The processes that control aging remain poorly understood. We have exploited mutants in the nematode, C. elegans, that compromise mitochondrial function and scavenging of reactive oxygen species (ROS) to understand their relation to lifespan. We discovered unanticipated roles and interactions of the mitochondrial superoxide dismutases (mtSODs), SOD-2 and SOD-3. Both SODs localize to mitochondrial supercomplex I:III:IV. Loss of SOD-2 specifically: 1) decreases the activities of complexes I and II; complexes III, and IV remain normal, 2) increases the lifespan of animals with a complex I defect, but not the lifespan of animals with a complex II defect, and kills an animal with a complex III defect, 3) induces a presumed pro-inflammatory response. Knockdown of a molecule that may be a pro-inflammatory mediator very markedly extends lifespan and health of certain mitochondrial mutants. The relationship between the electron transport chain, ROS and lifespan is complex, and defects in mitochondrial function have specific interactions with ROS scavenging mechanisms. We conclude that mtSODs are embedded within the supercomplex I:III:IV, and stabilize or locally protect it from reactive oxygen species (ROS) damage. The results call for a change in the usual paradigm for the interaction of electron transport chain function, ROS release, scavenging and compensatory responses.

Introduction Ubiquinone (UQ) is a redox active lipid that transfers electrons from complex I or II to complex III in the electron transport chain (ETC). The long-lived C. elegans mutant clk-1 is unable to synthesize its native ubiquinone, and accumulates high amounts of its precursor, 5-demethoxyubiquinone-9 (DMQ9). In clk-1, complex I-III activity is inhibited while complex II-III activity is normal. We asked whether the complex I-III defect in clk-1 was caused by: 1) a defect in the ETC; 2) an inhibitory effect of DMQ9; or 3) a decreased amount of ubiquinone. Methods We extracted the endogenous quinones from wildtype (N2) and clk-1 mitochondria, replenished them with exogenous ubiquinones, and measured ETC activities. Results Replenishment of extracted mutant and wildtype mitochondria resulted in equal enzymatic activities for complex I-III and II-III ETC assays. Blue native gels showed that supercomplex formation was indistinguishable between clk-1 and N2. Addition of a pentane extract from clk-1 mitochondria containing DMQ9 to wildtype mitochondria specifically inhibited complex I-III activity. UQ in clk-1 mitochondria was oxidized compared to N2. Discussion Our results show that no measurable intrinsic ETC defect exists in clk-1 mitochondria. The data indicate that DMQ9 specifically inhibits electron transfer from complex I to ubiquinone.

Introduction Neurotoxicity induced by early developmental exposure to volatile anesthetics is a characteristic of organisms across a wide range of species, extending from the nematode C. elegans to mammals. Prevention of anesthetic-induced neurotoxicity (AIN) will rely upon an understanding of its underlying mechanisms. However, no forward genetic screens have been undertaken to identify the critical pathways affected in AIN. By characterizing such pathways, we may identify mechanisms to eliminate isoflurane induced AIN in mammals. Methods Chemotaxis in adult C. elegans after larval exposure to isoflurane was used to measure AIN. We initially compared changes in chemotaxis indices between classical mutants known to affect nervous system development adding mutants in response to data. Activation of specific genes was visualized using fluorescent markers. Animals were then treated with rapamycin or preconditioned with isoflurane to test effects on AIN. Results Forty-four mutations, as well as pharmacologic manipulations, identified two pathways, highly conserved from invertebrates to humans, that regulate AIN in C. elegans. Activation of one stress-protective pathway (DAF-2 dependent) eliminates AIN, while activation of a second stress-responsive pathway (endoplasmic reticulum (ER) associated stress) causes AIN. Pharmacologic inhibition of the mechanistic Target of Rapamycin (mTOR) blocks ER-stress and AIN. Preconditioning with isoflurane prior to larval exposure also inhibited AIN. Discussion Our data are best explained by a model in which isoflurane acutely inhibits mitochondrial function causing activation of responses that ultimately lead to ER-stress. The neurotoxic effect of isoflurane can be completely prevented by manipulations at multiple points in the pathways that control this response. Endogenous signaling pathways can be recruited to protect organisms from the neurotoxic effects of isoflurane.