Gene-expression profile of the ageing brain in mice.

Ageing of the brain leads to impairments in cognitive and motor skills, and is the major risk factor for several common neurological disorders such as Alzheimer disease (AD) and Parkinson disease (PD). Recent studies suggest that normal brain ageing is associated with subtle morphological and functional alterations in specific neuronal circuits, as opposed to large-scale neuronal loss. In fact, ageing of the central nervous system in diverse mammalian species shares many features, such as atrophy of pyramidal neurons, synaptic atrophy, decrease of striatal dopamine receptors, accumulation of fluorescent pigments, cytoskeletal abnormalities, and reactive astrocytes and microglia. To provide the first global analysis of brain ageing at the molecular level, we used oligonucleotide arrays representing 6,347 genes to determine the gene-expression profile of the ageing neocortex and cerebellum in mice. Ageing resulted in a gene-expression profile indicative of an inflammatory response, oxidative stress and reduced neurotrophic support in both brain regions. At the transcriptional level, brain ageing in mice displays parallels with human neurodegenerative disorders. Caloric restriction, which retards the ageing process in mammals, selectively attenuated the age-associated induction of genes encoding inflammatory and stress responses.

Age-related changes in neural volume and microstructure associated with interleukin-6 are ameliorated by a calorie-restricted diet in old rhesus monkeys.

Systemic levels of proinflammatory cytokines such as interleukin-6 (IL-6) increase in old age and may contribute to neural atrophy in humans. We investigated IL-6 associations with age in T1-weighted segments and microstructural diffusion indices using MRI in aged rhesus monkeys (Macaca mulatta). Further, we determined if long-term 30% calorie restriction (CR) reduced IL-6 and attenuated its association with lower tissue volume and density. Voxel-based morphometry (VBM) and diffusion-weighted voxelwise analyses were conducted. IL-6 was associated with less global gray and white matter (GM and WM), as well as smaller parietal and temporal GM volumes. Lower fractional anisotropy (FA) was associated with higher IL-6 levels along the corpus callosum and various cortical and subcortical tracts. Higher IL-6 concentrations across subjects were also associated with increased mean diffusivity (MD) throughout many brain regions, particularly in corpus callosum, cingulum, and parietal, frontal, and prefrontal areas. CR monkeys had significantly lower IL-6 and less associated atrophy. An IL-6xCR interaction across modalities also indicated that CR mitigated IL-6 related changes in several brain regions compared to controls. Peripheral IL-6 levels were correlated with atrophy in regions sensitive to aging, and this relationship was decreased by CR.

Oxidative stress, caloric restriction, and aging.

Under normal physiological conditions, the use of oxygen by cells of aerobic organisms generates potentially deleterious reactive oxygen metabolites. A chronic state of oxidative stress exists in cells because of an imbalance between prooxidants and antioxidants. The amount of oxidative damage increases as an organism ages and is postulated to be a major causal factor of senescence. Support for this hypothesis includes the following observations: (i) Overexpression of antioxidative enzymes retards the age-related accrual of oxidative damage and extends the maximum life-span of transgenic Drosophila melanogaster. (ii) Variations in longevity among different species inversely correlate with the rates of mitochondrial generation of the superoxide anion radical (O2) and hydrogen peroxide. (iii) Restriction of caloric intake lowers steady-state levels of oxidative stress and damage, retards age-associated changes, and extends the maximum life-span in mammals.

Dietary restriction in mice beginning at 1 year of age: effect on life-span and spontaneous cancer incidence.

Lifelong dietary restriction beginning at 3 to 6 weeks of age in rodents is known to decelerate the rate of aging, increase mean and maximum life-spans, and inhibit the occurrence of many spontaneous cancers. Little is known about the effects of dietary restriction started in middle age. In the experiments now reported the food intake of 12- to 13-month-old mice of two long-lived strains was restricted by using nutrient-enriched diets in accordance with the concept of "undernutrition without malnutrition." The mice on the restricted diet averaged 10 to 20 percent increases in mean and maximum survival times compared to the control mice. Spontaneous lymphoma was inhibited by the food restriction.

Dietary restriction retards age-related loss of gamma crystallins in the mouse lens.

The soluble crystallins in lenses from diet-restricted and control mice of diverse ages (2, 11, or 30 months) were studied by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results obtained with both methods suggest that dietary restriction decelerates age-related loss of soluble gamma crystallins.

Gene expression profile of aging and its retardation by caloric restriction.

The gene expression profile of the aging process was analyzed in skeletal muscle of mice. Use of high-density oligonucleotide arrays representing 6347 genes revealed that aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were either completely or partially prevented by caloric restriction, the only intervention known to retard aging in mammals. Transcriptional patterns of calorie-restricted animals suggest that caloric restriction retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage.

Effect of antioxidants on androgen-induced AP-1 and NF-kappaB DNA-binding activity in prostate carcinoma cells.

BACKGROUND: Previous studies have suggested that male hormones (androgens) and certain forms of oxygen (reactive oxygen species) are linked to the development of prostate cancer. We hypothesized that androgens contribute to prostate carcinogenesis by increasing oxidative stress. We further hypothesized that antioxidants reduce prostate cancer risk by modulating androgen effects on cellular processes. METHODS: To test these hypotheses, we looked for 1) a change in the level of reactive oxygen species in the presence of androgens, 2) androgen-induced binding activity of transcriptional activators AP-1 and NF-kappaB, whose activities are known to be altered during cell proliferation, and 3) the effect of antioxidants on androgen-induced transcription factor binding. RESULTS: Physiologic concentrations (1 nM) of 5alpha-dihydrotestosterone or 1-10 nM R1881, a synthetic androgen, produced sustained elevation of AP-1 and NF-kappaB DNA-binding activity in LNCaP cells, an androgen-responsive human prostate carcinoma cell line. Androgen-independent DU145 cells (another human prostate carcinoma cell line) were unaffected by R1881 treatment. AP-1-binding activity increased 5 hours after 1 nM R1881 treatment; NF-kappaB DNA-binding activity increased after 36 hours. Both activities remained elevated for at least 120 hours. Nuclear AP-1 and NF-kappaB protein levels were not elevated. Antioxidant vitamins C plus E blocked both androgen-induced DNA-binding activity and production of reactive oxygen species. CONCLUSION: Physiologic concentrations of androgens induce production of reactive oxygen species and cause prolonged AP-1 and NF-kappaB DNA-binding activities, which are diminished by vitamins C and E.

Prostatic localization of spontaneous early invasive carcinoma in Lobund-Wistar rats.

Animal models of human prostate cancer are very limited in number but are of obvious importance to develop. Dr. Morris Pollard (M. Pollard, J. Natl. Cancer Inst., 51: 1235-1241, 1973) has reported that Lobund-Wistar rats develop spontaneous metastatic prostatic cancer when they become old (approximately 25% incidence after 25 months). A chemically induced form of the disease has also been described in Lobund-Wistar rats. However, recent reports suggest that most of the chemically induced adenocarcinomas are not prostatic in origin, with most arising in the seminal vesicle, and thereby raise questions about the origin of the spontaneous cancers. We herein report cancer spontaneously arising in the lateral lobes of the prostates in Lobund-Wistar rats. One of 8 rats killed at 16 months of age showed prostatic carcinoma in situ. Two of 39 rats killed at 20 months displayed early invasive adenocarcinomas with no signs of metastases. Because sectioning of the prostates in this study was limited to face sections from a single block for each rat, it is highly probable that the true incidence of dysplasias and carcinomas is underestimated by these data. Dysplastic or neoplastic changes were not seen in either the seminal vesicles or other portions of the prostatic complex. The nuclei of adenocarcinoma cells showed less labeling with antibody to the androgen hormone receptor than did normal cells. These data strongly support the validity of the Pollard model of spontaneous prostate cancer in Lobund-Wistar rats.

Dietary intervention at middle age: caloric restriction but not dehydroepiandrosterone sulfate increases lifespan and lifetime cancer incidence in mice.

Dietary manipulations to prevent cancer and other diseases of aging have drawn broad public and scientific attention. One indicator of this interest is that dehydroepiandrosterone (DHEA) supplements are widely consumed by those who hope that this hormone may keep them "younger longer." However, key data to support this belief are lacking. For example, the influence of DHEA treatment on spontaneous cancer and life span in healthy, long-lived strains of mice or rats is unknown. This is in contrast to the situation for caloric restriction (CR), which is known to oppose cancer development and increase maximum life span in rodents. To address this issue, we assigned 300 middle age (12-month-old) male C57BL/6 mice to one of four groups (n = 75 for each group) and evaluated them for longevity and spontaneous disease patterns. Two groups were fed a normal diet (ND), and two others were fed a calorie-restricted diet (RD). One ND group and one RD group were also given 25 microg/ml DHEA sulfate (DHEAS) in their drinking water. Although urine samples from DHEAS-treated mice contained 10-fold more DHEA and DHEAS than did samples from unsupplemented mice, DHEAS administration did not affect body weight, life span, or cancer patterns. The RD lowered body weight by 26% and increased maximum life span by approximately 15%. The incidence of the most prevalent cancer, plasma cell neoplasm, was higher in RD mice (66%) than in ND mice (41%). Thus, DHEAS, as administered here, influenced neither cancer nor longevity at two caloric intakes. In contrast, CR from middle age increased longevity, the age at which tumor-bearing mice died, and the percentage of mice dying with cancers, suggesting that CR may retard promotion and/or progression of existing lymphoid cancers.

Dietary restriction from middle age attenuates age-associated lymphoma development and interleukin 6 dysregulation in C57BL/6 mice.

Dietary restriction (DR) started in middle age profoundly reduces the occurrence of lymphoma in C57BL/6 mice. Here, we report immunocellular and molecular changes associated with this mode of cancer prevention. Twelve-month-old male C57BL/6 mice were either fed a control diet or subjected to moderate DR (approximately 25% < control intake). DR significantly reduced lymphoma development (incidence at 25 months, 19% of 72 control mice versus 5% of 60 DR mice). Flow cytometry of splenocytes showed that DR increased the percentage of CD4+ and CD8+ cells. Lymphomatous spleens displayed varied labeling patterns and high percentages of cells in S phase. Splenocyte c-myc expression tended to increase with age in controls and was reduced by DR. Lymphopenia and markedly reduced nucleated cell yields from peripheral lymphoid tissues were induced by DR. Serum interleukin 6 levels increased with age and were quite high (> 2500 pg/ml) in several mice with lymphoma and other histopathological findings. DR attenuated this age-associated increase. Immunohistochemical studies of lymphomatous spleens showed the presence of interleukin 6 in monocytic appearing cells but not in lymphoma cells. These observations support the possibility that an age-associated interleukin 6 dysregulation is important in lymphomagenesis.

Lifelong voluntary exercise in the mouse prevents age-related alterations in gene expression in the heart.

We present the first quantitative gene expression analysis of cardiac aging under conditions of sedentary and active lifestyles using high-density oligonucleotide arrays representing 11,904 cDNAs and expressed sequence tags (ESTs). With these data, we test the hypothesis that exercise attenuates the gene expression changes that normally occur in the aging heart. Male mice (Mus domesticus) were sampled from the 16th generation of selective breeding for high voluntary exercise. For the selective breeding protocol, breeders were chosen based on the maximum number of wheel revolutions run on days 5 and 6 of a test at 8 wk of age. For the colony sampled herein, mice were housed individually over their entire lifetimes (from weaning) either with or without access to running wheels. The hearts of these two treatment groups (active and sedentary) were assayed at middle age (20 mo) and old age (33 mo). Genes significantly affected by age in the hearts of the sedentary population by at least a 50% expression change (n = 137) were distributed across several major categories, including inflammatory response, stress response, signal transduction, and energy metabolism. Genes significantly affected by age in the active population were fewer (n = 62). Of the 42 changes in gene expression that were common to both treatment groups, 32 (72%) displayed smaller fold changes as a result of exercise. Thus exercise offset many age-related gene expression changes observed in the hearts of the sedentary animals. These results suggest that adaptive physiological mechanisms that are induced by exercise can retard many effects of aging on heart muscle at the transcriptional level.

Controlling caloric consumption: protocols for rodents and rhesus monkeys.

One approach for investigating biological aging is to compare control-fed animals with others restricted in calorie intake by 20% or more. Caloric restriction (CR) is the only intervention shown to extend the maximum lifespan of several invertebrates and vertebrates including spiders, fish, rats and mice. The capacity of CR to retard aging in nonhuman primates is now being explored. The rodent studies show that CR opposes the development of many age-associated pathophysiological changes, including changes to the brain and changes in learning and behavior. One goal of studying CR in rodent is to determine the mechanisms by which it retards aging to design interventions that duplicate those effects. The methods that we use for conducting CR studies on mice and rhesus monkeys are described. We employ procedures designed to achieve a high degree of caloric control for all animals in the study. As used in our studies, this control includes the following features: 1) animals are individually housed, and 2) all individuals in the control group eat the same number of calories (i.e., they are not fed ad lib). Although this method results in strict caloric control for all animals, there seems to be considerable procedural flexibility for the successful conduct of CR studies.

Failure of dietary restriction to retard age-related neurochemical changes in mice.

Age-sensitive neurochemical measures and estrous cyclicity were studied in female mice from the long-lived C3B10F1 strain fed either a control diet or subjected to dietary restriction (DR) from 3 weeks of age. Striatal dopaminergic D2 receptor density decreased by 25% from 9-10 months to 28-30 months of age in the control group. This decline was uninfluenced by DR. Anterior pituitary dopamine + dihydroxyphenylacetic acid content increased by 2.5 fold with age in the control group but DR failed to oppose this age-related change. In contrast to DR's lack of influence on these two neurochemical measures were findings on estrous cyclicity. Although mice on DR did not display estrous cycles, cyclicity was rapidly initiated when these mice were switched to the control diet at 12 and even at 22 months of age. Thus, limited aspects of neuroendocrine aging were retarded by DR in this long-lived mouse model.

Age-associated alterations of the mitochondrial genome.

Age-associated alterations of the mitochondrial genome occur in several different species; however, their physiological relevance remains unclear. The age-associated changes of mitochondrial DNA (mtDNA) include nucleotide point mutations and modifications, as well as deletions. In this review, we summarize the current literature on age-associated mtDNA mutations and deletions and comment on their abundance. A clear need exists for a more thorough evaluation of the total damage to the mitochondrial genome that accumulates in aged tissues.

Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila.

Drosophila melanogaster displays an age-associated increase in oxidative damage and a decrease in mitochondrial transcripts. To determine if these changes result in energy production deficiencies, we measured the electron transport system (ETS) enzyme activity, and ATP levels with age. No statistically significant influences of age on activities of complexes I and II or citrate synthase were observed. In contrast, from 2 to 45 days post-eclosion, declines were found in complex IV cytochrome c oxidase activity (COX, 40% decline) and ATP abundance (15%), while lipid peroxidation increased 71%. We next examined flies that were either genetically or chemically oxidatively stressed to determine the effect on levels of mitochondrial-encoded cytochrome oxidase I RNA (coxI) and COX activity. A catalase null mutant line had 48% of coxI RNA compared to the wild type. In Cu/Zn superoxide dismutase (cSOD) null flies, the rate of coxI RNA decline was greater than in controls. CoxI RNA also declined with increasing hydrogen peroxide (H2O2) treatment, which was reflected in reduced cytochrome c oxidase (COX) activity. These results show that oxidative stress is closely associated with reductions in mitochondrial transcript levels and support the hypothesis that oxidative stress may contribute to mitochondrial dysfunction and aging in D. melanogaster.

Restriction of energy intake, energy expenditure, and aging.

Energy restriction (ER), without malnutrition, increases maximum life span and retards the development of a broad array of pathophysiological changes in laboratory rodents. The mechanism responsible for the retardation of aging by ER is, however, unknown. One proposed explanation is a reduction in energy expenditure (EE). Reduced EE may increase life span by decreasing the number of oxygen molecules interacting with mitochondria, thereby lowering reactive oxygen species (ROS) production. As a step toward testing this hypothesis, it is important to determine the effect of ER on EE. Several whole-body, organ, and cellular studies have measured the influence of ER on EE. In general, whole-body studies have reported an acute decrease in mass-adjusted EE that disappears with long-term ER. Organ-specific studies have shown that decreases in EE of liver and gastrointestinal tract are primarily responsible for initial reductions in EE with ER. These data, however, do not determine whether cellular EE is altered with ER. Three major processes contributing to resting EE at the cellular level are mitochondrial proton leak, Na(+)-K(+)-ATPase activity, and protein turnover. Studies suggest that proton leak and Na(+)-K(+)-ATPase activity are decreased with ER, whereas protein turnover is either unchanged or slightly increased with ER. Thus, two of the three major processes contributing to resting EE at the cellular level may be decreased with ER. Although additional cellular measurements are needed, the current results suggest that a lowering of EE could be a mechanism for the action of ER.

Association of age-related mitochondrial abnormalities with skeletal muscle fiber atrophy.

The hypothesis that mitochondrial dysfunction contributes to the senescent loss of skeletal muscle was investigated in quadriceps from 2- to 39-year old rhesus monkeys. Histological approaches, both cross-sectional (a single cross-section of the muscle) and longitudinal (multiple cross-sections of individual fibers spanning a 350-1600 microm region), were used to identify muscle fibers with abnormal mitochondrial electron transport system (ETS) enzyme activities and mitochondrial DNA deletions. Fibers were examined for two ETS activities, succinate dehydrogenase (SDH, ETS complex II) and cytochrome c oxidase (COX, ETS complex IV). The number of individual fibers containing ETS abnormalities (predominately negative for cytochrome c oxidase activity and/or hyperreactive for succinate dehydrogenase) increased with age. Deletions of the mitochondrial genome were observed in 89% of these ETS abnormal fibers. Longitudinal analysis allowed characterization of the ETS abnormal phenotype along their length. A decrease in cross-sectional area in 14% of the ETS abnormal fibers supports the hypothesis that deleted mitochondrial genomes may contribute to age-related fiber atrophy.

Localization of 4-hydroxy-2-nonenal-modified proteins in kidney following iron overload.

Intraperitoneal (IP) injection of ferric nitrilotriacetate (Fe-NTA) to rats and mice results in iron-induced free radical injury and cancer in kidneys. We sought to clarify the exact localization of acute oxidative damage in Fe-NTA-induced nephrotoxicity by performing immunogold light and electron microscopic (EM) techniques using an antibody against 4-hydroxy-2-nonenal (HNE)-modified proteins. Biochemical assays were done to provide complementary quantitative data. Renal accumulation of lipid peroxidation-derived aldehydes, such as malondialdehyde (MDA) and 4-hydroxy-2-alkenals (4-HDA), increased in parallel with protein carbonyl content, an indicator of protein oxidation, 30 min after administration of Fe-NTA. Immunogold light microscopy showed that HNE-modified proteins increased at 30 min with positivity localized to proximal tubular cells. Immunogold EM demonstrated that HNE-modified proteins were mainly in the mitochondria and nuclei of the proximal tubular epithelium. The intensity of labeling at both the light and EM levels increased together with levels of biochemically measured lipid peroxidation products and protein carbonyl content. Our data suggest that the mechanism of acute nephrotoxicity of Fe-NTA involves mitochondrial and nuclear oxidative damage, findings that may help to define the mechanisms of iron-induced cell injury.

Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria.

The purpose of this study was to understand the nature of the causes underlying the senescence-related decline in skeletal muscle mass and performance. Protein and lipid oxidative damage to upper hindlimb skeletal muscle mitochondria was compared between mice fed ad libitum and those restricted to 40% fewer calories--a regimen that increases life span by approximately 30-40% and attenuates the senescence-associated decrement in skeletal muscle mass and function. Oxidative damage to mitochondrial proteins, measured as amounts of protein carbonyls and loss of protein sulfhydryl content, and to mitochondrial lipids, determined as concentration of thiobarbituric acid reactive substances, significantly increased with age in the ad libitum-fed (AL) C57BL/6 mice. The rate of superoxide anion radical generation by submitochondrial particles increased whereas the activities of antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidase in muscle homogenates remained unaltered with age in the AL group. In calorically-restricted (CR) mice there was no age-associated increase in mitochondrial protein or lipid oxidative damage, or in superoxide anion radical generation. Crossover studies, involving the transfer of 18- to 22-month-old mice fed on the AL regimen to the CR regimen, and vice versa, indicated that the mitochondrial oxidative damage could not be reversed by CR or induced by AL feeding within a time frame of 6 weeks. Results of this study indicate that mitochondria in skeletal muscles accumulate significant amounts of oxidative damage during aging. Although such damage is largely irreversible, it can be prevented by restriction of caloric intake.

Androgen-induced oxidative stress in human LNCaP prostate cancer cells is associated with multiple mitochondrial modifications.

We investigated the role of androgen-induced oxidative stress in prostate cancer using the androgen-responsive LNCaP human prostate cancer cell line exposed to a 1-nM concentration of the synthetic androgen R1881 (which correlates with serum androgen levels). Such exposure, which decreases growth rate and increases oxidative stress in LNCaP cells, induced statistically significant mitochondrial changes. A 40% increase in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, indicative of mitochondrial dehydrogenase activity, occurred 24 hr after androgen treatment. This change preceded 50-110% increases, 40-96 hr after R1881 exposure, in levels of cellular peroxides and hydroxyl radicals as measured by 2'7'-dicholorofluorescin diacetate (DCF) fluorescence. On the basis of electron microscopy measurements, R1881 treatment increased the area fraction of mitochondria per cell by approximately 100% at 72 hr. In agreement, mitochondrial mass at 96 hr, evaluated by the fluorescent dye nonyl acridine orange (NAO), was 80% higher in treated cells. R1881 exposure for 24 hr lowered the activities of electron transport system (ETS) complexes, I, II, and IV by 17-27% and ATP levels by 50%. The ETS inhibitors, rotenone and antimycin A, lowered androgen-induced DCF fluorescence readings to control levels thereby suggesting ETS involvement in androgen-induced oxidant production. Addition of alpha-tocopherol succinate abrogated R1881-induced elevations in MTT reduction. In sum, androgens may, directly or indirectly, contribute to oxidative stress in LNCaP cells by regulating mitochondrial number, activity, and oxidant production by mechanisms that are, at least in part, sensitive to an antioxidant.