mitochondrial dysfunction is a major source of reactive oxygen species (ROS) and oxidative modification to proteins. Mitochondrial electron transport chain (ETC) complexes I and III are the sites of ROS production and we hypothesize that proteins of the ETC complexes are primary targets of ROS-mediated modification which impairs their structure and function. The pectoralis, primarily an aerobic red muscle, and quadriceps, primarily an anaerobic white muscle, have different rates of respiration and oxygen-carrying capacity, and hence, different rates of ROS production. This raises the question of whether these muscles exhibit different levels of oxidative protein modification. Our studies reveal that the pectoralis shows a dramatic age-related decline in almost all complex activities that correlates with increased oxidative modification. Similar complex proteins were modified in the quadriceps, at a significantly lower level with less change in enzyme and ETC coupling function. We postulate that mitochondrial ROS causes damage to specific ETC subunits which increases with age and leads to further mitochondrial dysfunction. We conclude that physiological characteristics of the pectoralis vs quadriceps may play a role in age-associated rate of mitochondrial dysfunction and in the decline in tissue function.     

Age-related changes in the activities of respiratory chain complexes and mitochondrial morphology in Drosophila

Using Drosophila melanogaster, we examined changes in the activities of some of the respiratory enzyme complexes with age. The age-related decreases of enzyme activities were observed especially in complex I. We also examined changes in the ultrastructure of mitochondria in the flight muscles of thoraces. The results indicated that the mitochondrial size varied more widely in aged flies than in young ones, in addition to the slight increase in the average size with age. These changes had already appeared before the survival began to decrease, clearly indicating that the accumulation of such changes seriously damages mitochondrial function.     

Oxidatively damaged proteins of heart mitochondrial electron transport complexes

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.     

Effects of aging on activities of mitochondrial electron transport chain complexes and oxidative damage in rat heart

Mitochondrial dysfunction and accumulation of oxidative damage have been implicated to be the major factors of aging. However, data on age-related changes in activities of mitochondrial electron transport chain (ETC) complexes remain controversial and molecular mechanisms responsible for ETC dysfunction are still largely unknown. In this study, we examined the effect of aging on activities of ETC complexes and oxidative damage to proteins and lipids in cardiac mitochondria from adult (6-month-old), old (15-month-old) and senescent (26-month-old) rats. ETC complexes I-IV displayed different extent of inhibition with age. The most significant decline occurred in complex IV activity, whereas complex II activity was unchanged in old rats and was only slightly reduced in senescent rats. Compared to adult, old and senescent rat hearts had significantly higher levels of malondialdehyde, 4-hydroxynonenal (HNE) and dityrosine, while thiol group content was reduced. Despite marked increase in HNE content with age (25 and 76 % for 15- and 26-month-old rats, respectively) Western blot analysis revealed only few HNE-protein adducts. The present study suggests that non-uniform decline in activities of ETC complexes is due, at least in part, to mitochondrial oxidative damage; however, lipid peroxidation products appear to have a limited impact on enzyme functions.     

Age-associated decreased activities of mitochondrial electron transport chain complexes in heart and skeletal muscle: role of L-carnitine

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and aging. L-Carnitine (4-N-trimethylammonium-3-hydroxybutric acid) plays an important role in transport of fatty acid from cytoplasm to mitochondria for energy production. Previous studies in our laboratory reported L-carnitine as a free radical scavenger in aged rats. In the present study we focused the effect of L-carnitine on the activities of electron transport chain in young and aged rats. The activities of electron transport chain complexes were found to be significantly decreased in aged rats when compared to young control rats. Supplementation of carnitine to young and aged rats for 14 and 21 days improved the electron transport chain complexes levels in aged rats when compared with young rats in duration dependent manner. No significant changes were observed in young rats. Our result suggested that L-carnitine improved the activities of electron transport chain enzymes there by improving the energy status in aged rats.     

Membrane-potential-dependent changes in the stoichiometry of charge translocation by the mitochondrial electron transport chain

The charge/oxygen (q+/O) stoichiometry of mitochondria respiring on succinate was measured under conditions of high membrane potential (delta psi). The technique used was a variation of the steady-state method of Al-Shawi and Brand [(1981) Biochem. J. 200, 539-546]. We show that q+/O was about 2.7 at high values of delta psi (170 mV). As delta psi was lowered from 170 mV to 85 mV with the respiratory inhibitor malonate the q+/O stoichiometry increased to 6.0. A number of artefacts which could have led to an underestimation of the q+/O stoichiometry were eliminated. These included effects of any rapid change in mitochondrial volume, internal pH, activity of the endogenous K+/H+ exchanger or in H+ conductance due to changes in delta psi after the addition of inhibitor. The experiments presented here are the first direct demonstration that the stoichiometry of proton pumping by the mitochondrial respiratory chain changes as delta psi is varied.     

Age-related changes in mouse bone permeability

The determination of lacunar-canalicular permeability is essential for understanding local fluid flow in bone, which may indicate how bone senses changes in the mechanical environment to regulate mechano-adaptation. The estimates of lacunar-canalicular permeability found in the literature vary by up to eight orders of magnitude, and age-related permeability changes have not been measured in non-osteonal mouse bone. The objective of this study is to use a poroelastic approach based on nanoindentation data to characterize lacunar-canalicular permeability in murine bone as a function of age. Nine wild type C57BL/6 mice of different ages (2, 7 and 12 months) were used. Three tibiae from each age group were embedded in epoxy resin, cut in half and indented in the longitudinal direction in the mid-cortex using two spherical fluid indenter tips (R=238 μm and 500 μm). Results suggest that the lacunar-canalicular intrinsic permeability of mouse bone decreases from 2 to 7 months, with no significant changes from 7 to 12 months. The large indenter tip imposed larger contact sizes and sampled larger ranges of permeabilities, particularly for the old bone. This age-related difference in the distribution was not seen for indents with the smaller radius tip. We conclude that the small tip effectively measured lacunar-canalicular permeability, while larger tip indents were influenced by vascular permeability. Exploring the age-related changes in permeability of bone measured by nanoindentation will lead to a better understanding of the role of fluid flow in mechano-transduction. This understanding may help indicate alterations in bone adaptation and remodeling.     

Brain region-specific deficit in mitochondrial electron transport chain complexes in children with autism

Mitochondria play important roles in generation of free radicals, ATP formation, and in apoptosis. We studied the levels of mitochondrial electron transport chain (ETC) complexes, that is, complexes I, II, III, IV, and V, in brain tissue samples from the cerebellum and the frontal, parietal, occipital, and temporal cortices of subjects with autism and age-matched control subjects. The subjects were divided into two groups according to their ages: Group A (children, ages 4-10 years) and Group B (adults, ages 14-39 years). In Group A, we observed significantly lower levels of complexes III and V in the cerebellum (p<0.05), of complex I in the frontal cortex (p<0.05), and of complexes II (p<0.01), III (p<0.01), and V (p<0.05) in the temporal cortex of children with autism as compared to age-matched control subjects, while none of the five ETC complexes was affected in the parietal and occipital cortices in subjects with autism. In the cerebellum and temporal cortex, no overlap was observed in the levels of these ETC complexes between subjects with autism and control subjects. In the frontal cortex of Group A, a lower level of ETC complexes was observed in a subset of autism cases, that is, 60% (3/5) for complexes I, II, and V, and 40% (2/5) for complexes III and IV. A striking observation was that the levels of ETC complexes were similar in adult subjects with autism and control subjects (Group B). A significant increase in the levels of lipid hydroperoxides, an oxidative stress marker, was also observed in the cerebellum and temporal cortex in the children with autism. These results suggest that the expression of ETC complexes is decreased in the cerebellum and the frontal and temporal regions of the brain in children with autism, which may lead to abnormal energy metabolism and oxidative stress. The deficits observed in the levels of ETC complexes in children with autism may readjust to normal levels by adulthood.     

Measuring the quantity and activity of mitochondrial electron transport chain complexes in tissues of central nervous system using blue native polyacrylamide gel electrophoresis

Mitochondrial dysfunction and degeneration are associated with many neurodegenerative disorders. A dysfunctional mitochondrial electron transport chain (ETC) impairs ATP production and accelerates the generation of free radicals. To evaluate mitochondrial function, reliable methods are needed. Conventional spectrophotometric assays may not eliminate interference from nonspecific enzyme activities and do not measure quantities of specific ETC complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to resolve mitochondrial ETC complexes. Combined with histochemical staining, it has also been applied to measure ETC enzyme activities in muscles. The current study is to determine (1) whether BN-PAGE can be used to detect ETC complexes from different regions of the central nervous system (CNS) and (2) the quantitative range of BN-PAGE in measuring the amounts and activities of different ETC complexes. By systematically varying the protein amount and the time of histochemical reactions, we have found linear ranges comparable to spectrophotometric assays for measuring enzyme activities of several ETC complexes. In addition, we found linear ranges for measuring protein quantities in several ETC complexes. These results demonstrate that BN-PAGE can be used to measure the amount and activity of the ETC enzymes from the nerve tissues and, thus, can be applied to evaluate the functional changes of mitochondria in neurodegenerative disorders.     

Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Age-related changes in the functioning of the mitochondrial potassium-transporting system

Changes in the rate of respiration and functioning of the ATP-dependent potassium channel in liver and heart mitochondria of one-, three-, eight-, and 24-month-old Wistar male rats have been investigated. It was shown that the activity of the channel in the mitochondria of both tissues in 24-month-old animals decreases more than three times, and the content of potassium, 1.5–2 times compared with young one-month-old rats. The changes occur against the background of age-related decrease of energy supply in mitochondria, the respiratory complex-I undergoing the greatest changes upon aging. The decrease of channel activity may be the result of changes in channel sensitivity to modulators and a decrease in the expression of mitochondrial K+-transporting channel-protein with a molecular mass of 5.5 kDa upon aging found in this work. As a result, the functioning of not only the mitoK_ATP but also the whole mitochondrial potassium cycle is impaired.     

Significant modulation of mitochondrial electron transport system by nicotine in various rat brain regions

The mitochondrion is the organelle responsible for generation of most usable energy in a cell. It also plays an important role in a series of physiological processes such as apoptosis and proliferation. Although previous studies have demonstrated that nicotine modulates the morphology and function of mitochondria, the mechanism(s) underlying these effects is largely unknown. In this study, using a microarray consisting of 4793 clones derived from a mouse dopamine cDNA library, we profiled the gene expression patterns for six brain regions (amygdala, hippocampus, nucleus accumbens, prefrontal cortex, striatum and ventral tegmental area) of female Sprague-Dawley rats subjected to nicotine treatment for 7 days through osmotic minipump infusion. We identified a number of genes and pathways, including components of the electron transport system of mitochondria, such as cytochrome c oxidase subunit I (Mt-co1), Mt-co2, Mt-co3, cytochrome b (Mt-cyb), mitochondrial NADH dehydrogenase 4 (Mt-nd4), and Mt-nd6, that were significantly modulated by nicotine in multiple brain regions. Bioinformatics analysis provided evidence that Gene Ontology categories related to the electron transport system were overrepresented in each brain region. Finally, the results from the microarray analysis were verified by quantitative RT-PCR for four representative genes. Together, our findings imply that mitochondria are involved in neuronal adaptation to chronic nicotine exposure.     

Age-related changes in mutagen activation by rat tissues

Age-related changes in drug metabolism of the liver, lung and kidney of adult female Long-Evans rats were determined by measuring changes in mutagen formation. Activation of aflatoxin B1 (AFB1), 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF) to mutagenic derivatives was assayed using the Ames Salmonella test system. The promutagens were incubated with tissue fractions from rats ranging in age from 2.5 to 25 months. With all three compounds, hepatic, renal and pulmonary activation was lower in the senescent than in the young adult animals. The largest decrease, however, occurred prior to middle-age, i.e. before 9-13 months. In liver and kidney, little change was detectable between the middle-aged and the old (20-25 months) animals. However, pulmonary metabolism in the oldest animals was slightly higher than in the extracts from the middle-aged rats. The observed decline in mutagen activation may thus be a function of maturation rather than senescence.