Jan Gruber

Evidence for a trade-off between survival and fitness caused by resveratrol treatment of Caenorhabditis elegans

Abstract:  Resveratrol is a naturally occurring polyphenolic compound commonly found in plant‐derived products, including red wine. A large number of beneficial effects including anticarcinogenic action and protection from atherosclerotic disease have been attributed to resveratrol. Increased resveratrol intake has been suggested as an explanation for the beneficial effects of moderate red wine consumption. Resveratrol also consistently extends the mean and maximum life span in model organisms including nematode worms. It has been suggested that resveratrol exerts its life‐span–extending effect through calorie restriction or hormesis mimetic effects. We have characterized the effect of resveratrol on stress resistance, developmental rate, growth, and fecundity in the nematode worm Caenorhabditis elegans in order to determine whether the beneficial effects of resveratrol on life span are associated with trade‐offs in terms of early life fitness in nematodes. We find that resveratrol treatment increases stress resistance, specifically to oxidative stress, and causes a small but significant decrease in fecundity early in life without affecting overall fecundity. Resveratrol increased mean and maximum life span by delaying the onset of the exponential increase in mortality characterizing the “dying phase” in C. elegans, but did not affect the dying phase itself, suggesting that it did not act by directly affecting metabolism.

Mitochondria-targeted antioxidants and metabolic modulators as pharmacological interventions to slow ageing

Populations in many nations today are rapidly ageing. This unprecedented demographic change represents one of the main challenges of our time. A defining property of the ageing process is a marked increase in the risk of mortality and morbidity with age. The incidence of cancer, cardiovascular and neurodegenerative diseases increases non-linearly, sometimes exponentially with age. One of the most important tasks in biogerontology is to develop interventions leading to an increase in healthy lifespan (health span), and a better understanding of basic mechanisms underlying the ageing process itself may lead to interventions able to delay or prevent many or even all age-dependent conditions. One of the putative basic mechanisms of ageing is age-dependent mitochondrial deterioration, closely associated with damage mediated by reactive oxygen species (ROS). Given the central role that mitochondria and mitochondrial dysfunction play not only in ageing but also in apoptosis, cancer, neurodegeneration and other age-related diseases there is great interest in approaches to protect mitochondria from ROS-mediated damage. In this review, we explore strategies of targeting mitochondria to reduce mitochondrial oxidative damage with the aim of preventing or delaying age-dependent decline in mitochondrial function and some of the resulting pathologies. We discuss mitochondria-targeted and -localized antioxidants (e.g.: MitoQ, SkQ, ergothioneine), mitochondrial metabolic modulators (e.g. dichloroacetic acid), and uncouplers (e.g.: uncoupling proteins, dinitrophenol) as well as some alternative future approaches for targeting compounds to the mitochondria, including advances from nanotechnology.

Ageing in nematodes: do antioxidants extend lifespan in Caenorhabditis elegans?

Antioxidants are often investigated as a promising strategy for extending lifespan. Accordingly, there is significant interest in novel antioxidant compounds derived from natural sources such as plant extracts. However, because lifespan studies are laborious and expensive to conduct, candidate compounds are frequently selected based simply on their in vitro antioxidant efficacy, with the implicit assumption that in vitro antioxidants are also in vivo antioxidants, and that in vivo antioxidants will decrease functionally relevant oxidative damage and thereby extend lifespan. We investigated the validity of these assumptions in the model organism, Caenorhabditiselegans. Nematodes were exposed to 6 plant extracts, selected out of a total of 34 based on a simple in vitro antioxidant assay. We found no correlation between in vitro and in vivo antioxidant capacities. Antioxidant efficacies were also not predictive of lifespan benefits. Further studies into those extracts that produced significant lifespan extension indicated that a direct antioxidant effect is unlikely to be the main factor responsible for the modulation of nematode lifespan.

Deceptively simple but simply deceptive - Caenorhabditis elegans lifespan studies: Considerations for aging and antioxidant effects

The nematode worm Caenorhabditis elegans (C. elegans) is increasingly popular as a model organism for aging studies as well as for testing antioxidants and other compounds for effects on longevity. However, results in the literature are sometimes confusing and contradictory [1–4]. This review introduces C. elegans as a model organism, discusses aspects that make it attractive for aging and antioxidant research, and addresses some problems and potential artifacts.

Mitochondrial Changes in Ageing Caenorhabditis elegans – What Do We Learn from Superoxide Dismutase Knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the “vicious cycle” theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the “vicious cycle” theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the “vicious cycle” theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed.

The mitochondria-targeted antioxidant MitoQ extends lifespan and improves healthspan of a transgenic Caenorhabditis elegans model of Alzheimer disease.

β-Amyloid (Aβ)-induced toxicity and oxidative stress have been postulated to play critical roles in the pathogenic mechanism of Alzheimer disease (AD). We investigated the in vivo ability of a mitochondria-targeted antioxidant, MitoQ, to protect against Aβ-induced toxicity and oxidative stress in a Caenorhabditis elegans model overexpressing human Aβ. Impairment of electron transport chain (ETC) enzymatic activity and mitochondrial dysfunction are early features of AD. We show that MitoQ extends lifespan, delays Aβ-induced paralysis, ameliorates depletion of the mitochondrial lipid cardiolipin, and protects complexes IV and I of the ETC. Despite its protective effects on lifespan, healthspan, and ETC function, we find that MitoQ does not reduce DCFDA fluorescence, protein carbonyl levels or modulate steadystate ATP levels or oxygen consumption rate. Moreover, MitoQ does not attenuate mitochondrial DNA (mtDNA) oxidative damage. In agreement with its design, the protective effects of MitoQ appear to be targeted specifically to the mitochondrial membrane and our findings suggest that MitoQ may have therapeutic potential for Aβ- and oxidative stress-associated neurodegenerative disorders, particularly AD.

Is uric acid protective or deleterious in acute ischemic stroke? A prospective cohort study

Contrasting observations have been made between serum urate and ischemic stroke outcomes in studies involving Caucasian populations. To assess the hypothesis that urate is associated with stroke outcomes, a prospective follow-up study was performed in a cohort of Asian patients with ischemic stroke. Patients diagnosed with transient ischemic attack, first or recurrent ischemic stroke were included in this study. Serum urate, measured using high-performance liquid chromatography, was correlated with 12-month functional and vascular stroke outcomes. Poor functional outcome was defined as a modified Rankin scale exceeding 2 and vascular outcome was defined as a composite of recurrent stroke, myocardial infarction or vascular death during the study period. A total of 503 patients of mean age 63 (SD 12) years were included. A U-shaped relationship between urate quartiles and poor functional outcomes was demonstrated. More patients with low (<280microM) and high (>410microM) urate levels had poor functional outcomes (36% and 27% respectively), compared to those with urate levels between 340 and 410microM (14%). No significant relationship was observed between urate and vascular outcomes. Depending on its level, serum urate may exhibit protective and deleterious effects on stroke outcomes.

The effect of dichloroacetate on health- and lifespan in C. elegans

Aging is associated with increased vulnerability to chronic, degenerative diseases and death. Strategies for promoting healthspan without necessarily affecting lifespan or aging rate have gained much interest. The mitochondrial free radical theory of aging suggests that mitochondria and, in particular, age-dependent mitochondrial decline play a central role in aging, making compounds that affect mitochondrial function a possible strategy for the modulation of healthspan and possibly the aging rate. Here we tested such a “metabolic tuning” approach in nematodes using the mitochondrial modulator dichloroacetate (DCA). We explored DCA as a proof-of-principle compound to alter mitochondrial parameters in wild-type animals and tested whether this approach is suitable for reducing reactive oxygen species (ROS) production and for improving organismal health- and lifespan. In parallel, we addressed the potential problem of operator bias by running both unblinded and blinded lifespan studies. We found that DCA treatment (1) increased ATP levels without elevating oxidative protein damage and (2) reduced ROS production in adult C. elegans. DCA treatment also significantly prolonged nematode health- and lifespan, but did not strongly impact mortality doubling time. Operator blinding resulted in considerably smaller lifespan-extending effects of DCA. Our data illustrate the promise of a “metabolic tuning” intervention strategy, emphasize the importance of mitochondria in nematode aging and highlight operator bias as a potential confounder in lifespan studies.

Hydrogen Sulfide Is an Endogenous Regulator of Aging in Caenorhabditis elegans

AIMS To investigate the role of endogenous hydrogen sulfide (H2S) in the control of aging and healthspan of Caenorhabditis elegans. RESULTS We show that the model organism, C. elegans, synthesizes H2S. Three H2S-synthesizing enzymes are present in C. elegans, namely cystathionine γ lyase (CSE), cystathionine β synthetase, and 3-mercaptopyruvate transferase (MPST or 3-MST). Genetic deficiency of mpst-1 (3-MST orthologue 1), but not cth-2 (CSE orthologue), reduced the lifespan of C. elegans. This effect was reversed by a pharmacological H2S donor (GYY4137). GYY4137 also reduced detrimental age-dependent changes in a range of physiological indices, including pharyngeal contraction and defecation. Treatment of C. elegans with GYY4137 increased the expression of several age-related, stress response, and antioxidant genes, whereas MitoSOX Red fluorescence, indicative of reactive oxygen species generation, was increased in mpst-1 knockouts and decreased by GYY4137 treatment. GYY4137 additionally increased the lifespan in short-lived mev-1 mutants with elevated oxidative stress and protected wild-type C. elegans against paraquat poisoning. The lifespan-prolonging and health-promoting effects of H2S in C. elegans are likely due to the antioxidant action of this highly cell-permeable gas. INNOVATION The possibility that novel pharmacological agents based on the principle of H2S donation may be able to retard the onset of age-related disease by slowing the aging process warrants further study. CONCLUSION Our results show that H2S is an endogenous regulator of oxidative damage, metabolism, and aging in C. elegans and provide new insight into the mechanisms, which control aging in this model organism.

Stochastic drift in mitochondrial DNA point mutations: a novel perspective ex silico.

The mitochondrial free radical theory of aging (mFRTA) implicates Reactive Oxygen Species (ROS)-induced mutations of mitochondrial DNA (mtDNA) as a major cause of aging. However, fifty years after its inception, several of its premises are intensely debated. Much of this uncertainty is due to the large range of values in the reported experimental data, for example on oxidative damage and mutational burden in mtDNA. This is in part due to limitations with available measurement technologies. Here we show that sample preparations in some assays necessitating high dilution of DNA (single molecule level) may introduce significant statistical variability. Adding to this complexity is the intrinsically stochastic nature of cellular processes, which manifests in cells from the same tissue harboring varying mutation load. In conjunction, these random elements make the determination of the underlying mutation dynamics extremely challenging. Our in silico stochastic study reveals the effect of coupling the experimental variability and the intrinsic stochasticity of aging process in some of the reported experimental data. We also show that the stochastic nature of a de novo point mutation generated during embryonic development is a major contributor of different mutation burdens in the individuals of mouse population. Analysis of simulation results leads to several new insights on the relevance of mutation stochasticity in the context of dividing tissues and the plausibility of ROS ”vicious cycle” hypothesis.