Ubiquinone (UQ), a.k.a. coenzyme Q, is a redox-active lipid that participates in several cellular processes, in particular mitochondrial electron transport. Primary UQ deficiency is a rare but severely debilitating condition. Mclk1 (a.k.a. Coq7) encodes a conserved mitochondrial enzyme that is necessary for UQ biosynthesis. We engineered conditional Mclk1 knockout models to study pathogenic effects of UQ deficiency and to assess potential therapeutic agents for the treatment of UQ deficiencies. We found that Mclk1 knockout cells are viable in the total absence of UQ. The UQ biosynthetic precursor DMQ9 accumulates in these cells and can sustain mitochondrial respiration, albeit inefficiently. We demonstrated that efficient rescue of the respiratory deficiency in UQ-deficient cells by UQ analogues is side chain length dependent, and that classical UQ analogues with alkyl side chains such as idebenone and decylUQ are inefficient in comparison with analogues with isoprenoid side chains. Furthermore, Vitamin K2, which has an isoprenoid side chain, and has been proposed to be a mitochondrial electron carrier, had no efficacy on UQ-deficient mouse cells. In our model with liver-specific loss of Mclk1, a large depletion of UQ in hepatocytes caused only a mild impairment of respiratory chain function and no gross abnormalities. In conjunction with previous findings, this surprisingly small effect of UQ depletion indicates a nonlinear dependence of mitochondrial respiratory capacity on UQ content. With this model, we also showed that diet-derived UQ10 is able to functionally rescue the electron transport deficit due to severe endogenous UQ deficiency in the liver, an organ capable of absorbing exogenous UQ.
The mitochondrial free radical theory of aging (MFRTA) proposes that aging is caused by damage to macromolecules from mitochondrial reactive oxygen species (ROS). This is based on the observed association of the rate of aging and the aged phenotype with the generation of ROS and with oxidative damage. The theory has led to the strong conviction in the general public that the consumption of antioxidants is crucial to health and beneficial to lifespan. However, a variety of recent findings convincingly demonstrate that ROS generation and oxidative damage cannot be the cause of aging. Here we propose that ROS play a role in mediating a stress response to age-dependent damage, which could generate the observed correlation between aging and ROS without implying that ROS cause aging.
PMID: 21824781 CAMSID: cams4495
The nematode worm Caenorhabditis elegans has been used to identify hundreds of genes that influence longevity and thereby demonstrate the strong influence of genetics on lifespan determination. In order to simplify lifespan studies in worms, many researchers have employed 5-fluoro-2′-deoxyuridine (FUdR) to inhibit the development of progeny. While FUdR has little impact on the lifespan of wild-type worms, we demonstrate that FUdR causes a dramatic, dose-dependent, two-fold increase in the lifespan of the mitochondrial mutant gas-1. Thus, the concentration of FUdR employed in a lifespan study can determine whether a particular strain is long-lived or short-lived compared to wild-type.
PMID: 21893079 CAMSID: cams4494
Lifespan; Caenorhabditis elegans; FUdR; gas-1; genetics of aging
Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a redox-active lipid present in all cellular membranes where it functions in a variety of cellular processes. The best known functions of UQ are to act as a mobile electron carrier in the mitochondrial respiratory chain and to serve as a lipid soluble antioxidant in cellular membranes. All eukaryotic cells synthesize their own UQ. Most of the current knowledge on the UQ biosynthetic pathway was obtained by studying Escherichia coli and S. cerevisiae UQ-deficient mutants. The orthologues of all the genes known from yeast studies to be involved in UQ biosynthesis have subsequently been found in higher organisms. Animal mutants with different genetic defects in UQ biosynthesis display very different phenotypes, despite the fact that in all these mutants the same biosynthetic pathway is affected. This review summarizes the present knowledge of the eukaryotic biosynthesis of UQ, with focus on the biosynthetic genes identified in animals, including C. elegans, rodents and humans. Moreover, we review the phenotypes of mutants in these genes and discuss the functional consequences of UQ deficiency in general.
PMID: 23190198 CAMSID: cams4455
ubiquinone biosynthesis; coenzyme Q; ubiquinone deficiency; COQ genes; unicellular model systems; metazoan model systems; human patients
To achieve a long lifespan, animals must be resistant to various injuries as well as be able to avoid or delay lethality from age-dependent diseases. Here we show that long-lived Mclk1+/− mutants have enhanced resistance to neurological damage following global cerebral ischemia/reperfusion (I/R) injury induced by transient bilateral common carotid artery occlusion (BCCAO). Both young (~100 days old) and relatively aged (~450 days old) mutants display increased resistance as indicated by a significant decrease in the amount of degenerating cells observed in forebrain cortex and in hippocampal areas after ischemia and reperfusion. Furthermore, less oxidative damage resulting from the procedure was measured in the brain of young Mclk1+/− animals. The finding that both young and old mutants are protected indicates that this is a basic phenotype of these mutants and not a secondary consequence of their slow rate of aging. Thus, the partial resistance to I/R injury by enhancing recovery from age-dependent vascular accidents is likely part of what allows for the increased lifespan of Mclk1+/− mutants. By relating this neuroprotective effect to previously reported characteristics of the Mclk1+/− phenotype, including altered mitochondrial metabolism and increased HIF-1α expression, this study establishes these mutants as useful models to analyze the mechanisms underlying tolerance to ischemia, particularly those associated with ischemic preconditioning, as well as to clarify the relation between aging and age-dependent diseases.
PMID: 20170652 CAMSID: cams4458
Mclk1; Ischemia; Reperfusion; Aging; Age-related diseases; Ischemic tolerance; BCCAO; Oxidative stress
According to the widely acknowledged mitochondrial free radical theory of aging (MFRTA), the macromolecular damage that results from the production of toxic reactive oxygen species (ROS) during cellular respiration is THE cause of aging. However, although it is clear that oxidative damage increases during aging, the fundamental question regarding whether mitochondrial oxidative stress is in any way causal to the aging process remains unresolved. An increasing number of studies on long-lived vertebrate species, mutants and transgenic animals have seriously challenged the pervasive MFRTA. Here, we describe some of these new results, including those pertaining to the phenotype of the long-lived Mclk1+/− mice, which appear irreconcilable with the MFRTA. Thus, we believe that it is reasonable to now consider the MFRTA as refuted and that it is time to use the insight gained by many years of testing this theory to develop new views as to the physiological causes of aging.
PMID: 19730800 CAMSID: cams4457
Caenorhabditis elegans CEP-1 and its mammalian homolog p53 are critical for responding to diverse stress signals. In this study, we found that cep-1 inactivation suppressed the prolonged lifespan of electron transport chain (ETC) mutants, such as isp-1 and nuo-6, but rescued the shortened lifespan of other ETC mutants, such as mev-1 and gas-1. We compared the CEP-1-regulated transcriptional profiles of the long-lived isp-1 and the short-lived mev-1 mutants and, to our surprise, found that CEP-1 regulated largely similar sets of target genes in the two mutants despite exerting opposing effects on their longevity. Further analyses identified a small subset of CEP-1-regulated genes that displayed distinct expression changes between the isp-1 and mev-1 mutants. Interestingly, this small group of differentially regulated genes are enriched for the “aging” Gene Ontology term, consistent with the hypothesis that they might be particularly important for mediating the distinct longevity effects of CEP-1 in isp-1 and mev-1 mutants. We further focused on one of these differentially regulated genes, ftn-1, which encodes ferritin in C. elegans, and demonstrated that it specifically contributed to the extended lifespan of isp-1 mutant worms but did not affect the mev-1 mutant lifespan. We propose that CEP-1 responds to different mitochondrial ETC stress by mounting distinct compensatory responses accordingly to modulate animal physiology and longevity. Our findings provide insights into how mammalian p53 might respond to distinct mitochondrial stressors to influence cellular and organismal responses.
Perturbing different components of the mitochondrial electron transport chain (ETC) can affect the lifespan of Caenorhabditis elegans in divergent ways. ETC dysfunction can either attenuate or extend C. elegans lifespan. Here we demonstrate that the C. elegans homolog of mammalian p53, CEP-1, is key in mediating differential survival of distinct ETC mutants. Inactivating cep-1 in two long-lived ETC mutants shortened their lifespans, whereas cep-1 deletion in two short-lived ETC mutants restored their lifespans. Because how CEP-1 does this is unknown, we compared the genome-wide expression profiles of the long-lived isp-1 and short-lived mev-1 ETC mutants upon cep-1 removal. We found that CEP-1 is able to differentially regulate a small subset of genes depending on the specific ETC mutations. We validated one of the genes, the iron transporter ferritin (ftn-1), to be an important target through which CEP-1 mediates distinct lifespan outcome in specific ETC mutants. We also identified numerous other candidate CEP-1 targets that merit future analysis, which will further elucidate how CEP-1 differentially responds to the distinct ETC dysfunction of various mitochondrial mutants. Our study provides new insights that will inform how mammalian p53, a critical tumor suppressor, might respond to mitochondrial and metabolic stresses.
MCLK1 and COQ3 are mitochondrial enzymes necessary for ubiquinone biosynthesis, but only MCLK1 also regulates the partitioning of ubiquinone between mitochondrial membranes and affects longevity in mice.
Mclk1 (also known as Coq7) and Coq3 code for mitochondrial enzymes implicated in the biosynthetic pathway of ubiquinone (coenzyme Q or UQ). Mclk1+/− mice are long-lived but have dysfunctional mitochondria. This phenotype remains unexplained, as no changes in UQ content were observed in these mutants. By producing highly purified submitochondrial fractions, we report here that Mclk1+/− mice present a unique mitochondrial UQ profile that was characterized by decreased UQ levels in the inner membrane coupled with increased UQ in the outer membrane. Dietary-supplemented UQ10 was actively incorporated in both mitochondrial membranes, and this was sufficient to reverse mutant mitochondrial phenotypes. Further, although homozygous Coq3 mutants die as embryos like Mclk1 homozygous null mice, Coq3+/− mice had a normal lifespan and were free of detectable defects in mitochondrial function or ubiquinone distribution. These findings indicate that MCLK1 regulates both UQ synthesis and distribution within mitochondrial membranes.
The mitochondria of slowly aging Mclk1+/− mutant mice produce high levels of reactive oxygen species (ROS). These animals display enhanced immune reactivity in response to lipopolysaccharide, Salmonella, and tumor-cell grafts, experience limited damage from these treatments and are partially protected from infection and tumorigenesis. We propose that the activation of the immune system by mitochondrial ROS reduces the rate of aging.
aging; biomarkers of aging; Mclk1; mitochondria; reactive oxygen species; T-cell cytotoxicity; xenografts
C. elegans is a model used to study cholesterol metabolism and the functions of its metabolites. Several studies have reported that, in worms, cholesterol is not a structural component of the membrane as it is in vertebrates. However, as in other animals, it is used for the synthesis of steroid hormones that regulate physiological processes such as dauer formation, molting and defecation. After cholesterol is taken up by the gut, mechanisms of transport of cholesterol between tissues in C. elegans involve lipoproteins, as in mammals. A recent study shows that both cholesterol uptake and lipoprotein metabolism in C. elegans are regulated by molecules whose activities, biosynthesis, and secretion strongly resemble those of mammalian bile acids, which are metabolites of cholesterol that act on metabolism in a variety of ways. Importantly, it was found that oxidative stress upsets the regulation of the synthesis of these molecules. Given the known function of mammalian bile acids as metabolic regulators of lipid and glucose homeostasis, future investigations of the biology of C. elegans bile acid-like molecules could provide information on the etiology of human metabolic disorders that are characterized by elevated oxidative stress.
C. elegans; clk-1; bile acids; cholesterol; dafachronic acids; dauer formation; defecation; lipoproteins; metabolic syndrome; oxidative stress; steroid hormones
The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1+/− mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1+/− mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1+/− mutants, as well as during spontaneous tumorigenesis in Mclk1+/−
Trp53+/− double mutants. Tumour latency was increased by the Mclk1+/− genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8+ T lymphocytes from Mclk1+/− graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1+/− mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.
Rationale: Acute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown.
Objectives: To determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury.
Methods: Exposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival.
Measurements and Main Results: We found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D–deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK.
Conclusions: Mitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.
cell death; epithelium; Bcl-2 proteins; acute respiratory distress syndrome
Mammalian bile acids (BAs) are oxidized metabolites of cholesterol whose amphiphilic properties serve in lipid and cholesterol uptake. BAs also act as hormone-like substances that regulate metabolism. The Caenorhabditis elegans clk-1 mutants sustain elevated mitochondrial oxidative stress and display a slow defecation phenotype that is sensitive to the level of dietary cholesterol. We found that: 1) The defecation phenotype of clk-1 mutants is suppressed by mutations in tat-2 identified in a previous unbiased screen for suppressors of clk-1. TAT-2 is homologous to ATP8B1, a flippase required for normal BA secretion in mammals. 2) The phenotype is suppressed by cholestyramine, a resin that binds BAs. 3) The phenotype is suppressed by the knock-down of C. elegans homologues of BA–biosynthetic enzymes. 4) The phenotype is enhanced by treatment with BAs. 5) Lipid extracts from C. elegans contain an activity that mimics the effect of BAs on clk-1, and the activity is more abundant in clk-1 extracts. 6) clk-1 and clk-1;tat-2 double mutants show altered cholesterol content. 7) The clk-1 phenotype is enhanced by high dietary cholesterol and this requires TAT-2. 8) Suppression of clk-1 by tat-2 is rescued by BAs, and this requires dietary cholesterol. 9) The clk-1 phenotype, including the level of activity in lipid extracts, is suppressed by antioxidants and enhanced by depletion of mitochondrial superoxide dismutases. These observations suggest that C. elegans synthesizes and secretes molecules with properties and functions resembling those of BAs. These molecules act in cholesterol uptake, and their level of synthesis is up-regulated by mitochondrial oxidative stress. Future investigations should reveal whether these molecules are in fact BAs, which would suggest the unexplored possibility that the elevated oxidative stress that characterizes the metabolic syndrome might participate in disease processes by affecting the regulation of metabolism by BAs.
Cholesterol metabolism, in particular the transport of cholesterol in the blood by lipoproteins, is an important determinant of human cardiovascular health. Bile acids are breakdown products of cholesterol that have detergent properties and are secreted into the gut by the liver. Bile acids carry out three distinct roles in cholesterol metabolism: 1) Their synthesis from cholesterol participates in cholesterol elimination. 2) They act as detergents in the uptake of dietary cholesterol from the gut. 3) They regulate many aspects of metabolism, including cholesterol metabolism, by molecular mechanisms similar to that of steroid hormones. We have found that cholesterol uptake and lipoprotein metabolism in the nematode Caenorhabditis elegans are regulated by molecules whose activities, biosynthesis, and secretion strongly resemble that of bile acids and which might be bile acids. Most importantly we have found that oxidative stress upsets the regulation of the synthesis of these molecules. The metabolic syndrome is a set of cardiovascular risk factors that include obesity, high blood cholesterol, hypertension, and insulin resistance. Given the function of bile acids as metabolic regulators, our findings with C. elegans suggest the unexplored possibility that the elevated oxidative stress that characterizes the metabolic syndrome may participate in mammalian disease processes by affecting the regulation of bile acid synthesis.
Impairments of various aspects of mitochondrial function have been associated with increased lifespan in various model organisms ranging from Caenorhabditis elegans to mice. For example, disruption of the function of the ‘Rieske’ iron-sulfur protein (RISP) of complex III of the mitochondrial electron transport chain can result in increased lifespan in the nematode worm C. elegans. However, the mechanisms by which impaired mitochondrial function affects aging remain under investigation, including whether or not they require decreased electron transport. We have generated knock-in mice with a loss-of-function Risp mutation that is homozygous lethal. However, heterozygotes (Risp+/P224S) were viable and had decreased levels of RISP protein and complex III enzymatic activity. This decrease was sufficient to impair mitochondrial respiration and to decrease overall metabolic rate in males, but not females. These defects did not appear to exert an overtly deleterious effect on the health of the mutants, since young Risp+/P224S mice are outwardly normal, with unaffected performance and fertility. Furthermore, biomarkers of oxidative stress were unaffected in both young and aged animals. Despite this, the average lifespan of male Risp+/P224S mice was shortened and aged Risp+/P224S males showed signs of more rapidly deteriorating health. In spite of these differences, analysis of Gompertz mortality parameters showed that Risp heterozygosity decreased the rate of increase of mortality with age and increased the intrinsic vulnerability to death in both sexes. However, the intrinsic vulnerability was increased more dramatically in males, which resulted in their shortened lifespan. For females, the slower acceleration of age-dependent mortality results in significantly increased survival of Risp+/P224S mice in the second half of lifespan. These results demonstrate that even relatively small perturbations of the mitochondrial electron transport chain can have significant physiological effects in mammals, and that the severity of those effects can be sex-dependent.
The study of long-lived C. elegans mutants suggests that mitochondrial oxidants can actually help reduce aging by acting as stress signals, rather than acting solely as toxic molecules.
The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O⋅–), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response.
An unequivocal demonstration that mitochondria are important for lifespan comes from studies with the nematode Caenorhabditis elegans. Mutations in mitochondrial proteins such as ISP-1 and NUO-6, which function directly in mitochondrial electron transport, lead to a dramatic increase in the lifespan of this organism. One theory proposes that toxicity of mitochondrial reactive oxygen species (ROS) is the cause of aging and predicts that the generation of the ROS superoxide should be low in these mutants. Here we have measured superoxide generation in these mutants and found that it is in fact elevated, rather than reduced. Furthermore, we found that this elevation is necessary and sufficient for longevity, as it is abolished by antioxidants and induced by mild treatment with oxidants. This suggests that superoxide can act as a signal triggering cellular changes that attenuate the effects of aging. This idea suggests a new model for the well-documented correlation between ROS and the aged phenotype. We propose that a gradual increase of molecular damage during aging triggers a concurrent, gradually intensifying, protective superoxide response.
The editorial board of Aging reviews research papers published in 2009, which they
believe have or will have a significant impact on aging research. Among many
others, the topics include genes that accelerate aging or in contrast promote
longevity in model organisms, DNA damage responses and telomeres, molecular
mechanisms of life span extension by calorie restriction and pharmacologic
interventions into aging. The emerging message in 2009 is that aging is not
random but determined by a genetically-regulated longevity network and can be
decelerated both genetically and pharmacologically.
aging; senescence; signal transduction; genes for longevity
Mice with a single copy of Mclk1 (a.k.a. Coq7), a gene that encodes a mitochondrial enzyme required for the biosynthesis of ubiquinone and other functions, live longer than wild-type mice. The prolonged survival implies a decreased mortality from age-dependent lethal pathologies. Atherosclerosis is one of the main age-dependent pathologies in humans and can be modeled in mice that lack Apolipoprotein E (ApoE-/-) or mice that lack the Low Density Lipoprotein Receptor (LDLr-/-) in addition to being fed an atherosclerosis-inducing diet. We sought to determine if Mclk1 heterozygosity protects against atherosclerosis and dyslipidemia in these models.
We found that Mclk1 heterozygosity did not protect against dyslipidemia, oxidative stress, or atherosclerosis in young (6 or 10 months) or older (18 months) mice. Furthermore, the absence of ApoE suppressed the lifespan-promoting effects of Mclk1 heterozygosity.
These findings indicate that although Mclk1 heterozygosity can extend lifespan of mice, it does not necessarily protect against atherosclerosis. Moreover, in the presence of hyperlipidemia and chronic inflammation, Mclk1 heterozygosity is incapable of extending lifespan.
The oxidative stress theory of aging postulates that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS) generated during normal metabolism. Superoxide dismutases (SODs) counteract this process by detoxifying superoxide. It has previously been shown that elimination of either cytoplasmic or mitochondrial SOD in yeast, flies, and mice results in decreased lifespan. In this experiment, we examine the effect of eliminating each of the five individual sod genes present in Caenorhabditis elegans. In contrast to what is observed in other model organisms, none of the sod deletion mutants shows decreased lifespan compared to wild-type worms, despite a clear increase in sensitivity to paraquat- and juglone-induced oxidative stress. In fact, even mutants lacking combinations of two or three sod genes survive at least as long as wild-type worms. Examination of gene expression in these mutants reveals mild compensatory up-regulation of other sod genes. Interestingly, we find that sod-2 mutants are long-lived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on known pathways of lifespan extension reveals a clear interaction with genes that affect mitochondrial function: sod-2 deletion markedly increases lifespan in clk-1 worms while clearly decreasing the lifespan of isp-1 worms. Combined with the mitochondrial localization of SOD-2 and the fact that sod-2 mutant worms exhibit phenotypes that are characteristic of long-lived mitochondrial mutants—including slow development, low brood size, and slow defecation—this suggests that deletion of sod-2 extends lifespan through a similar mechanism. This conclusion is supported by our demonstration of decreased oxygen consumption in sod-2 mutant worms. Overall, we show that increased oxidative stress caused by deletion of sod genes does not result in decreased lifespan in C. elegans and that deletion of sod-2 extends worm lifespan by altering mitochondrial function.
In this paper, we examine the oxidative stress theory of aging using C. elegans as a model system. This theory proposes that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS). To test this theory, we examined the effect of deleting each of the five individual superoxide dismutase (SOD) genes on lifespan and sensitivity to oxidative stress. Since SOD acts to detoxify ROS, the oxidative stress theory predicts that deletion of sod genes should increase oxidative stress and decrease lifespan. However, in contrast to yeast, flies, and mice, where loss of either cytoplasmic or mitochondrial SOD results in decreased lifespan, we find that none of the sod deletion mutants in C. elegans exhibits a shortened lifespan despite increased sensitivity to oxidative stress. Surprisingly, we find that sod-2 mutant worms have extended lifespan and even worms with the primary cytoplasmic, mitochondrial, and extracellular sod genes deleted can live longer than wild-type worms. By examining genetic interactions with other genes known to extend lifespan and by comparing the phenotype of worms lacking sod-2 to that of known long-lived mitochondrial mutants such as clk-1 or isp-1, we provide evidence that the loss of sod-2 extends lifespan through alteration of mitochondrial function.
Two mutant mouse models of longevity in which the loss of only one copy of the gene leads to a significantly increased lifespan have recently been described: Igf1r+/- and mclk1+/-. Igf1r encodes a transmembrane receptor kinase for the insulin-like growth factor-1, and mclk1 encodes a hydroxylase that is necessary for the biosynthesis of ubiquinone. Interestingly, the motivation for testing the longevity of both of these mutants came from observations in the nematode Caenorhabditis elegans. IGF-1R protein is homologous to DAF-2 and mCLK1 is the mouse orthologue of the C. elegans enzyme CLK-1. In worms, the homozygous inactivation of both of these longevity genes is viable and no dominant mutations are known. In addition to aging slowly, old mclk1+/- mice were found to undergo loss-of-heterozygosity at the mclk1 locus, which results in clones of mclk1-/- cells in the liver, presumably because mclk1-/- cells can outcompete mclk1+/- cells under certain conditions. We will discuss how these observations suggest novel directions of research, but also call for some caution in the interpretation of past and future results.
aging; evolutionary conservation; loss-of-heterozygosity; mclk1; mouse models of longevity; ubiquinone
clk-1 encodes a demethoxyubiquinone (DMQ) hydroxylase that is necessary for ubiquinone biosynthesis. When Caenorhabditis elegans clk-1 mutants are grown on bacteria that synthesize ubiquinone (UQ), they are viable but have a pleiotropic phenotype that includes slowed development, behaviors, and aging. However, when grown on UQ-deficient bacteria, the mutants arrest development transiently before growing up to become sterile adults. We identified nine suppressors of the missense mutation clk-1(e2519), which harbors a Glu-to-Lys substitution. All suppress the mutant phenotypes on both UQ-replete and UQ-deficient bacteria. However, each mutant suppresses a different subset of phenotypes, indicating that most phenotypes can be uncoupled from each other. In addition, all suppressors restore the ability to synthesize exceedingly small amounts of UQ, although they still accumulate the precursor DMQ, suggesting that the presence of DMQ is not responsible for the Clk-1 phenotypes. We cloned six of the suppressors, and all encode tRNAGlu genes whose anticodons are altered to read the substituted Lys codon of clk-1(e2519). To our knowledge, these suppressors represent the first missense suppressors identified in any metazoan. The pattern of suppression we observe suggests that the individual members of the tRNAGlu family are expressed in different tissues and at different levels.
Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as
Drosophila Nipped-B, a regulator of
Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of
Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in
C. elegans embryos. Using antisense morpholino oligonucleotides to knock down
Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.
A complex previously found only in yeast is described in metazoa, where it functions both in chromatid cohesion and in migration during development.