Matricide in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> as an example of programmed death of an animal organism: The role of mitochondrial oxidative stress

Nematode Caenorhabditis elegans is a widely used model for studying genetic and molecular mechanisms of lifespan regulation. The choice between two life strategies—normal aging and matricide (programmed death)-made by an adult hermaphroditic C. elegans organism is based on food availability and is also affected by different kinds of stress. We have tested a hypothesis concerning an increase in matricide probability as a result of oxidative stress; this hypothesis is based on the phenoptosis theory. High concentrations of paraquat, a strong mitochondrial stressor, were shown to increase matricide propensity. Mutants with a decreased antioxidant potential of mitochondria (nnt) were more sensitive to the reagent. On the other hand, paraquat concentrations required for this effect to be observed are toxic for the progeny, whereas at low paraquat concentration matricide frequency is the same in wild-type worms and nnt mutants. Therefore, one can assume that the molecular mechanisms of matricide initiation under normal conditions are not necessarily connected to mitochondrial oxidative stress.     

Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long‐lived animals

Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (López‐Otín et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematode Caenorhabditis elegans also exhibits age‐dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role in C. elegans aging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age‐dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age‐dependent functional decline commonly observed in C. elegans. Computational modeling of mtDNA dynamics in C. elegans indicates that the lifespan of short‐lived animals such as C. elegans is likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long‐lived animals such as humans and rhesus monkey and possibly in rodents.     

Reproductive fitness and dietary choice behavior of the genetic model organism Caenorhabditis elegans under semi-natural conditions

Laboratory breeding conditions of the model organism C. elegans do not correspond with the conditions in its natural soil habitat. To assess the consequences of the differences in environmental conditions, the effects of air composition, medium and bacterial food on reproductive fitness and/or dietary-choice behavior of C. elegans were investigated. The reproductive fitness of C. elegans was maximal under oxygen deficiency and not influenced by a high fractional share of carbon dioxide. In media approximating natural soil structure, reproductive fitness was much lower than in s tandard laboratory media. I n seminatural media, the reproductive fitness of C. elegans was low with the standard laboratory food bacterium E. coli (γ-Proteobacteria), but significantly higher with C. arvensicola (Bacteroidetes) and B. tropica (β-Proteobacteria) as food. Dietary-choice experiments in semi-natural media revealed a low preference of C. elegans for E. coli but significantly higher preferences for C. arvensicola and B. tropica (among other bacteria). Dietary-choice experiments under quasi-natural conditions, which were feasible by fluorescence in situ hybridization (FISH) of bacteria, showed a high preference of C. elegans for Cytophaga-Flexibacter-Bacteroides, Firmicutes, and β-Proteobacteria, but a low preference for γ-Proteobacteria. The results show that data on C. elegans under standard laboratory conditions have to be carefully interpreted with respect to their biological significance.     

Autophagy activity contributes to programmed cell death in Caenorhabditis elegans

The physiological relationship between autophagy and programmed cell death during C. elegans development is poorly understood. In C. elegans, 131 somatic cells and a large number of germline cells undergo programmed cell death. Autophagy genes function in the removal of somatic cell corpses during embryogenesis. Here we demonstrated that autophagy activity participates in germ-cell death induced by genotoxic stress. Upon γ ray treatment, fewer germline cells execute the death program in autophagy mutants. Autophagy also contributes to physiological germ-cell death and post-embryonic cell death in ventral cord neurons when ced-3 caspase activity is partially compromised. Our study reveals that autophagy activity contributes to programmed cell death during C. elegans development.     

Grooming behaviors of black‐tailed prairie dogs are influenced by flea parasitism,conspecifics, and proximity to refuge

Grooming is a common animal behavior that aids in ectoparasite defense. Ectoparasites can stimulate grooming, and natural selection can also favor endogenous mechanisms that evoke periodic bouts of “programmed” grooming to dislodge or kill ectoparasites before they bite or feed. Moreover, grooming can function as a displacement or communication behavior. We compared the grooming behaviors of adult female black‐tailed prairie dogs (Cynomys ludovicianus) on colonies with or without flea control via pulicide dust. Roughly 91% of the prairie dogs sampled on the non‐dusted colony carried at least one flea, whereas we did not find fleas on two dusted colonies. During focal observations, prairie dogs on the non‐dusted colony groomed at higher frequencies and for longer durations than prairie dogs on the dusted colonies, lending support to the hypothesis that fleas stimulated grooming. However, the reduced amount of time spent grooming on the dusted colonies suggested that approximately 25% of grooming might be attributed to factors other than direct stimulation from ectoparasites. Non‐dusted colony prairie dogs rarely autogroomed when near each other. Dusted colony prairie dogs autogroomed for shorter durations when far from a burrow opening (refuge), suggesting a trade‐off between self‐grooming and antipredator defense. Allogrooming was detected only on the non‐dusted colony and was limited to adult females grooming young pups. Grooming appears to serve an antiparasitic function in C. ludovicianus. Antiparasitic grooming might aid in defense against fleas that transmit the plague bacterium Yersinia pestis. Plague was introduced to North America ca. 1900 and now has a strong influence on most prairie dog populations, suggesting a magnified effect of grooming on prairie dog fitness.     

A deuterohemin peptide extends lifespan and increases stress resistance in Caenorhabditis elegans

Abstract

This group has invented a novel deuterohemin containing peptide deuterohemin-AlaHisThrValGluLys (DhHP-6), which has various biological activities including protection of murine ischemia reperfusion injury, improving cell survival and preventing apoptosis. It was hypothesized that DhHP-6 is beneficial on the lifespan of Caenorhabditis elegans (C. elegans) and increases their resistance to heat and oxidative stress. C. elegans were treated with different concentrations of DhHP-6. Survival time and sensitivity to heat and paraquat were investigated. The data demonstrated that the mean survival time of C. elegans was significantly increased (p < 0.05) in the DhHP-6 treated group compared with the control group. The maximum lifespan was not affected by DhHP-6 treatment. DhHP-6 improved the survival rate of C. elegans in the acute heat stress (35°C) and rescued the C. elegans' sensitivity to paraquat in acute oxidative stress. Superoxide dismutase 3 (SOD-3) protein was up-regulated by DhHP-6 treatment. It was further demonstrated that stress resistance genes such as hsp-16.1, hsp-16.49 and sir-2.1 were regulated by DhHP-6. DAF-16 and SIR-2.1 genes are essential for the beneficial effect of DhHP-6. Therefore, the investigation into the beneficial effect of DhHP-6 on C. elegans' lifespan has the potential to develop novel drugs to prevent ageing.     

A pharmacological network for lifespan extension in Caenorhabditis elegans

One goal of aging research is to find drugs that delay the onset of age‐associated disease. Studies in invertebrates, particularly Caenorhabditis elegans, have uncovered numerous genes involved in aging, many conserved in mammals. However, which of these encode proteins suitable for drug targeting is unknown. To investigate this question, we screened a library of compounds with known mammalian pharmacology for compounds that increase C. elegans lifespan. We identified 60 compounds that increase longevity in C. elegans, 33 of which also increased resistance to oxidative stress. Many of these compounds are drugs approved for human use. Enhanced resistance to oxidative stress was associated primarily with compounds that target receptors for biogenic amines, such as dopamine or serotonin. A pharmacological network constructed with these data reveal that lifespan extension and increased stress resistance cluster together in a few pharmacological classes, most involved in intercellular signaling. These studies identify compounds that can now be explored for beneficial effects on aging in mammals, as well as tools that can be used to further investigate the mechanisms underlying aging in C. elegans.     

Genetic interaction with temperature is an important determinant of nematode longevity

As in other poikilotherms, longevity in C. elegans varies inversely with temperature; worms are longer‐lived at lower temperatures. While this observation may seem intuitive based on thermodynamics, the molecular and genetic basis for this phenomenon is not well understood. Several recent reports have argued that lifespan changes across temperatures are genetically controlled by temperature‐specific gene regulation. Here, we provide data that both corroborate those studies and suggest that temperature‐specific longevity is more the rule than the exception. By measuring the lifespans of worms with single modifications reported to be important for longevity at 15, 20, or 25 °C, we find that the effect of each modification on lifespan is highly dependent on temperature. Our results suggest that genetics play a major role in temperature‐associated longevity and are consistent with the hypothesis that while aging in C. elegans is slowed by decreasing temperature, the major cause(s) of death may also be modified, leading to different genes and pathways becoming more or less important at different temperatures. These differential mechanisms of age‐related death are not unlike what is observed in humans, where environmental conditions lead to development of different diseases of aging.     

Beneficial and harmful effects of alcohol exposure on Caenorhabditis elegans worms

Alcoholic beverages are consumed widely throughout the world. While the harmful effects of alcoholism are well recognized, the beneficial effects of moderate alcohol consumption to human health remain debatable. In this study, we investigated the effects of long-term ethanol exposure on nematode Caenorhabditis elegans worms. At high concentrations (?4%), ethanol significantly impaired mobility, reduced fertility, and shortened lifespan. Interestingly, at low concentrations (1–2%), it extended lifespan, accompanied with a slower decline of mobility during aging, although it slightly impaired development, fertility, and chemotaxis. The lifespan-prolonging effects of ethanol at the low concentrations were seen in normal worms exposed to ethanol from egg, young larva, and young adult stages but were not observed in age-1 and sir-2.1 mutant worms. Our study demonstrated hormetic effects of ethanol and further established C. elegans as a suitable animal model to study ethanol related problems.     

Age‐dependent changes and biomarkers of aging in Caenorhabditis elegans

Caenorhabditis elegans is an exceptionally valuable model for aging research because of many advantages, including its genetic tractability, short lifespan, and clear age‐dependent physiological changes. Aged C. elegans display a decline in their anatomical and functional features, including tissue integrity, motility, learning and memory, and immunity. Caenorhabditis elegans also exhibit many age‐associated changes in the expression of microRNAs and stress‐responsive genes and in RNA and protein quality control systems. Many of these age‐associated changes provide information on the health of the animals and serve as valuable biomarkers for aging research. Here, we review the age‐dependent changes in C. elegans and their utility as aging biomarkers indicative of the physiological status of aging.     

Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.     

Caenorhabditis elegans orthologs of human genes differentially expressed with age are enriched for determinants of longevity

We report a systematic RNAi longevity screen of 82 Caenorhabditis elegans genes selected based on orthology to human genes differentially expressed with age. We find substantial enrichment in genes for which knockdown increased lifespan. This enrichment is markedly higher than published genomewide longevity screens in C. elegans and similar to screens that preselected candidates based on longevity‐correlated metrics (e.g., stress resistance). Of the 50 genes that affected lifespan, 46 were previously unreported. The five genes with the greatest impact on lifespan (>20% extension) encode the enzyme kynureninase (kynu‐1), a neuronal leucine‐rich repeat protein (iglr‐1), a tetraspanin (tsp‐3), a regulator of calcineurin (rcan‐1), and a voltage‐gated calcium channel subunit (unc‐36). Knockdown of each gene extended healthspan without impairing reproduction. kynu‐1(RNAi) alone delayed pathology in C. elegans models of Alzheimer's disease and Huntington's disease. Each gene displayed a distinct pattern of interaction with known aging pathways. In the context of published work, kynu‐1, tsp‐3, and rcan‐1 are of particular interest for immediate follow‐up. kynu‐1 is an understudied member of the kynurenine metabolic pathway with a mechanistically distinct impact on lifespan. Our data suggest that tsp‐3 is a novel modulator of hypoxic signaling and rcan‐1 is a context‐specific calcineurin regulator. Our results validate C. elegans as a comparative tool for prioritizing human candidate aging genes, confirm age‐associated gene expression data as valuable source of novel longevity determinants, and prioritize select genes for mechanistic follow‐up.     

New label‐free automated survival assays reveal unexpected stress resistance patterns during C. elegans aging

Caenorhabditis elegans is an excellent model for high‐throughput experimental approaches but lacks an automated means to pinpoint time of death during survival assays over a short time frame, that is, easy to implement, highly scalable, robust, and versatile. Here, we describe an automated, label‐free, high‐throughput method using death‐associated fluorescence to monitor nematode population survival (dubbed LFASS for label‐free automated survival scoring), which we apply to severe stress and infection resistance assays. We demonstrate its use to define correlations between age, longevity, and severe stress resistance, and its applicability to parasitic nematodes. The use of LFASS to assess the effects of aging on susceptibility to severe stress revealed an unexpected increase in stress resistance with advancing age, which was largely autophagy‐dependent. Correlation analysis further revealed that while severe thermal stress resistance positively correlates with lifespan, severe oxidative stress resistance does not. This supports the view that temperature‐sensitive protein‐handling processes more than redox homeostasis underpin aging in C. elegans. That the ages of peak resistance to infection, severe oxidative stress, heat shock, and milder stressors differ markedly suggests that stress resistance and health span do not show a simple correspondence in C. elegans.     

<Emphasis Type="Italic">C. elegans</Emphasis> behavior of preference choice on bacterial food

Caenorhabditis elegans is a free living soil nematode and thus in its natural habitat, C. elegans encounters many different species of soil bacteria. Although some soil bacteria may be excellent sources of nutrition for the worm, others may be pathogenic. Thus, we undertook a study to understand how C. elegans can identify their preferred food using a simple behavioral assay. We found that there are various species of soil bacteria that C. elegans prefers in comparison to the standard laboratory E. coli strain OP50. In particular, two bacterial strains, Bacillus mycoides and Bacillus soli, were preferred strains. Interestingly, the sole feeding of these bacteria to wild type animals results in extended lifespan through the activation of the autophagic process. Further studies will be required to understand the precise mechanism controlling the behavior of identification and selection of food in C. elegans.