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Controlling protein activity and localization is a key tool in modern biology. Mammalian steroid receptor ligand-binding domain (LBD) fusions have been used in a range of organisms and cell types to inactivate proteins of interest until the cognate steroid ligand is applied. Here, we demonstrate that the glucocorticoid receptor LBD confers ligand-gated control of a heterologous gene expression system (Q system) and the DAF-16 transcription factor in Caenorhabditis elegans These experiments provide a powerful tool for temporal control of protein activity, and will bolster existing tools used to modulate gene expression and protein activity in this animal.

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.

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 ?-ketoglutarate (?-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit ? is identified as a novel binding protein of ?-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 ?-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by ?-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 ?-KG requires ATP synthase subunit ? and is dependent on target of rapamycin (TOR) downstream. Endogenous ?-KG levels are increased on starvation and ?-KG does not extend the lifespan of dietary-restricted animals, indicating that ?-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 FROM AUTHOR]

Neurotransmitter signaling in the mature nervous system is well understood, but the functions of transmitters in the immature nervous system are less clear. Although transmitters released during embryogenesis regulate neuronal proliferation and migration, little is known about their role in regulating early neuronal differentiation. Here, we show that GABA and glutamate drive calcium-dependent embryonic electrical activity that regulates transmitter specification. The number of neurons expressing different transmitters changes when GABA or glutamate signaling is blocked chronically, either using morpholinos to knock down transmitter-synthetic enzymes or applying pharmacological receptor antagonists during a sensitive period of development. We find that calcium spikes are triggered by metabotropic GABA and glutamate receptors, which engage protein kinases A and C. The results reveal a novel role for embryonically expressed neurotransmitters. [ABSTRACT FROM AUTHOR]