Robyn Branicky

clk-1, mitochondria, and physiological rates.

Mutations in the C. elegans maternal-effect gene clk-1 are highly pleiotropic, affecting the duration of diverse developmental and behavioral processes. They result in an average slowing of embryonic and post-embryonic development, adult rhythmic behaviors, reproduction, and aging.(1) CLK-1 is a highly conserved mitochondrial protein,(2,3) but even severe clk-1 mutations affect mitochondrial respiration only slightly.(3) Here, we review the evidence supporting the regulatory role of clk-1 in physiological timing. We also discuss possible models for the action of CLK-1, in particular, one proposing that CLK-1 is involved in the coordination of mitochondrial and nuclear function. BioEssays 22:48-56, 2000.

The Anti-neurodegeneration Drug Clioquinol Inhibits the Aging-associated Protein CLK-1

The development of neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington disease is strongly age-dependent. Discovering drugs that act on the high rate of aging in older individuals could be a means of combating these diseases. Reduction of the activity of the mitochondrial enzyme CLK-1 (also known as COQ7) slows down aging in Caenorhabditis elegans and in mice. Clioquinol is a metal chelator that has beneficial effects in several cellular and animal models of neurodegenerative diseases as well as on Alzheimer disease patients. Here we show that clioquinol inhibits the activity of mammalian CLK-1 in cultured cells, an inhibition that can be blocked by iron or cobalt cations, suggesting that chelation is involved in the mechanism of action of clioquinol on CLK-1. We also show that treatment of nematodes and mice with clioquinol mimics a variety of phenotypes produced by mutational reduction of CLK-1 activity in these organisms. These results suggest that the surprising action of clioquinol on several age-dependent neurodegenerative diseases with distinct etiologies might result from a slowing down of the aging process through action of the drug on CLK-1. Our findings support the hypothesis that pharmacologically targeting aging-associated proteins could help relieve age-dependent diseases.

Why only time will tell

The nematode Caenorhabditis elegans has become a model system for the study of the genetic basis of aging. In particular, many mutations that extend life span have been identified in this organism. When loss-of-function mutations in a gene lead to life span extension, it is a necessary conclusion that the gene normally limits life span in the wild type. The effect of a given mutation depends on a number of environmental and genetic conditions. For example, the combination of two mutations can result in additive, synergistic, subtractive, or epistatic effects on life span. Valuable insight into the processes that determine life span can be obtained from such genetic analyses, especially when interpreted with caution, and when molecular information about the interacting genes is available. Thus, genetic and molecular analyses have implicated several genes classes (daf, clk and eat) in life span determination and have indicated that aging is affected by alteration of several biological processes, namely dormancy, physiological rates, food intake, and reproduction.

Uncoupling the Pleiotropic Phenotypes of clk-1 with tRNA Missense Suppressors in Caenorhabditis elegans

ABSTRACT 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.

Lipid Transport and Signaling in Caenorhabditis elegans

The strengths of the Caenorhabditis elegans model have been recently applied to the study of the pathways of lipid storage, transport, and signaling. As the lipid storage field has recently been reviewed, in this minireview we (1) discuss some recent studies revealing important physiological roles for lipases in mobilizing lipid reserves, (2) describe various pathways of lipid transport, with a particular focus on the roles of lipoproteins, (3) debate the utility of using C. elegans as a model for human dyslipidemias that impinge on atherosclerosis, and (4) describe several systems where lipids affect signaling, highlighting the particular properties of lipids as information‐carrying molecules. We conclude that the study of lipid biology in C. elegans exemplifies the advantages afforded by a whole‐animal model system where interactions between tissues and organs, and functions such as nutrient absorption, distribution, and storage, as well as reproduction can all be studied simultaneously. Developmental Dynamics 239:1365–1377, 2010.

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.

Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling

Superoxide dismutases (SODs) are universal enzymes of organisms that live in the presence of oxygen. They catalyze the conversion of superoxide into oxygen and hydrogen peroxide. Superoxide anions are the intended product of dedicated signaling enzymes as well as the byproduct of several metabolic processes including mitochondrial respiration. Through their activity, SOD enzymes control the levels of a variety of reactive oxygen species (ROS) and reactive nitrogen species, thus both limiting the potential toxicity of these molecules and controlling broad aspects of cellular life that are regulated by their signaling functions. All aerobic organisms have multiple SOD proteins targeted to different cellular and subcellular locations, reflecting the slow diffusion and multiple sources of their substrate superoxide. This compartmentalization also points to the need for fine local control of ROS signaling and to the possibility for ROS to signal between compartments. In this review, we discuss studies in model organisms and humans, which reveal the dual roles of SOD enzymes in controlling damage and regulating signaling.

Evolutionary conservation of drug action on lipoprotein metabolism-related targets.

Genetic analysis has shown that the slower than normal rhythmic defecation behavior of the clk-1 mutants of Caenorhabditis elegans is the result of altered lipoprotein metabolism. We show here that this phenotype can be suppressed by drugs that affect lipoprotein metabolism, including drugs that affect HMG-CoA reductase activity, reverse cholesterol transport, or HDL levels. These pharmacological effects are highly specific, as these drugs affect defecation only in clk-1 mutants and not in the wild-type and do not affect other behaviors of the mutants. Furthermore, drugs that affect processes not directly related to lipid metabolism show no or minimal activity. Based on these findings, we carried out a compound screen that identified 190 novel molecules that are active on clk-1 mutants, 15 of which also specifically decrease the secretion of apolipoprotein B (apoB) from HepG2 hepatoma cells. The other 175 compounds are potentially active on lipid-related processes that cannot be targeted in cell culture. One compound, CHGN005, was tested and found to be active at reducing apoB secretion in intestinal Caco-2 cells as well as in HepG2 cells. This compound was also tested in a mouse model of dyslipidemia and found to decrease plasma cholesterol and triglyceride levels. Thus, target processes for pharmacological intervention on lipoprotein synthesis, transport, and metabolism are conserved between nematodes and vertebrates, which allows the use of C. elegans for drug discovery.

Specification of muscle neurotransmitter sensitivity by a Paired-like homeodomain protein in Caenorhabditis elegans

The effects of neurotransmitters depend on the receptors expressed on the target cells. In Caenorhabditis elegans, there are two types of GABA receptors that elicit opposite effects: excitatory receptors that open cation-selective channels, and inhibitory receptors that open anion-selective channels. The four non-striated enteric muscle cells required for the expulsion step of the defecation behavior are all sensitive to GABA: the sphincter muscle expresses a classical GABA-sensitive chloride channel (UNC-49) and probably relaxes in response to GABA, while the other three cells express a cation-selective channel (EXP-1) and contract. Here we show that the expression of the exp-1 gene is under the control of dsc-1, which encodes a Paired-like homeodomain protein, a class of transcription factors previously associated with the terminal differentiation of neurons in C. elegans. dsc-1 mutants have anatomically normal enteric muscles but are expulsion defective. We show that this defect is due to the lack of expression of exp-1 in the three cells that contract in response to GABA. In addition, dsc-1, but not exp-1, affects the periodicity of the behavior, revealing an unanticipated role for the enteric muscles in regulating this ultradian rhythm.

Making a splash with splicing

In a recent Nature paper, Heintz et al. identify a splicing factor (SFA-1) that is crucial for the longevity conferred by dietary restriction and the TORC1 pathway modulation in C. elegans.