Signaling via the FLP-14/FRPR-19 neuropeptide pathway sustains nociceptive response to repeated noxious stimuli in C. elegans

In order to thrive in constantly changing environments, animals must adaptively respond to threatening events. Noxious stimuli are not only processed according to their absolute intensity, but also to their context. Adaptation processes can cause animals to habituate at different rates and degrees in response to permanent or repeated stimuli. Here, we used a forward genetic approach in Caenorhabditis elegans to identify a neuropeptidergic pathway, essential to prevent fast habituation and maintain robust withdrawal responses to repeated noxious stimuli. This pathway involves the FRPR-19A and FRPR-19B G-protein coupled receptor isoforms produced from the frpr-19 gene by alternative splicing. Loss or overexpression of each or both isoforms can impair withdrawal responses caused by the optogenetic activation of the polymodal FLP nociceptor neuron. Furthermore, we identified FLP-8 and FLP-14 as FRPR-19 ligands in vitro. flp-14, but not flp-8, was essential to promote withdrawal response and is part of the same genetic pathway as frpr-19 in vivo. Expression and cell-specific rescue analyses suggest that FRPR-19 acts both in the FLP nociceptive neurons and downstream interneurons, whereas FLP-14 acts from interneurons. Importantly, genetic impairment of the FLP-14/FRPR-19 pathway accelerated the habituation to repeated FLP-specific optogenetic activation, as well as to repeated noxious heat and harsh touch stimuli. Collectively, our data suggest that well-adjusted neuromodulation via the FLP-14/FRPR-19 pathway contributes to promote nociceptive signals in C. elegans and counteracts habituation processes that otherwise tend to rapidly reduce aversive responses to repeated noxious stimuli.     

Systemic regulation of autophagy in Caenorhabditis elegans

When no supply of environmental nutrients is available, cells induce autophagy, thereby generating a source of emergency metabolic substrates and energy to maintain the basal cellular activity needed for survival. This autophagy response to starvation has been well characterized in various multicellular organisms, including worms, flies, and mice. Although prosurvival effects of autophagy in response to starvation are well known in animals, the mechanisms by which animals regulate and coordinate autophagy systemically remain elusive. Using C. elegans as a model system, we found that specific amino acids could regulate starvation-induced autophagy, and that MGL-1 and MGL-2, Caenorhabditis elegans homologs of metabotropic glutamate receptors, were involved. MGL-1 and MGL-2 specifically acted in AIY and AIB neurons, respectively, to modulate the autophagy response in other tissues such as pharyngeal muscle. Our recent study suggests that the autophagy response to starvation, previously thought to be cell-autonomous, can be systemically regulated, and that there is a specific sensor for monitoring systemic amino acids levels in Caenorhabditis elegans.     

Co-transmission of neuropeptides and monoamines choreograph the C. elegans escape response

Co-localization and co-transmission of neurotransmitters and neuropeptides is a core property of neural signaling across species. While co-transmission can increase the flexibility of cellular communication, understanding the functional impact on neural dynamics and behavior remains a major challenge. Here we examine the role of neuropeptide/monoamine co-transmission in the orchestration of the C. elegans escape response. The tyraminergic RIM neurons, which coordinate distinct motor programs of the escape response, also co-express the neuropeptide encoding gene flp-18. We find that in response to a mechanical stimulus, flp-18 mutants have defects in locomotory arousal and head bending that facilitate the omega turn. We show that the induction of the escape response leads to the release of FLP-18 neuropeptides. FLP-18 modulates the escape response through the activation of the G-protein coupled receptor NPR-5. FLP-18 increases intracellular calcium levels in neck and body wall muscles to promote body bending. Our results show that FLP-18 and tyramine act in different tissues in both a complementary and antagonistic manner to control distinct motor programs during different phases of the C. elegans flight response. Our study reveals basic principles by which co-transmission of monoamines and neuropeptides orchestrate in arousal and behavior in response to stress.     

Initial Phases of Starvation and Activity of Bacteria at Surfaces

The activity of the hydrophilic Vibrio sp. strain DW1 and the hydrophobic Pseudomonas sp. strain S9, which both undergo starvation-induced responses, was examined at nutrient-enriched and nutrient-deficient interfaces. The initial period of response to a starvation regime (“dwarfing” phase) is a sequence of two processes: fragmentation and continuous size reduction of the fragmented cells. This dwarfing phase is also one of intense metabolic activity as supported by O₂ uptake measurements of the endogenous metabolism and the use of inhibitors of the proton flow, the electron transport chain, and membrane-bound ATPase. Hydrophilic bacteria become even smaller at nutrient-deficient surfaces than in the liquid phase upon starvation, and this is reflected in a higher endogenous metabolism exhibited by surface-associated cells compared with those in the liquid phase. On the other hand, hydrophobic bacteria dwarfing at surfaces did not exhibit a greater size reduction and exhibited an endogenous metabolism that was only slightly higher than that of cells in the liquid phase. Bacterial scavenging of surface-localized nutrients is related to the degree of irreversible binding of dwarf and starved bacteria, which in turn may be related to the degree of cell surface hydrophobicity.     

The Perilipin Homologue,Lipid Storage Droplet 2, Regulates Sleep Homeostasis and Prevents Learning Impairments Following Sleep Loss

Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. Recently, two genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), have been shown to modulate the response to starvation. bmm mutants have excess fat and are resistant to starvation, whereas Lsd2 mutants are lean and sensitive to starvation. Thus, we hypothesized that bmm and Lsd2 may play a role in sleep regulation. Indeed, bmm mutant flies display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking.     

Drosophila larvae lacking the bcl-2 gene, buffy, are sensitive to nutrient stress, maintain increased basal target of rapamycin (Tor) signaling and exhibit characteristics of altered basal energy metabolism

Background

B cell lymphoma 2 (Bcl-2) proteins are the central regulators of apoptosis. The two bcl-2 genes in Drosophila modulate the response to stress-induced cell death, but not developmental cell death. Because null mutants are viable, Drosophila provides an optimum model system to investigate alternate functions of Bcl-2 proteins. In this report, we explore the role of one bcl-2 gene in nutrient stress responses.

Results

We report that starvation of Drosophila larvae lacking the bcl-2 gene, buffy, decreases survival rate by more than twofold relative to wild-type larvae. The buffy null mutant reacted to starvation with the expected responses such as inhibition of target of rapamycin (Tor) signaling, autophagy initiation and mobilization of stored lipids. However, the autophagic response to starvation initiated faster in larvae lacking buffy and was inhibited by ectopic buffy. We demonstrate that unusually high basal Tor signaling, indicated by more phosphorylated S6K, was detected in the buffy mutant and that removal of a genomic copy of S6K, but not inactivation of Tor by rapamycin, reverted the precocious autophagy phenotype. Instead, Tor inactivation also required loss of a positive nutrient signal to trigger autophagy and loss of both was sufficient to activate autophagy in the buffy mutant even in the presence of enforced phosphoinositide 3-kinase (PI3K) signaling. Prior to starvation, the fed buffy mutant stored less lipid and glycogen, had high lactate levels and maintained a reduced pool of cellular ATP. These observations, together with the inability of buffy mutant larvae to adapt to nutrient restriction, indicate altered energy metabolism in the absence of buffy.

Conclusions

All animals in their natural habitats are faced with periods of reduced nutrient availability. This study demonstrates that buffy is required for adaptation to both starvation and nutrient restriction. Thus, Buffy is a Bcl-2 protein that plays an important non-apoptotic role to promote survival of the whole organism in a stressful situation.     

Effects of starvation and feeding upon corpus allatum activity and oöcyte growth in adult female Periplaneta americana

Direct radiochemical determinations of juvenile hormone (JH) biosynthesis by corpora allata (CA) isolated from starved and re-fed Periplanteta americana have been employed to elucidate the humoral mechanisms involved in the modulation of reproductive activity in response to food availability. When starvation was initiated in mature adult females at the time of formation of an oötheca the next oötheca was normally deposited 5 to 6 days later, a delay of 2–3 days, and a third oötheca was formed by only 50% of starved females. The terminal oöcytes in the remaining females were either resorbed or maintained in an arrested state. Ovarian development had effectively ceased after 2 weeks of starvation but recommenced within 3 days of re-feeding. The CA of most starved females exhibited 2 activity cycles following food withdrawal. The first peak occurred on day 1 of the starvation period and was coincident with the timing for fed controls. The second peak was delayed by about 2 days and the activity of the CA then declined to the extent that glands from animals starved for more than 11 days were completely inactive. Feeding, after 10 or 16 days starvation, resulted in a resumption of CA activity which was detectable in some animals within 24 hr, and very high rates of JH biosynthesis were found 4 or 5 days later. The results suggest that P. americana can readily and efficiently modulate egg production in response to food supply, and that control is effected through alterations in JH production by the CA. The use of farnesenic acid as a biochemical probe indicates that CA inactivity after long periods of starvation does not arise because malnutrition has caused complete metabolic shut-down in the glands, and that JH biosynthesis is basically modulated at a control point prior to the last two enzymic stages in the pathway.     

Nutritional factors of inflammation induction or lipid mechanism of endotoxin transport

Integral parameters of bacterial lipopolysaccharide concentration and antiendotoxin immunity activity were determined in the blood serum of subjects with two extreme variants of eating disorders: obesity and anorexia nervosa (in its clinical model, long-term starvation). The results of the study showed that obese patients had initially higher endotoxin concentrations (2.41 ± 0.11 vs 1.13 ± 0.05 EU/mL in the control), which significantly decreased as a result taking a course of orlistat or after starvation (for no longer than 20 days) (to 1.34 ± 0.04 and 1.01 ± 0.09 EU/mL, respectively). The decrease in the endotoxin serum level was associated with an increase in the concentration of high-density lipoproteins (HDLs) and a decrease in the concentration of low-density lipoproteins (LDLs). This allowed us to assume that the complex of the latter with the endotoxin is the LDL fraction that may function as a depot of the hydrophobic form of the lipopolysaccharide molecule in the bloodstream. Long-term starvation (starting from day 20) led to the appearance of symptoms characteristic of septic states, which was preceded by a twofold increase in the concentration of the endotoxin and antibodies against the enterobacterial common antigen and subsequent increase in the level of anti-glycolipid antibodies (from 133.2 ± 2.6 to 355.6 ± 15.0 optical density arbitrary units) starting from day 25?C27 of starvation, which is characteristic for the initial phase of the systemic inflammatory response syndrome.     

Exogenous strigolactones impact metabolic profiles and phosphate starvation signalling in roots

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2′-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2′-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2′-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

LIN-35/Rb Causes Starvation-Induced Germ Cell Apoptosis via CED-9/Bcl2 Downregulation in Caenorhabditis elegans

Apoptosis is an important mechanism for maintaining germ line health. In Caenorhabditis elegans, germ cell apoptosis occurs under normal conditions to sustain gonad homeostasis and oocyte quality. Under stress, germ cell apoptosis can be triggered via different pathways, including the following: (i) the CEP-1/p53 pathway, which induces germ cell apoptosis when animals are exposed to DNA damage; (ii) the mitogen-activated protein kinase kinase (MAPKK) pathway, which triggers germ cell apoptosis when animals are exposed to heat shock, oxidative stress, or osmotic stress; and (iii) an unknown mechanism that triggers germ cell apoptosis during starvation. Here, we address how starvation induces germ cell apoptosis. Using polysomal profiling, we found that starvation for 6 h reduces the translationally active ribosomes, which differentially affect the mRNAs of the core apoptotic machinery and some of its regulators. During starvation, lin-35/Rb mRNA increases its expression, resulting in the accumulation of this protein. As a consequence, LIN-35 downregulates the expression of the antiapoptotic gene ced-9/Bcl-2. We observed that the reduced translation of ced-9/Bcl-2 mRNA during food deprivation together with its downregulation drastically affects its protein accumulation. We propose that CED-9/Bcl-2 downregulation via LIN-35/Rb triggers germ cell apoptosis in C. elegans in response to starvation.     

Candida albicans the chameleon: transitions and interactions between multiple phenotypic states confer phenotypic plasticity

The ability of microbial cells to exist in multiple states is a ubiquitous property that promotes adaptation and survival. This phenomenon has been extensively studied in the opportunistic pathogen Candida albicans, which can transition between multiple phenotypic states in response to environmental signals. C. albicans normally exists as a commensal in the human body, but can also cause debilitating mucosal infections or life-threatening systemic infections. The ability to switch between cellular forms contributes to C. albicans’ capacity to infect different host niches, and strictly regulates the program of sexual mating. We review the unique properties associated with different phenotypic states, as well as how interactions between cells in different states can further augment microbial behavior.     

A Sexually Conditioned Switch of Chemosensory Behavior in C. elegans

In sexually reproducing animals, mating is essential for transmitting genetic information to the next generation and therefore animals have evolved mechanisms for optimizing the chance of successful mate location. In the soil nematode C. elegans, males approach hermaphrodites via the ascaroside pheromones, recognize hermaphrodites when their tails contact the hermaphrodites'' body, and eventually mate with them. These processes are mediated by sensory signals specialized for sexual communication, but other mechanisms may also be used to optimize mate location. Here we describe associative learning whereby males use sodium chloride as a cue for hermaphrodite location. Both males and hermaphrodites normally avoid sodium chloride after associative conditioning with salt and starvation. However, we found that males become attracted to sodium chloride after conditioning with salt and starvation if hermaphrodites are present during conditioning. For this conditioning, which we call sexual conditioning, hermaphrodites are detected by males through pheromonal signaling and additional cue(s). Sex transformation experiments suggest that neuronal sex of males is essential for sexual conditioning. Altogether, these results suggest that C. elegans males integrate environmental, internal and social signals to determine the optimal strategy for mate location.     

Role of laccase in the biology and virulence of Cryptococcus neoformans

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.     

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

The green alga Hematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high light leads to the accumulation of carbohydrates and fatty acids as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways has been well investigated, little is known about the systemic effects of the stress response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high light. The data were integrated into a putative genome-scale model of the green alga to in silico test hypotheses of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large scale integrative approaches to pinpoint metabolic adjustment to changing environments.     

OLA-1, an Obg-like ATPase,integrates hunger with temperature information in sensory neurons in C. elegans

Animals detect changes in both their environment and their internal state and modify their behavior accordingly. Yet, it remains largely to be clarified how information of environment and internal state is integrated and how such integrated information modifies behavior. Well-fed C. elegans migrates to past cultivation temperature on a thermal gradient, which is disrupted when animals are starved. We recently reported that the neuronal activities synchronize between a thermosensory neuron AFD and an interneuron AIY, which is directly downstream of AFD, in well-fed animals, while this synchrony is disrupted in starved animals. However, it remained to be determined whether the disruption of the synchrony is derived from modulation of the transmitter release from AFD or from the modification of reception or signal transduction in AIY. By performing forward genetics on a transition of thermotaxis behavior along starvation, we revealed that OLA-1, an Obg-like ATPase, functions in AFD to promote disruption of AFD-AIY synchrony and behavioral transition. Our results suggest that the information of hunger is delivered to the AFD thermosensory neuron and gates transmitter release from AFD to disrupt thermotaxis, thereby shedding light onto a mechanism for the integration of environmental and internal state to modulate behavior.