Alex Bokov

The role of oxidative damage and stress in aging

The Free Radical/Oxidative Stress Theory of Aging, which was first proposed in 1956, is currently one of the most popular explanations for how aging occurs at the biochemical/molecular level. However, most of the evidence in support of this theory is correlative, e.g., oxidative damage to various biomolecules increases with age, and caloric restriction, which increases life span and retards aging, reduces the age-related increase in oxidative damage to biomolecules. The most direct test of the Free Radical/Oxidative Stress Theory of Aging is to specifically alter the age-related increase in oxidative damage and determine how this alteration affects life span. For the first time, investigators can use genetically altered animals to test directly the role of oxidative damage in aging. In this manuscript, we critically review the past research in this area and discuss potential future research directions in testing the Free Radical/Oxidative Theory of Aging.

Is the Oxidative Stress Theory of Aging Dead

Currently, the oxidative stress (or free radical) theory of aging is the most popular explanation of how aging occurs at the molecular level. While data from studies in invertebrates (e.g., C. elegans and Drosophila) and rodents show a correlation between increased lifespan and resistance to oxidative stress (and in some cases reduced oxidative damage to macromolecules), direct evidence showing that alterations in oxidative damage/stress play a role in aging are limited to a few studies with transgenic Drosophila that overexpress antioxidant enzymes. Over the past eight years, our laboratory has conducted an exhaustive study on the effect of under- or overexpressing a large number and wide variety of genes coding for antioxidant enzymes. In this review, we present the survival data from these studies together. Because only one (the deletion of the Sod1 gene) of the 18 genetic manipulations we studied had an effect on lifespan, our data calls into serious question the hypothesis that alterations in oxidative damage/stress play a role in the longevity of mice.

The overexpression of major antioxidant enzymes does not extend the lifespan of mice

We evaluated the effect of overexpressing antioxidant enzymes on the lifespans of transgenic mice that overexpress copper zinc superoxide dismutase (CuZnSOD), catalase, or combinations of either CuZnSOD and catalase or CuZnSOD and manganese superoxide dismutase (MnSOD). Our results show that the overexpression of these major antioxidant enzymes, which are known to scavenge superoxide and hydrogen peroxide in the cytosolic and mitochondrial compartments, is insufficient to extend lifespan in mice.

Mice Deficient in Both Mn Superoxide Dismutase and Glutathione Peroxidase-1 Have Increased Oxidative Damage and a Greater Incidence of Pathology but No Reduction in Longevity

To test the impact of increased mitochondrial oxidative stress as a mechanism underlying aging and age-related pathologies, we generated mice with a combined deficiency in two mitochondrial-localized antioxidant enzymes, Mn superoxide dismutase (MnSOD) and glutathione peroxidase-1 (Gpx-1). We compared life span, pathology, and oxidative damage in Gpx1(-/-), Sod2(+/-)Gpx1(+/-), Sod2(+/-)Gpx1(-/-), and wild-type control mice. Oxidative damage was elevated in Sod2(+/-)Gpx1(-/-) mice, as shown by increased DNA oxidation in liver and skeletal muscle and increased protein oxidation in brain. Surprisingly, Sod2(+/-)Gpx1(-/-) mice showed no reduction in life span, despite increased levels of oxidative damage. Consistent with the important role for oxidative stress in tumorigenesis during aging, the incidence of neoplasms was significantly increased in the older Sod2(+/-)Gpx1(-/-) mice (28-30 months). Thus, these data do not support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice and do not support the oxidative stress theory of aging.

CBP gene transfer increases BDNF levels and ameliorates learning and memory deficits in a mouse model of Alzheimer's disease

Cognitive dysfunction and memory loss are common features of Alzheimers disease (AD). Abnormalities in the expression profile of immediate early genes that play a critical role in memory formation, such as the cAMP-response element binding protein (CREB), have been reported in the brains of AD patients. Here we show that amyloid-β (Aβ) accumulation, which plays a primary role in the cognitive deficits of AD, interferes with CREB activity. We further show that restoring CREB function via brain viral delivery of the CREB-binding protein (CBP) improves learning and memory deficits in an animal model of AD. Notably, such improvements occur without changes in Aβ and tau pathology, and instead are linked to an increased level of brain-derived neurotrophic factor. The resulting data suggest that Aβ-induced learning and memory deficits are mediated by alterations in CREB function, based on the finding that restoring CREB activity by directly modulating CBP levels in the brains of adult mice is sufficient to ameliorate learning and memory. Therefore, increasing CBP expression in adult brains may be a valid therapeutic approach not only for AD, but also for various brain disorders characterized by alterations in immediate early genes, further supporting the concept that viral vector delivery may be a viable therapeutic approach in neurodegenerative diseases.

Rapamycin Extends Life and Health in C57BL/6 Mice

Target of rapamycin inhibition by rapamycin feeding has previously been shown to extend life in genetically heterogeneous mice. To examine whether it similarly affected mouse health, we fed encapsulated rapamycin or a control diet to C57BL/6Nia mice of both sexes starting at 19 months of age. We performed a range of health assessments 6 and 12 months later. Rapamycin feeding significantly reduced mTOR activity in most but not all tissues. It also reduced total and resting metabolic rate during the light (inactive) phase of the light:dark cycle in females only but had no effect on spontaneous activity or metabolism during the dark (active) phase of either sex. Males only had less fragmented sleep when fed rapamycin, whereas stride length and rotarod performance were improved in both sexes. Survival was also improved by this late-life rapamycin feeding, and some pathological lesions were delayed. We found no adverse health consequences associated with rapamycin treatment.

Lack of methionine sulfoxide reductase A in mice increases sensitivity to oxidative stress but does not diminish life span.

Methionine sulfoxide reductase A (MsrA) repairs oxidized methionine residues within proteins and may also function as a general antioxidant. Previous reports have suggested that modulation of MsrA in mice and mammalian cell culture can affect the accumulation of oxidized proteins and may regulate resistance to oxidative stress. Thus, under the oxidative stress theory of aging, these results would predict that MsrA regulates the aging process in mammals. We show here that MsrA−/− mice are more susceptible to oxidative stress induced by paraquat. Skin‐derived fibroblasts do not express MsrA, but fibroblasts cultured from MsrA−/− mice were, nevertheless, also more susceptible to killing by various oxidative stresses. In contrast to previous reports, we find no evidence for neuromuscular dysfunction in MsrA−/− mice in either young adult or in older animals. Most important, we found no difference between MsrA−/− and control mice in either their median or maximum life span. Thus, our results show that MsrA regulates sensitivity to oxidative stress in mice but has no effect on aging, as determined by life span.—Salmon, A. B., Pérez, V. I., Bokov, A., Jernigan, A., Kim, G., Zhao, H., Levine, R. L., Richardson, A. Lack of methionine sulfoxide reductase A in mice increases sensitivity to oxidative stress but does not diminish life span. FASEB J. 23, 3601–3608 (2009). www.fasebj.org

Does reduced IGF-1R signaling in Igf1r+/- mice alter aging?

Mutations in insulin/IGF-1 signaling pathway have been shown to lead to increased longevity in various invertebrate models. Therefore, the effect of the haplo- insufficiency of the IGF-1 receptor (Igf1r+/−) on longevity/aging was evaluated in C57Bl/6 mice using rigorous criteria where lifespan and end-of-life pathology were measured under optimal husbandry conditions using large sample sizes. Igf1r+/− mice exhibited reductions in IGF-1 receptor levels and the activation of Akt by IGF-1, with no compensatory increases in serum IGF-1 or tissue IGF-1 mRNA levels, indicating that the Igf1r+/− mice show reduced IGF-1 signaling. Aged male, but not female Igf1r+/− mice were glucose intolerant, and both genders developed insulin resistance as they aged. Female, but not male Igf1r+/− mice survived longer than wild type mice after lethal paraquat and diquat exposure, and female Igf1r+/− mice also exhibited less diquat-induced liver damage. However, no significant difference between the lifespans of the male Igf1r+/− and wild type mice was observed; and the mean lifespan of the Igf1r+/− females was increased only slightly (less than 5%) compared to wild type mice. A comprehensive pathological analysis showed no significant difference in end-of-life pathological lesions between the Igf1r+/− and wild type mice. These data show that the Igf1r+/− mouse is not a model of increased longevity and delayed aging as predicted by invertebrate models with mutations in the insulin/IGF-1 signaling pathway.

Long-Lived Ames Dwarf Mice Are Resistant to Chemical Stressors

To probe the connection between longevity and stress resistance, we compared the sensitivity of Ames long-lived dwarf mice and control littermates with paraquat, diquat, and dobutamine. In young adult animals, 95% of male and 39% of female controls died after paraquat administration, but no dwarf animals died. When the experiment was repeated at an older age or a higher dosage of paraquat, dwarf mice still showed greater resistance. Dwarf mice also were more resistant to diquat; 80% of male and 60% of female controls died compared with 40% and 20% of dwarf mice, despite greater sensitivity of dwarf liver to diquat. Dwarf mice were also less sensitive to dobutamine-induced cardiac stress and had lower levels of liver and lung F(2)-isoprostanes. This is the first direct in vivo evidence that long-lived Ames dwarf mice have enhanced resistance to chemical insult, particularly oxidative stressors.

Mice Fed Rapamycin Have an Increase in Lifespan Associated with Major Changes in the Liver Transcriptome

Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycins longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (<300 transcripts) were observed in transcriptome of rapamycin-fed males; however, a large number of transcripts (>4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity.