Synaptic removal of diacylglycerol by DGKζ and PSD‐95 regulates dendritic spine maintenance

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKζ is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD‐95. Overexpression of DGKζ in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD‐95 binding. Conversely, DGKζ knockdown reduces spine density. Mice deficient in DGKζ expression show reduced spine density and excitatory synaptic transmission. Time‐lapse imaging indicates that DGKζ is required for spine maintenance but not formation. We propose that PSD‐95 targets DGKζ to synaptic DAG‐producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.     

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.     

Loss of NDG‐4 extends lifespan and stress resistance in Caenorhabditis elegans

NDG‐4 is a predicted transmembrane acyltransferase protein that acts in the distribution of lipophilic factors. Consequently, ndg‐4 mutants lay eggs with a pale appearance due to lack of yolk, and they are resistant to sterility caused by dietary supplementation with the long‐chain omega‐6 polyunsaturated fatty acid dihommogamma‐linolenic acid (DGLA). Two other proteins, NRF‐5 and NRF‐6, a homolog of a mammalian secreted lipid binding protein and a NDG‐4 homolog, respectively, have previously been shown to function in the same lipid transport pathway. Here, we report that mutation of the NDG‐4 protein results in increased organismal stress resistance and lifespan. When NDG‐4 function and insulin/IGF‐1 signaling are reduced simultaneously, maximum lifespan is increased almost fivefold. Thus, longevity conferred by mutation of ndg‐4 is partially overlapping with insulin signaling. The nuclear hormone receptor NHR‐80 (HNF4 homolog) is required for longevity in germline less animals. We find that NHR‐80 is also required for longevity of ndg‐4 mutants. Moreover, we find that nrf‐5 and nrf‐6 mutants also have extended lifespan and increased stress resistance, suggesting that altered lipid transport and metabolism play key roles in determining lifespan.     

Reducing sphingolipid synthesis orchestrates global changes to extend yeast lifespan

Studies of aging and longevity are revealing how diseases that shorten life can be controlled to improve the quality of life and lifespan itself. Two strategies under intense study to accomplish these goals are rapamycin treatment and calorie restriction. New strategies are being discovered including one that uses low‐dose myriocin treatment. Myriocin inhibits the first enzyme in sphingolipid synthesis in all eukaryotes, and we showed recently that low‐dose myriocin treatment increases yeast lifespan at least in part by down‐regulating the sphingolipid‐controlled Pkh1/2‐Sch9 (ortholog of mammalian S6 kinase) signaling pathway. Here we show that myriocin treatment induces global effects and changes expression of approximately forty percent of the yeast genome with 1252 genes up‐regulated and 1497 down‐regulated (P < 0.05) compared with untreated cells. These changes are due to modulation of evolutionarily conserved signaling pathways including activation of the Snf1/AMPK pathway and down‐regulation of the protein kinase A (PKA) and target of rapamycin complex 1 (TORC1) pathways. Many processes that enhance lifespan are regulated by these pathways in response to myriocin treatment including respiration, carbon metabolism, stress resistance, protein synthesis, and autophagy. These extensive effects of myriocin match those of rapamycin and calorie restriction. Our studies in yeast together with other studies in mammals reveal the potential of myriocin or related compounds to lower the incidence of age‐related diseases in humans and improve health span.     

Drosophila insulin‐like peptide dilp1 increases lifespan and glucagon‐like Akh expression epistatic to dilp2

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF‐like peptide (dilp) paralogs, including tandem‐encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 levels. It has been shown that dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause. Here, we find that dilp1 is also highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1‐dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 ? dilp2 double mutants. On the other hand, dilp1 and dilp2 redundantly or synergistically interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. These interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro‐longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. Our data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro‐longevity insulin‐like peptide.     

Movement decline across lifespan of Caenorhabditis elegans mutants in the insulin/insulin‐like signaling pathway

Research in aging biology has identified several pathways that are molecularly conserved across species that extend lifespan when mutated. The insulin/insulin‐like signaling (IIS) pathway is one of the most widely studied of these. It has been assumed that extending lifespan also extends healthspan (the period of life with minimal functional loss). However, data supporting this assumption conflict and recent evidence suggest that life extension may, in and of itself, extend the frail period. In this study, we use Caenorhabditis elegans to further probe the link between lifespan and healthspan. Using movement decline as a measure of health, we assessed healthspan across the entire lifespan in nine IIS pathway mutants. In one series of experiments, we studied healthspan in mass cultures, and in another series, we studied individuals longitudinally. We found that long‐lived mutants display prolonged mid‐life movement and do not prolong the frailty period. Lastly, we observed that early‐adulthood movement was not predictive of late‐life movement or survival, within identical phenotypes. Overall, these observations show that extending lifespan does not prolong the period of frailty. Both genotype and a stochastic component modulate aging, and movement late in life is more variable than early‐life movement.     

Behavioural response to combined insecticide and temperature stress in natural populations of Drosophila melanogaster

Insecticide resistance evolves extremely rapidly, providing an illuminating model for the study of adaptation. With climate change reshaping species distribution, pest and disease vector control needs rethinking to include the effects of environmental variation and insect stress physiology. Here, we assessed how both long‐term adaptation of populations to temperature and immediate temperature variation affect the genetic architecture of DDT insecticide response in Drosophila melanogaster. Mortality assays and behavioural assays based on continuous activity monitoring were used to assess the interaction between DDT and temperature on three field‐derived populations from climate extremes (Raleigh for warm temperate, Tasmania for cold oceanic and Queensland for hot tropical). The Raleigh population showed the highest mortality to DDT, whereas the Queensland population, epicentre for derived alleles of the resistance gene Cyp6g1, showed the lowest. Interaction between insecticide and temperature strongly affected mortality, particularly for the Tasmanian population. Activity profiles analysed using self‐organizing maps show that the insecticide promoted an early response, whereas elevated temperature promoted a later response. These distinctive early or later activity phases revealed similar responses to temperature and DDT dose alone but with more or less genetic variance depending on the population. This change in genetic variance among populations suggests that selection particularly depleted genetic variance for DDT response in the Queensland population. Finally, despite similar (co)variation between traits in benign conditions, the genetic responses across population differed under stressful conditions. This showed how stress‐responsive genetic variation only reveals itself in specific conditions and thereby escapes potential trade‐offs in benign environments.     

Reproductive interference by male Drosophila subobscura on female D. persimilis: A laboratory experiment

While females often reject courtship attempts by heterospecific males, reproductive interference by harassment from such males can nonetheless occur, potentially reducing female fitness. Such effects may be profound following a range expansion, when males from a new species may suddenly encounter (and perhaps even become abundant relative to) females of related native species. Drosophila subobscura recently invaded North America and may impact native species through reproductive interference and other processes. We test for the potential for reproductive interference by D. subobscura males on D. persimilis females in the laboratory. D. subobscura males aggressively copulated with D. persimilis females, including many females that exhibit rejection behaviors. Despite females attempting to dismount the males, the heterospecific copulations are on average longer than conspecific copulations, and females exhibit some reluctance to remate with conspecific males following this harassment. Females confined with both conspecific and heterospecific males produce fewer adult progeny than those with either conspecific males only or with conspecific males and distantly related D. simulans males that do not engage in female harassment. Overall, our results illustrate how reproductive interference by an invasive species can have negative effects on resident natural populations.