Very low (nano- and subnanomolar) concentrations of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

The identity of neuronal cell types is established and maintained by the expression of neuronal genes coding for ion channels, neurotransmitters, and neuropeptides, among others. Some of these genes have been shown to affect lifespan; however, their role in lifespan control remains largely unclear. The Drosophila melanogaster gene Lim3 encodes a transcription factor involved in complicated motor neuron specification networks. We previously identified Lim3 as a candidate gene affecting lifespan. To obtain direct evidence of the involvement of Lim3 in lifespan control, Lim3 overexpression and RNAi knockdown were induced in the nervous system and muscles of Drosophila using the GAL4-UAS binary system. We demonstrated that Lim3 knockdown in the nervous system increased survival at an early age and that Lim3 knockdown in muscles both increased survival at an early age and extended median lifespan, directly establishing the involvement of Lim3 in lifespan control. Lim3 overexpression in nerves and muscles was deleterious and led to lethality and decreased lifespan, respectively. Lim3 misexpression in both nerves and muscles increased locomotion regardless of changes in lifespan, which indicated that the effects of Lim3 on lifespan and locomotion can be uncoupled. Decreased synaptic activity was observed in the neuromuscular junctions of individuals with Lim3 overexpression in muscles, in association with decreased lifespan. However, no changes in NMJ activity were associated with the positive shift in locomotion observed in all misexpression genotypes. Our data suggested that modifications in the microtubule network may be induced by Lim3 misexpression in muscles and cause an increase in locomotion.

Previously, extremely low, nanomolar concentrations of the mitochondria-targeted plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) were shown to prolong the lifespan of male and female Drosophila melanogaster by about 10 % (Anisimov et al., Biochemistry (Moscow) 73:1329-1342, 2008). Using long-term monitoring of SkQ1 effects on the Drosophila lifespan, we analyzed different integral parameters of Drosophila survival and mortality under SkQ1 treatment. Meta-analysis was used to evaluate the average SkQ1 effect measured in terms of standard deviation. The effect appeared to be 0.25 for females and 0.18 for males, which corresponds to a low effect by Cohen's "Rules-of-Thumb". The SkQ1 effects on the Drosophila lifespan were reproducible over six years and showed no relationship to fluctuations in the mean lifespan of the w(1118) line used in the experiments, methods of preparation and administration of the drug, seasons, or calendar years. Adding SkQ1 to fly food was associated with a reduction in early mortality and a decrease in random variation in lifespan. All survival curves were fitted by Gompertz function. Analysis of the Gompertz function parametric plane demonstrated significant differences between points corresponding to experimental and control cohorts. The Strehler-Mildvan correlations for 11 experiments with females and for 7 experiments with males were calculated. The significant increase in the slope of the regression lines indicated that feeding flies SkQ1 reduced the rate of fall of fly vitality and, consequently, slowed aging. These findings indicated that the SkQ1 effect on lifespan was associated with both elevation of life quality and slowing of aging.

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It was previously found that 20 pM SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) solution increased lifespan of virgin females and males of the fruit fly Drosophila melanogaster with maximal and highly reproducible effect on early survival of females. In this paper we demonstrate that SkQ1 solution of the same concentration does not increase lifespan of mated females and males, early effect on female survival being absent, whereas early fertility and the total number of progeny are elevated in treated flies. Increase in fertility observed in young mated females instead of increase of survival typical for young virgin females might illustrate the trade-off between the fly's lifespan and reproduction.

Lim3 encodes an RNA polymerase II transcription factor with a key role in neuron specification. It was also identified as a candidate gene that affects lifespan. These pleiotropic effects indicate the fundamental significance of the potential interplay between neural development and lifespan control. The goal of this study was to analyze the causal relationships between Lim3 structural variations, and gene expression and lifespan changes, and to provide insights into regulatory pathways controlling lifespan. Fifty substitution lines containing second chromosomes from a Drosophila natural population were used to analyze the association between lifespan and sequence variation in the 5′-regulatory region, and first exon and intron of Lim3A, in which we discovered multiple transcription start sites (TSS). The core and proximal promoter organization for Lim3A and a previously unknown mRNA named Lim3C were described. A haplotype of two markers in the Lim3A regulatory region was significantly associated with variation in lifespan. We propose that polymorphisms in the regulatory region affect gene transcription, and consequently lifespan. Indeed, five polymorphic markers located within 380 to 680 bp of the Lim3A major TSS, including two markers associated with lifespan variation, were significantly associated with the level of Lim3A transcript, as evaluated by real time RT-PCR in embryos, adult heads, and testes. A naturally occurring polymorphism caused a six-fold change in gene transcription and a 25% change in lifespan. Markers associated with long lifespan and intermediate Lim3A transcription were present in the population at high frequencies. We hypothesize that polymorphic markers associated with Lim3A expression are located within the binding sites for proteins that regulate gene function, and provide general rather than tissue-specific regulation of transcription, and that intermediate levels of Lim3A expression confer a selective advantage and longer lifespan.

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The nervous system has long been suggested as a key tissue that defines life span. The identity of neuronal cell types is established during development and maintained throughout adulthood due to the expression of specific neuronal genes coding for ion channels, neurotransmitters and neuropeptides, G-protein-coupled receptors, motor proteins, recognition and adhesion molecules. In this paper, we review data on the role of neuronal genes in Drosophila melanogaster life span control. Several pathways responsible for life span regulation are also important for the development of the nervous system. Genes involved in insulin-like, Target of Rapamycin, Janus Kinase/Signal Transducer and Activator of Transcription and cell polarity pathways, a number of global regulators and transcription factors play key roles both in aging and longevity control and in shaping the nervous system as a network of specialized neuronal cells in early development. Is their impact on life span related, at least partially, to their developmental functions or is it explained by other pleiotropic influences later in life? In this paper, we address this question based on the published data and our own findings.