Reactive oxygen species (ROS) are produced by a variety of processes in all cellular compartments. The best known ROS are superoxide and hydrogen peroxide, which are toxic agents that damage macromolecules and have been implicated in a number of diseases and in aging. ROS are detoxified by specialized enzymes specific to each cellular compartment. For example, there are mitochondrial, cytoplasmic and extracellular superoxide dismutases. Numerous in vitro studies have shown that ROS also affect gene expression via effects on components of signal transduction pathways and via effects on ROS-sensitive transcription factors. In addition, some ROS-damaged macromolecules affect signal transduction via specialized receptors (e.g. oxidized low density lipoproteins). We have been studying mutants with slow physiological rates, including slow embryonic and post-embryonic development, slow behaviors, slow reproduction and an increased lifespan. The molecular identity of the mutant genes frequently suggested that they might alter ROS metabolism. For example,
isp-1 encodes a subunit of mitochondrial complex III, and
clk-1 encodes an enzyme that is necessary for the biosynthesis of ubiquinone, a redox active lipid that is involved in the production of most cellular superoxide. We have shown that several of the phenotypes of
clk-1 mutants can be suppressed by disrupting the expression of enzymes that detoxify ROS. For example, RNAi knockdown of SOD-1, the cytoplasmic SOD, suppresses the slow embryonic, post-embryonic and germline development of
clk-1 mutants but not the slow defecation cycle. In contrast, a knockout mutation of
sod-4, which encodes an extracellular SOD, does suppress the slow defecation cycle. As the only known function of the SODs is the detoxification of superoxide, these findings indicate that the phenotype of
clk-1 mutants is due to low levels of superoxide and that superoxide regulates physiological rates in C. elegans. We have further extended these findings by showing that the effect of the ras pathway on vulva formation is modulated by ROS and that the oxidation of lipoproteins affected germline development via IP3 signaling. We are seeking to further determine the molecular nature as well as the cellular and sub-cellular origins of the ROS that affect physiological rates and aging in worms. For this we are characterizing more
clk-1-like and
isp-1-like mutants, as well as using suppressor mutations and RNAi against ROS-detoxifying enzymes. We are also attempting to show that the phenotypic effects of altered ROS production can be uncoupled from other effects of altered mitochondrial respiratory chain function.