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.

The mitochondrial genome of Physarum polycephalum encodes five tRNAs, four of which are edited by nucleotide insertion. Two of these tRNAs, tRNA met1 and tRNA met2 , contain predicted mismatches at the beginning (proximal end) of the acceptor stem. In addition, the putative 59 end of tRNA met2 overlaps the 39 end of a small, abundant, noncoding RNA, which we term ppoRNA. These anomalies led us to hypothesize that these two Physarum mitochondrial tRNAs undergo additional editing events. Here, we show that tRNA met1 and tRNA met2 each has a nonencoded G at its 59 end. In contrast to the other nucleotides that are added to Physarum mitochondrial RNAs, these extra G residues are likely added post-transcriptionally based on (1) the absence of added G in precursor transcripts containing inserted C and AA residues, (2) the presence of potential intermediates characteristic of 59 replacement editing, and (3) preferential incorporation of GTP into tRNA molecules under conditions that do not support transcription. This is the first report of both post-transcriptional nucleotide insertions and the addition of single Gs in P. polycephalum mitochondrial transcripts. We postulate that tRNA met1 and tRNA met2 are acted upon by an activity similar to that present in the mitochondria of certain other amoebozoons and chytrid fungi, suggesting that enzymes that repair the 59 end of tRNAs may be widespread.

Summary Volatile anesthetics (VAs) cause profound neurological effects, including reversible loss of consciousness and immobility. Despite their widespread use, the mechanism of action of VAs remains one of the unsolved puzzles of neuroscience [1,2]. Genetic studies in C. elegans [3, 4], Drosophila [3,5], and mice [6–9] indicate that ion channels controlling the neuronal resting membrane potential (RMP) also control anesthetic sensitivity. Leak channels selective for K+ [10–13] or permeable to Na+ [14], are critical for establishing RMP. We hypothesized that halothane, a VA, caused immobility by altering the neuronal RMP. In C. elegans, halothane induced immobility is acutely and completely reversed by channelrhodopsin-2 based depolarization of the RMP when expressed specifically in cholinergic neurons. Furthermore, hyperpolarizing cholinergic neurons via halorhodopsin activation increases sensitivity to halothane. The sensitivity of C. elegans to halothane can be altered by 25-fold by either manipulation of membrane conductance with optogenetic methods or generation of mutations in leak channels that set the RMP. Immobility induced by another VA, isoflurane, is not affected by these treatments, thereby excluding the possibility of non-specific hyperactivity. The sum of our data indicates that leak channels and the RMP are important determinants of halothane-induced general anesthesia.

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ;9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.