Clk-1 Mutants Research Articles

Clock genes regulate mating activity rhythms in the vector mosquitoes, Aedes albopictus and Culex quinquefasciatus.

Endogenous circadian rhythms result from genetically-encoded molecular clocks, whose components and downstream output factors cooperate to generate cyclic changes in activity. Mating is an important activity of mosquitoes, however, the key aspects of mating rhythm patterns and their regulatory mechanisms in two vector mosquito species, Aedes albopictus and Culex quinquefasciatus, remain unclear. We determined and compared the diel mating activity rhythms of these two mosquito species and discovered that Ae. albopictus had mating peaks in the light/dark transition periods (ZT0-3 and ZT9-12), while Cx. quinquefasciatus only had a mating peak at ZT12-15. Knockouts of the clock (clk) orthologous genes (Aalclk and Cxqclk) resulted in phase delay or phase reversal of the mating peaks in Ae. albopictus and Cx. quinquefasciatus, respectively. In addition, the temporal expression pattern of the desaturase orthologous genes, desat1, in both mosquito species was also different in respective wild-type strains and showed phase changes similar to the mating rhythms in clk mutant strains. Inhibition of desat1 expression resulted in decreased mating activity in male mosquitoes of both species but not females. In addition, desat1 regulated cuticular hydrocarbons' synthesis in both species. Silencing desat1 in male Ae. albopictus resulted in decreases of nonadecane and tricosane, which promoted mating, with concomitant increases of heptacosane, which inhibited mating. Silencing desat1 in male Cx. quinquefasciatus also resulted in decreases of tricosane, which promoted mating. These results suggest that Aalclk and Cxqclk have significant roles in the mating activity rhythms in both Ae. albopictus and Cx. quinquefasciatus by regulating the temporal expression of the desat1 gene under LD cycles, which affects sex pheromone synthesis and mating. This work provides insights into the molecular regulatory mechanism of distinct mating rhythm of Ae. albopictus and Cx. quinquefasciatus and may provide a basis for the control of these two important vector mosquitoes.

Elevated mitochondrial DNA copy number found in ubiquinone-deficient clk-1 mutants is not rescued by ubiquinone precursor 2-4-dihydroxybenzoate

Inside mitochondria reside semi-autonomous genomes, called mtDNA. mtDNA is multi-copy per cell and mtDNA copy number can vary from hundreds to thousands of copies per cell. The variability of mtDNA copy number between tissues, combined with the lack of variability of copy number within a tissue, suggest a homeostatic copy number regulation mechanism. Mutations in the gene encoding the Caenorhabditis elegans hydroxylase, CLK-1, result in elevated mtDNA. CLK-1’s canonical role in ubiquinone biosynthesis results in clk-1 mutants lacking ubiquinone. Importantly, clk-1 mutants also exhibit slowed biological timing phenotypes (pharyngeal pumping, defecation, development) and an activated stress response (UPRmt). These biological timing and stress phenotypes have been attributed to ubiquinone deficiency; however, it is unknown whether the mtDNA phenotype is also due to ubiquinone deficiency. To test this, in animals carrying the uncharacterized clk-1 (ok1247) mutant allele, we supplemented with an exogenous ubiquinone precursor 2-4-dihydroxybenzoate (DHB), which has previously been shown to restore ubiquinone biosynthesis. We measured phenotypes as a function of DHB across a log-scale range. Unlike the biological timing and stress phenotypes, the elevated mtDNA phenotype was not rescued. Since CLK-1’s canonical role is in ubiquinone biosynthesis and DHB does not rescue mtDNA copy number, we infer CLK-1 has an additional function in homeostatic mtDNA copy number regulation.

Early Exposure is Necessary for the Lifespan Extension Effects of Cocoa in C. elegans.

Background:We previously showed that cocoa, a rich source of polyphenols improved the age-associated health and extended the lifespan in C. elegans when supplemented starting from L1 stage.Aim:In this study, we aimed to find out the effects of timing of cocoa exposure on longevity improving effects and the mechanisms and pathways involved in lifespan extension in C. elegans.Methods:The standard E. coli OP50 diet of wild type C. elegans was supplemented with cocoa powder starting from different larval stages (L1, L2, L3, and L4) till the death, from L1 to adult day 1 and from adult day 1 till the death. For mechanistic studies, different mutant strains of C. elegans were supplemented with cocoa starting from L1 stage till the death. Survival curves were plotted, and mean lifespan was reported.Results:Cocoa exposure starting from L1 stage till the death and till adult day 1 significantly extended the lifespan of worms. However, cocoa supplementation at other larval stages as well as at adulthood could not extend the lifespan, instead the lifespan was significantly reduced. Cocoa could not extend the lifespan of daf-16, daf-2, sir-2.1, and clk-1 mutants.Conclusion:Early-start supplementation is essential for cocoa-mediated lifespan extension which is dependent on insulin/IGF-1 signaling pathway and mitochondrial respiration.

Activation of AMPK/mTORC1-Mediated Autophagy by Metformin Reverses Clk1 Deficiency-Sensitized Dopaminergic Neuronal Death

The autophagy-lysosome pathway (ALP) plays a critical role in the pathology of Parkinson's disease (PD). Clk1 (coq7) is a mitochondrial hydroxylase that is essential for coenzyme Q (ubiquinone) biosynthesis. We have reported previously that Clk1 regulates microglia activation via modulating microglia metabolic reprogramming, which contributes to dopaminergic neuronal survival. This study explores the direct effect of Clk1 on dopaminergic neuronal survival. We demonstrate that Clk1 deficiency inhibited dopaminergic neuronal autophagy in cultured MN9D dopaminergic neurons and in the substantia nigra pars compacta of Clk+/- mutant mice and consequently sensitized dopaminergic neuron damage and behavioral defects. These mechanistic studies indicate that Clk1 regulates the AMP-activated protein kinase (AMPK)/rapamycin complex 1 pathway, which in turn impairs the ALP and TFEB nuclear translocation. As a result, Clk1 deficiency promotes dopaminergic neuronal damage in vivo and in vitro, which ultimately contributes to sensitizing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuronal death and behavioral impairments in Clk1-deficient mice. Moreover, we found that activation of autophagy by the AMPK activator metformin increases dopaminergic neuronal survival in vitro and in the MPTP-induced PD model in Clk1 mutant mice. These results reveal that Clk1 plays a direct role in dopaminergic neuronal survival via regulating ALPs that may contribute to the pathologic development of PD. Modulation of Clk1 activity may represent a potential therapeutic target for PD.

N-acetyl-l-cysteine mimics the effect of dietary restriction on lifespan and reduces amyloid beta-induced toxicity in Caenorhabditis elegans.

The effects of antioxidants on lifespan have been widely studied. Our previous study showed supplementation with N-acetyl-l-cysteine (NAC) extends the lifespan of Caenorhabditis elegans. Here we aimed to determine the lifespan-extending mechanism involved with NAC and the effect of NAC on Alzheimer's disease (AD). NAC further increased the lifespan of age-1 and clk-1 mutants, which have increased lifespan owing to reduced insulin/IGF-1-like signaling and mitochondrial function, respectively. There was no additional lifespan extension in eat-2 background, a genetic model of dietary restriction (DR), by NAC. Gene knockdown experiments revealed that the effect of NAC is not dependent on SKN-1, a protein-sensing DR status, whereas DAF-16, a transcription factor regulating stress-responsive genes, is required for lifespan extension by NAC. NAC delayed paralysis caused by amyloid beta. Our results show that NAC mimics the effect of DR on lifespan, possibly through the induction of DAF-16 nuclear localization and may retard the incidence of AD.

A single biochemical activity underlies the pleiotropy of the aging-related protein CLK-1

The Caenorhabditis elegans clk-1 gene and the orthologous mouse gene Mclk1 encode a mitochondrial hydroxylase that is necessary for the biosynthesis of ubiquinone (UQ). Mutations in these genes produce broadly pleiotropic phenotypes in both species, including a lengthening of animal lifespan. A number of features of the C. elegans clk-1 mutants, including a maternal effect, particularly extensive pleiotropy, as well as unexplained differences between alleles have suggested that CLK-1/MCLK1 might have additional functions besides that in UQ biosynthesis. In addition, a recent study suggested that a cryptic nuclear localization signal could lead to nuclear localization in cultured mammalian cell lines. Here, by using immunohistochemical techniques in worms and purification techniques in mammalian cells, we failed to detect any nuclear enrichment of the MCLK1 or CLK-1 proteins and any biological activity of a C. elegans CLK-1 protein devoid of a mitochondrial localization sequence. In addition, and most importantly, by pharmacologically restoring UQ biosynthesis in clk-1 null mutants we show that loss of UQ biosynthesis is responsible for all phenotypes resulting from loss of CLK-1, including behavioral phenotypes, altered expression of mitochondrial quality control genes, and lifespan.

Connecting Circadian Genes to Neurodegenerative Pathways in Fruit Flies

Connecting Circadian Genes to Neurodegenerative Pathways in Fruit Flies

Extension of the established period of diacetyl adaptation by oxygen intermediates in the nematode Caenorhabditis elegans

After pre-exposure to the odorant diacetyl, the nematode Caenorhabditis elegans showed a decline in chemotactic responses to diacetyl, a phenomenon known as diacetyl adaptation. In the present study, we found that the established period of diacetyl adaptation in nematodes increased with the breeding temperature. When wild-type (N2) nematodes were bred at 15°C, adaptation was observed from the young adult (YA) to the 3-day-old adult that is reached 3days after the YA stage. On breeding nematodes at 20°C and 25°C, adaptation was observed between the YA and 5-day-old adult and between the YA and the 7-day-old adult, respectively. Breeding temperature has been shown to correlate with the rate of aging in nematodes, which is related to the level of oxygen consumption. Accordingly, long-lived isp-1 and clk-1 mutants that demonstrate decreased levels of oxygen consumption showed a shorter established period of adaptation than N2 nematodes, whereas short-lived gas-1 and mev-1 mutants that have a hypersensitive response to oxygen showed a longer period of adaptation than the N2. Moreover, the established period of diacetyl adaptation in N2 nematodes was shortened by the antioxidant α-lipoic acid. These results suggest that oxygen intermediates, which are produced by oxygen consumption, play a significant role in diacetyl adaptation. Although this is only one of many factors that regulate diacetyl adaptation, such as the release of neurotransmitters and changes in intracellular conditions, the acquisition of this adaptation requires an increase in the intensity of moderate oxygen signals.

Mitochondrial and cytoplasmic ROS have opposing effects on lifespan.

Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms.

A Long Noncoding RNA on the Ribosome Is Required for Lifespan Extension

The biogenesis of ribosomes and their coordination of protein translation consume an enormous amount of cellular energy. As such, it has been established that the inhibition of either process can extend eukaryotic lifespan. Here, we used next-generation sequencing to compare ribosome-associated RNAs from normal strains of Caenorhabditis elegans to those carrying the life-extending daf-2 mutation. We found a long noncoding RNA (lncRNA), transcribed telomeric sequence 1 (tts-1), on ribosomes of the daf-2 mutant. Depleting tts-1 in daf-2 mutants increases ribosome levels and significantly shortens their extended lifespan. We find tts-1 is also required for the longer lifespan of the mitochondrial clk-1 mutants but not the feeding-defective eat-2 mutants. In line with this, the clk-1 mutants express more tts-1 and fewer ribosomes than the eat-2 mutants. Our results suggest that the expression of tts-1 functions in different longevity pathways to reduce ribosome levels in a way that promotes life extension.

Promotion of growth by Coenzyme Q10 is linked to gene expression in C. elegans

Coenzyme Q (CoQ, ubiquinone) is an essential component of the respiratory chain, a cofactor of pyrimidine biosynthesis and acts as an antioxidant in extra mitochondrial membranes. More recently CoQ has been identified as a modulator of apoptosis, inflammation and gene expression. CoQ deficient Caenorhabditis elegans clk-1 mutants show several phenotypes including a delayed postembryonic growth. Using wild type and two clk-1 mutants, here we established an experimental set-up to study the consequences of endogenous CoQ deficiency or exogenous CoQ supply on gene expression and growth. We found that a deficiency of endogenous CoQ synthesis down-regulates a cluster of genes that are important for growth (i.e., RNA polymerase II, eukaryotic initiation factor) and up-regulates oxidation reactions (i.e., cytochrome P450, superoxide dismutase) and protein interactions (i.e., F-Box proteins). Exogenous CoQ supply partially restores the expression of these genes as well as the growth retardation of CoQ deficient clk-1 mutants. On the other hand exogenous CoQ supply does not alter the expression of a further sub-set of genes. These genes are involved in metabolism (i.e., succinate dehydrogenase complex), cell signalling or synthesis of lectins. Thus, our work provides a comprehensive overview of genes which can be modulated in their expression by endogenous or exogenous CoQ. As growth retardation in CoQ deficiency is linked to the gene expression profile we suggest that CoQ promotes growth via gene expression.

TAF-4 is required for the life extension of isp-1, clk-1 and tpk-1 Mit mutants

While numerous life-extending manipulations have been discovered in the nematode Caenorhabditis elegans, one that remains most enigmatic is disruption of oxidative phosphorylation. In order to unravel how such an ostensibly deleterious manipulation can extend lifespan, we sought to identify the ensemble of nuclear transcription factors that are activated in response to defective mitochondrial electron transport chain (ETC) function. Using a feeding RNAi approach, we targeted over 400 transcription factors and identified 15 that, when reduced in function, reproducibly and differentially altered the development, stress response, and/or fecundity of isp-1(qm150) Mit mutants relative to wild-type animals. Seven of these transcription factors--AHA-1, CEH-18, HIF-1, JUN-1, NHR-27, NHR-49 and the CREB homolog-1 (CRH-1)-interacting protein TAF-4--were also essential for isp-1 life extension. When we tested the involvement of these seven transcription factors in the life extension of two other Mit mutants, namely clk-1(qm30) and tpk-1(qm162), TAF-4 and HIF-1 were consistently required. Our findings suggest that the Mit phenotype is under the control of multiple transcriptional responses, and that TAF-4 and HIF-1 may be part of a general signaling axis that specifies Mit mutant life extension.

The Role of Oxygen Intermediates in the Retention Time of Diacetyl Adaptation in the Nematode Caenorhabditis elegans

Continuous presentation of the odorant diacetyl to the nematode Caenorhabditis elegans causes a decrease in the level of chemotactic response to diacetyl. This decline in response is caused by diacetyl adaptation. When wild-type nematodes were maintained at 15°C after pre-exposure to diacetyl, diacetyl adaptation did not continue up to 2 hr. Adaptation continued up to 6 hr in nematodes bred at 20°C, and it continued beyond 12 hr in nematodes bred at 25°C. These results indicate that the retention time of diacetyl adaptation is dependent on the environmental breeding temperature and suggest that moderate oxygen signals are required for maintaining the attenuated response to diacetyl because of the correlation between breeding temperature and production of oxygen intermediates. When isp-1 and clk-1 mutants, which show reduced rates of oxygen intermediate production, were maintained at 20 and 25°C after pre-exposure to diacetyl, the mutants showed a shorter retention time of diacetyl adaptation compared with that of wild-type nematodes. When gas-1 and mev-1 mutants, which have a hypersensitive response to oxidative stress, were maintained at 15 and 20°C, they showed a longer retention time of adaptation, that is, adaptation continued beyond 2 and 12 hr, respectively. When wild-type nematodes were maintained on plates that included 0.05% α-lipoic acid, which suppresses production of oxygen intermediates, the retention time of adaptation did not continue up to 6 hr in nematodes bred at 20°C and up to 12 hr in nematodes bred at 25°C. These results support the possibility that oxygen intermediates contribute to retention time for diacetyl adaptation in the nematode C. elegans.

Neuropeptide Signaling: From the Gut

In the defecation motor program of Caenorhabditis elegans, a pacemaker rhythm generated by the intestine leads to the activation of motor neurons controlling enteric muscle contraction. A new study demonstrates that this signal is conveyed by a neuropeptide that is released from intestinal cells and acutely depolarizes the motorneurons, acting much like a classical neurotransmitter.

A MAP kinase gene,Clk1, is required for conidiation and pathogenicity in the phytopathogenic fungusCurvularia lunata

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways, which play a wide variety of important roles in extracellular signal transduction. The first MAPK gene of the maize pathogen Curvularia lunata, Clk1, was isolated via a PCR-based approach with a primer pair designed on the basis of conserved regions of known MAPKs. Southern blot analysis showed that the gene existed in the genome as a single copy. The predicted amino acid sequence (352 amino acids) was highly homologous with MAP kinases of other phytopathogenic fungi. Flanking regions of Clk1 were obtained through RACE and genomic walking technology. To understand the role of Clk1 in C. lunata, targeted gene disruption was adopted to construct Clk1 mutants. It was found that mutants lacking functional domain of Clk1 were not able to produce conidia but tended to form a few special chlamydospore-shaped structures. Clk1 mutants grew slower in adverse environments (at 24°C), produced less cell degrading enzymes (CWDEs) than the wild type, and they were almost unable to infect maize leaves via artificial wounds.

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5′ and 3′, respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q10. Here we show that the Q9 content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q9 content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.

Dopamine acts through Cryptochrome to promote acute arousal in Drosophila

The fruit fly, Drosophila melanogaster, is generally diurnal, but a few mutant strains, such as the circadian clock mutant Clk(Jrk), have been described as nocturnal. We report here that increased nighttime activity of Clk mutants is mediated by high levels of the circadian photoreceptor CRYPTOCHROME (CRY) in large ventral lateral neurons (l-LN(v)s). We found that CRY expression is also required for nighttime activity in mutants that have high dopamine signaling. In fact, dopamine signaling is elevated in Clk(Jrk) mutants and acts through CRY to promote the nocturnal activity of this mutant. Notably, dopamine and CRY are required for acute arousal upon sensory stimulation. Because dopamine signaling and CRY levels are typically high at night, this may explain why a chronic increase in levels of these molecules produces sustained nighttime activity. We propose that CRY has a distinct role in acute responses to sensory stimuli: (1) circadian responses to light, as previously reported, and (2) noncircadian effects on arousal, as shown here.

Mitochondrial Oxidative Stress Alters a Pathway in Caenorhabditis elegans Strongly Resembling That of Bile Acid Biosynthesis and Secretion in Vertebrates

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.

Restoration of the behavioral rates and lifespan in clk-1 mutant nematodes in response to exogenous coenzyme Q10

The clk-1 gene encodes demethoxyubiquinone mono-oxygenase that is necessary for the biosynthesis of coenzyme Q (CoQ), which is an electron transporter in the respiratory chain of mitochondria. Therefore, clk-1 mutant nematodes that have loss-of-function mutations in the clk-1 gene lack endogenous CoQ9 and exhibit slowed behavioral rates and an extended lifespan compared with wild-type animals when they are fed standard bacteria containing endogenous CoQ8. This finding suggests that clk-1 regulates behavioral rates and the lifespan through CoQ in nematodes; however, the effects of exogenous CoQ on the regulation of these biological processes have been incompletely evaluated. In this study, we found that adding 10μM water-soluble CoQ10 to the culture medium of clk-1 mutant nematodes that were fed a diet of standard bacteria restored the pharyngeal pumping, defecation and the lifespan to levels that were comparable to those of wild-type animals. The results indicate that both behavioral rates and lifespan are regulated by the clk-1 gene through the action of CoQ in nematodes.

Phylogenetic ubiquity of the effects of altered ubiquinone biosynthesis on survival

The widespread use of unicellular and invertebrate model systems has revealed that the molecular mechanisms underlying cellular functions are exceedingly well conserved. The expanding research into the molecular mechanisms of aging keeps yielding this answer too [1, 2]. A perfect example of this is a study by Gonidakis, Finkel and Longo appearing in the present issue of Aging, which shows that a disruption of the biosynthesis of ubiquinone leads to increased survival of E. coli during stationary phase, associated with an increased resistance to treatments that induce oxidative stress [3]. These effects are dependent upon the presence of an intact arcA gene, which encodes the regulatory component of the ArcA/ArcB system, a hypoxia-inducible system of transcriptional regulation. To understand why these findings might be an example of evolutionary conservation a little background is needed. Interventions that disrupt mitochondrial function can increase lifespan in a variety of organisms including yeast [4], Caenorhabditis elegans[5-10], Drosophila [11], and mice [12-14]. One type of intervention is the disruption of mitochondrial function through the reduction of the expression of mitochondrial genes by RNAi, which increases lifespan in worms [5, 10] and in flies [11], possibly via a mitochondria-specific stress response [9]. Another type of intervention is a specific alteration of mitochondrial electron transport that alters the generation of reactive oxygen species (ROS) [15, 16]. For example, C. elegans isp-1 and nuo-6 mutants, which carry point mutations in subunits of the mitochondrial electron transport chain (ETC) display an elevated generation of mitochondrial superoxide which appears to be causal to their increased lifespan. Indeed, antioxidants suppress the mutants’ longevity and pro-oxidant treatment of the wild type phenocopies it [16]. clk-1 (called Mclk1 in mice) is another gene that has been studied in this context. It encodes a mitochondrial hydroxylase that is necessary for the biosynthesis of ubiquinone [6, 17, 18]. Ubiquinone (a.k.a. co-enzyme Q) is an electron transporter and antioxidant that is ubiquitous in the membranes of all organisms [19]. C. elegans clk-1 mutants [20] and mouse Mclk1+/− mutants [12, 13] are long-lived and have been shown to have elevated generation of mitochondrial ROS [16, 21]. In cultured vertebrate cells mitochondrial ROS have been shown to help stabilize and thus induce the protective activity of the hypoxia-inducible factor 1α (HIF-1α) [22-24]. It is striking therefore that HIF-1α has been tentatively implicated in the mechanisms of longevity of both C. elegans clk-1 [25] and mouse Mclk1+/− mutants [26]. Of course bacteria have no mitochondria; however, as the evolutionary ancestors of mitochondria, they have a plasma membrane ETC partly homologous to that of the organelle. As in mitochondria, the bacterial ETC appears to produce significant amounts of ROS [27]. Like eukaryotic cells, they have transcription factors sensitive to hypoxia. The ArcA/ArcB two-component system is one of the key pathways up-regulated in response to anaerobic conditions. Although not genetically homologous to eukaryotic HIF-1α, there are interesting parallels between the systems. While activated by the redox state of the bacterial quinone pool rather than by ROS [28-30], Arc activation is required for the resistance of E. coli to induced oxidative stress [31]. Thus, the work of Longo and co-workers in E. coli suggests that there might be a truly universal link between ubiquinone, ROS generation, hypoxia-sensitive transcription factors and cellular survival.