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Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Abstract Background Studies with the nematode model Caenorhabditis elegans have identified conserved biochemical pathways that act to modulate life span. Life span can also be influenced by the composition of the intestinal microbiome, and C. elegans life span can be dramatically influenced by its diet of Escherichia coli. Although C. elegans is typically fed the standard OP50 strain of E. coli, nematodes fed E. coli strains rendered respiratory deficient, either due to a lack coenzyme Q or the absence of ATP synthase, show significant life span extension. Here we explore the mechanisms accounting for the enhanced nematode life span in response to these diets. Results The intestinal load of E. coli was monitored by determination of worm-associated colony forming units (cfu/worm or coliform counts) as a function of age. The presence of GFP-expressing E. coli in the worm intestine was also monitored by fluorescence microscopy. Worms fed the standard OP50 E. coli strain have high cfu and GFP-labeled bacteria in their guts at the L4 larval stage, and show saturated coliform counts by day five of adulthood. In contrast, nematodes fed diets of respiratory deficient E. coli lacking coenzyme Q lived significantly longer and failed to accumulate bacteria within the lumen at early ages. Animals fed bacteria deficient in complex V showed intermediate coliform numbers and were not quite as long-lived. The results indicate that respiratory deficient Q-less E. coli are effectively degraded in the early adult worm, either at the pharynx or within the intestine, and do not accumulate in the intestinal tract until day ten of adulthood. Conclusions The findings of this study suggest that the nematodes fed the respiratory deficient E. coli diet live longer because the delay in bacterial colonization of the gut subjects the worms to less stress compared to worms fed the OP50 E. coli diet. This work suggests that bacterial respiration can act as a virulence factor, influencing the ability of bacteria to colonize and subsequently harm the animal host. Respiratory deficient bacteria may pose a useful model for probing probiotic relationships within the gut microbiome in higher organisms.

Animal development requires temporal coordination between recurrent processes and sequential events, but the underlying timing mechanisms are not yet understood. The molting cycle of C. elegans provides an ideal system to study this basic problem. We recently characterized LIN-42, which is related to the circadian clock protein PERIOD, as a key component of the developmental timer underlying rhythmic molting cycles. In this context, LIN-42 coordinates epithelial stem cell dynamics with progression of the molting cycle. Repeated actions of LIN-42 may enable the reprogramming of seam cell temporal fates, while stage-specific actions of LIN-42 and other heterochronic genes select fates appropriate for upcoming, rather than passing, life stages. Here, we discuss the possible configuration of the molting timer, which may include interconnected positive and negative regulatory loops among lin-42, conserved nuclear hormone receptors such as NHR-23 and -25, and the let-7 family of microRNAs. Physiological and environmental conditions may modulate the activities of particular components of this molting timer. Finding that LIN-42 regulates both a sleep-like behavioral state and epidermal stem cell dynamics further supports the model of functional conservation between LIN-42 and mammalian PERIOD proteins. The molting timer may therefore represent a primitive form of a central biological clock and provide a general paradigm for the integration of rhythmic and developmental processes.

The molting cycle of nematodes involves the periodic synthesis and removal of a collagen-rich exoskeleton, but the underlying molecular mechanisms are not well understood. Here, we describe the mlt-10 gene of Caenorhabditis elegans, which emerged from a genetic screen for molting-defective mutants sensitized by low cholesterol. MLT-10 defines a large family of nematode-specific proteins comprised of DUF644 and tandem P-X(2)-L-(S/T)-P repeats. Conserved nuclear hormone receptors promote expression of the mlt-10 gene in the hypodermis whenever the exoskeleton is remade. Further, a MLT-10::mCherry fusion protein is released from the hypodermis to the surrounding matrices and fluids during molting. The fusion protein is also detected in strands near the surface of animals. Both loss-of-function and gain-of-function mutations of mlt-10 impede the removal of old cuticles. However, the substitution mutation mlt-10(mg364), which disrupts the proline-rich repeats, causes the most severe phenotype. Mutations of mlt-10 are also associated with abnormalities in the exoskeleton and improper development of the epidermis. Thus, mlt-10 encodes a secreted protein involved in three distinct but interconnected aspects of the molting cycle. We propose that the molting cycle of C. elegans involves the dynamic assembly and disassembly of MLT-10 and possibly the paralogs of MLT-10.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development.

The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. Here, we show that the paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family. [ABSTRACT FROM AUTHOR]