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Reductions in brain kynurenic acid levels, a neuroinhibitory metabolite, improve cognitive function in diverse organisms. Thus, modulation of kynurenic acid levels is thought to have therapeutic potential in a range of brain disorders. Here we report that the steroid 5-androstene 3β, 17β-diol (ADIOL) reduces kynurenic acid levels and promotes associative learning in Caenorhabditis elegans We identify the molecular mechanisms through which ADIOL links peripheral metabolic pathways to neural mechanisms of learning capacity. Moreover, we show that in aged animals, which normally experience rapid cognitive decline, ADIOL improves learning capacity. The molecular mechanisms that underlie the biosynthesis of ADIOL as well as those through which it promotes kynurenic acid reduction are conserved in mammals. Thus, rather than a minor intermediate in the production of sex steroids, ADIOL is an endogenous hormone that potently regulates learning capacity by causing reductions in neural kynurenic acid levels.

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.

Caenorhabditis elegans serves as a model for understanding adiposity and its connections to aging. Current methodologies do not distinguish between fats serving the energy needs of the parent, akin to mammalian adiposity, from those that are distributed to the progeny, making it difficult to accurately interpret the physiological implications of fat content changes induced by external perturbations. Using spectroscopic coherent Raman imaging, we determine the protein content, chemical profiles and dynamics of lipid particles in live animals. We find fat particles in the adult intestine to be diverse, with most destined for the developing progeny. In contrast, the skin-like epidermis contains fats that are the least heterogeneous, the least dynamic and have high triglyceride content. These attributes are most consistent with stored somatic energy reservoirs. These results challenge the prevailing practice of assessing C. elegans adiposity by measurements that are dominated by the intestinal fat content.

Kynurenic acid (KynA) levels link peripheral metabolic status to neural functions including learning and memory. Since neural KynA levels dampen learning capacity, KynA reduction has been proposed as a therapeutic strategy for conditions of cognitive deficit such as neurodegeneration. While KynA is generated locally within the nervous system, its precursor, kynurenine (Kyn), is largely derived from peripheral resources. The mechanisms that import Kyn into the nervous system are poorly understood. Here, we provide genetic, anatomical, biochemical, and behavioral evidence showing that in C. elegans an ortholog of the human LAT1 transporter, AAT-1, imports Kyn into sites of KynA production.

The ability to coordinate behavioral responses with metabolic status is fundamental to the maintenance of energy homeostasis. In numerous species including Caenorhabditis elegans and mammals, neural serotonin signaling regulates a range of food-related behaviors. However, the mechanisms that integrate metabolic information with serotonergic circuits are poorly characterized. Here, we identify metabolic, molecular, and cellular components of a circuit that links peripheral metabolic state to serotonin-regulated behaviors in C. elegans. We find that blocking the entry of fatty acyl coenzyme As (CoAs) into peroxisomal β-oxidation in the intestine blunts the effects of neural serotonin signaling on feeding and egg-laying behaviors. Comparative genomics and metabolomics revealed that interfering with intestinal peroxisomal β-oxidation results in a modest global transcriptional change but significant changes to the metabolome, including a large number of changes in ascaroside and phospholipid species, some of which affect feeding behavior. We also identify body cavity neurons and an ether-a-go-go (EAG)-related potassium channel that functions in these neurons as key cellular components of the circuitry linking peripheral metabolic signals to regulation of neural serotonin signaling. These data raise the possibility that the effects of serotonin on satiety may have their origins in feedback, homeostatic metabolic responses from the periphery.

The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases are key contributors to the maintenance of protein homeostasis with age. We previously reported that expression of granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, enhance the accumulation and toxicity of TAR DNA binding protein 43 (TDP-43) in Caenorhabditis elegans (C. elegans). In this study we show that C. elegans granulins are produced in an age- and stress-dependent manner. Granulins localize to the endolysosomal compartment where they impair lysosomal protease expression and activity. Consequently, protein homeostasis is disrupted, promoting the nuclear translocation of the lysosomal transcription factor HLH-30/TFEB, and prompting cells to activate a compensatory transcriptional program. The three C. elegans granulin peptides exhibited distinct but overlapping functional effects in our assays, which may be due to amino acid composition that results in distinct electrostatic and hydrophobicity profiles. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease.

Mutations in the microtubule-associated protein tau (MAPT) underlie multiple neurodegenerative disorders, yet the pathophysiological mechanisms are unclear. A novel variant in MAPT resulting in an alanine to threonine substitution at position 152 (A152T tau) has recently been described as a significant risk factor for both frontotemporal lobar degeneration and Alzheimer's disease. Here we use complementary computational, biochemical, molecular, genetic and imaging approaches in Caenorhabditis elegans and mouse models to interrogate the effects of the A152T variant on tau function. In silico analysis suggests that a threonine at position 152 of tau confers a new phosphorylation site. This finding is borne out by mass spectrometric survey of A152T tau phosphorylation in C. elegans and mouse. Optical pulse-chase experiments of Dendra2-tau demonstrate that A152T tau and phosphomimetic A152E tau exhibit increased diffusion kinetics and the ability to traverse across the axon initial segment more efficiently than wild-type (WT) tau. A C. elegans model of tauopathy reveals that A152T and A152E tau confer patterns of developmental toxicity distinct from WT tau, likely due to differential effects on retrograde axonal transport. These data support a role for phosphorylation of the variant threonine in A152T tau toxicity and suggest a mechanism involving impaired retrograde axonal transport contributing to human neurodegenerative disease.

The widely conserved heat-shock response, regulated by heat-shock transcription factors, is not only essential for cellular stress resistance and adult longevity, but also for proper development. However, the genetic mechanisms by which heat-shock transcription factors regulate development are not well understood. In Caenorhabditis elegans, we conducted an unbiased genetic screen to identify mutations that could ameliorate the developmental-arrest phenotype of a heat-shock factor mutant. Here, we show that loss of the conserved translational activator rsks-1/S6 kinase, a downstream effector of mechanistic Target of Rapamycin (mTOR) kinase, can rescue the developmental-arrest phenotype of hsf-1 partial loss-of-function mutants. Unexpectedly, we show that the rescue is not likely caused by reduced translation, nor by activation of any of a variety of stress-protective genes and pathways. Our findings identify an as-yet unexplained regulatory relationship between the heat-shock transcription factor and the mTOR pathway during C. elegans development.

A general feature of animal aging is decline in learning and memory. Here we show that in Caenorhabditis elegans, a significant portion of this decline is due to accumulation of kynurenic acid (KYNA), an endogenous antagonist of neural N-methyl-D-aspartate receptors (NMDARs). We show that activation of a specific pair of interneurons either through genetic means or by depletion of KYNA significantly improves learning capacity in aged animals even when the intervention is applied in aging animals. KYNA depletion also improves memory. We show that insulin signaling is one factor in KYNA accumulation.

In species ranging from humans to Caenorhabditis elegans, dietary restriction (DR) grants numerous benefits, including enhanced learning. The precise mechanisms by which DR engenders benefits on processes related to learning remain poorly understood. As a result, it is unclear whether the learning benefits of DR are due to myriad improvements in mechanisms that collectively confer improved cellular health and extension of organismal lifespan or due to specific neural mechanisms. Using an associative learning paradigm in C. elegans, we investigated the effects of DR as well as manipulations of insulin, mechanistic target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and autophagy pathways-processes implicated in longevity-on learning. Despite their effects on a vast number of molecular effectors, we found that the beneficial effects on learning elicited by each of these manipulations are fully dependent on depletion of kynurenic acid (KYNA), a neuroinhibitory metabolite. KYNA depletion then leads, in an N-methyl D-aspartate receptor (NMDAR)-dependent manner, to activation of a specific pair of interneurons with a critical role in learning. Thus, fluctuations in KYNA levels emerge as a previously unidentified molecular mechanism linking longevity and metabolic pathways to neural mechanisms of learning. Importantly, KYNA levels did not alter lifespan in any of the conditions tested. As such, the beneficial effects of DR on learning can be attributed to changes in a nutritionally sensitive metabolite with neuromodulatory activity rather than indirect or secondary consequences of improved health and extended longevity.

BackgroundReliance on just one drug to treat the prevalent tropical disease, schistosomiasis, spurs the search for new drugs and drug targets. Inhibitors of human cyclic nucleotide phosphodiesterases (huPDEs), including PDE4, are under development as novel drugs to treat a range of chronic indications including asthma, chronic obstructive pulmonary disease and Alzheimer's disease. One class of huPDE4 inhibitors that has yielded marketed drugs is the benzoxaboroles (Anacor Pharmaceuticals).Methodology/principal findingsA phenotypic screen involving Schistosoma mansoni and 1,085 benzoxaboroles identified a subset of huPDE4 inhibitors that induced parasite hypermotility and degeneration. To uncover the putative schistosome PDE4 target, we characterized four PDE4 sequences (SmPDE4A-D) in the parasite's genome and transcriptome, and cloned and recombinantly expressed the catalytic domain of SmPDE4A. Among a set of benzoxaboroles and catechol inhibitors that differentially inhibit huPDE4, a relationship between the inhibition of SmPDE4A, and parasite hypermotility and degeneration, was measured. To validate SmPDE4A as the benzoxaborole molecular target, we first generated Caenorhabditis elegans lines that express a cDNA for smpde4a on a pde4(ce268) mutant (hypermotile) background: the smpde4a transgene restored mutant worm motility to that of the wild type. We then showed that benzoxaborole inhibitors of SmPDE4A that induce hypermotility in the schistosome also elicit a hypermotile response in the C. elegans lines that express the smpde4a transgene, thereby confirming SmPDE4A as the relevant target.Conclusions/significanceThe orthogonal chemical, biological and genetic strategies employed identify SmPDE4A's contribution to parasite motility and degeneration, and its potential as a drug target. Transgenic C. elegans is highlighted as a potential screening tool to optimize small molecule chemistries to flatworm molecular drug targets.

An overview of Caenorhabditis elegans as an experimental organism for studying energy balance is presented. Some of the unresolved questions that complicate the interpretation of lipid measurements from C. elegans are highlighted. We review studies that show that both lipid synthesis and lipid breakdown pathways are activated and needed for the longevity of hermaphrodites that lack their germlines. These findings illustrate the heterogeneity of triglyceride-rich lipid particles in C. elegans and reveal specific lipid signals that promote longevity. Finally, we provide a brief overview of feeding behavioral responses of C. elegans to varying nutritional conditions and highlight an unanticipated metabolic pathway that allows the incorporation of experience in feeding behavior.

© 2015 Yee et al.Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may par

The abnormal accumulation of fat increases the lifespans of nematodes that lack sex cells.

The kynurenine pathway of tryptophan metabolism is involved in the pathogenesis of several brain diseases, but its physiological functions remain unclear. We report that kynurenic acid, a metabolite in this pathway, functions as a regulator of food-dependent behavioral plasticity in C. elegans. The experience of fasting in C. elegans alters a variety of behaviors, including feeding rate, when food is encountered post-fast. Levels of neurally produced kynurenic acid are depleted by fasting, leading to activation of NMDA-receptor-expressing interneurons and initiation of a neuropeptide-y-like signaling axis that promotes elevated feeding through enhanced serotonin release when animals re-encounter food. Upon refeeding, kynurenic acid levels are eventually replenished, ending the elevated feeding period. Because tryptophan is an essential amino acid, these findings suggest that a physiological role of kynurenic acid is in directly linking metabolism to activity of NMDA and serotonergic circuits, which regulate a broad range of behaviors and physiologies.

Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.

The regulation of energy balance involves an intricate interplay between neural mechanisms that respond to internal and external cues of energy demand and food availability. Compelling data have implicated the neurotransmitter dopamine as an important part

The regulation of energy balance involves an intricate interplay between neural mechanisms that respond to internal and external cues of energy demand and food availability. Compelling data have implicated the neurotransmitter dopamine as an important part of body weight regulation. However, the precise mechanisms through which dopamine regulates energy homeostasis remain poorly understood. Here, we investigate mechanisms through which dopamine modulates energy storage. We showed that dopamine signaling regulates fat reservoirs in Caenorhabditis elegans. We found that the fat reducing effects of dopamine were dependent on dopaminergic receptors and a set of fat oxidation enzymes. Our findings reveal an ancient role for dopaminergic regulation of fat and suggest that dopamine signaling elicits this outcome through cascades that ultimately mobilize peripheral fat depots.

Metazoan transcription factors control distinct networks of genes in specific tissues, yet understanding how these networks are integrated into physiology, development, and homeostasis remains challenging. Inactivation of the nuclear hormone receptor nhr-25 ameliorates developmental and metabolic phenotypes associated with loss of function of an acyl-CoA synthetase gene, acs-3. ACS-3 activity prevents aberrantly high NHR-25 activity. Here, we investigated this relationship further by examining gene expression patterns following acs-3 and nhr-25 inactivation. Unexpectedly, we found that the acs-3 mutation or nhr-25 RNAi resulted in similar transcriptomes with enrichment in innate immunity and stress response gene expression. Mutants of either gene exhibited distinct sensitivities to pathogens and environmental stresses. Only nhr-25 was required for wild-type levels of resistance to the bacterial pathogen P. aeruginosa and only acs-3 was required for wild-type levels of resistance to osmotic stress and the oxidative stress generator, juglone. Inactivation of either acs-3 or nhr-25 compromised lifespan and resistance to the fungal pathogen D. coniospora. Double mutants exhibited more severe defects in the lifespan and P. aeruginosa assays, but were similar to the single mutants in other assays. Finally, acs-3 mutants displayed defects in their epidermal surface barrier, potentially accounting for the observed sensitivities. Together, these data indicate that inactivation of either acs-3 or nhr-25 causes stress sensitivity and increased expression of innate immunity/stress genes, most likely by different mechanisms. Elevated expression of these immune/stress genes appears to abrogate the transcriptional signatures relevant to metabolism and development.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved master regulator of metabolism and a therapeutic target in type 2 diabetes. As an energy sensor, AMPK activity is responsive to both metabolic inputs, for instance the ratio of AMP to ATP,

Metazoan transcription factors control distinct networks of genes in specific tissues, yet understanding how these networks are integrated into physiology, development, and homeostasis remains challenging. Inactivation of the nuclear hormone receptor nhr-25 ameliorates developmental and metabolic phenotypes associated with loss of function of an acyl-CoA synthetase gene, acs-3. ACS-3 activity prevents aberrantly high NHR-25 activity. Here, we investigated this relationship further by examining gene expression patterns following acs-3 and nhr-25 inactivation. Unexpectedly, we found that the acs-3 mutation or nhr-25 RNAi resulted in similar transcriptomes with enrichment in innate immunity and stress response gene expression. Mutants of either gene exhibited distinct sensitivities to pathogens and environmental stresses. Only nhr-25 was required for wild-type levels of resistance to the bacterial pathogen P. aeruginosa and only acs-3 was required for wild-type levels of resistance to osmotic stress and the oxidative stress generator, juglone. Inactivation of either acs-3 or nhr-25 compromised lifespan and resistance to the fungal pathogen D. coniospora. Double mutants exhibited more severe defects in the lifespan and P. aeruginosa assays, but were similar to the single mutants in other assays. Finally, acs-3 mutants displayed defects in their epidermal surface barrier, potentially accounting for the observed sensitivities. Together, these data indicate that inactivation of either acs-3 or nhr-25 causes stress sensitivity and increased expression of innate immunity/stress genes, most likely by different mechanisms. Elevated expression of these immune/stress genes appears to abrogate the transcriptional signatures relevant to metabolism and development.

Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.

The AGC family serine-threonine kinases Akt and Sgk are similar in primary amino acid sequence and in vitro substrate specificity, and both kinases are thought to directly phosphorylate and inhibit FoxO transcription factors. In the nematode Caenorhabditis elegans, it is well established that AKT-1 controls dauer arrest and lifespan by regulating the subcellular localization of the FoxO transcription factor DAF-16. SGK-1 is thought to act similarly to AKT-1 in lifespan control by phosphorylating and inhibiting the nuclear translocation of DAF-16/FoxO. Using sgk-1 null and gain-of-function mutants, we now provide multiple lines of evidence indicating that AKT-1 and SGK-1 influence C. elegans lifespan, stress resistance, and DAF-16/FoxO activity in fundamentally different ways. Whereas AKT-1 shortens lifespan, SGK-1 promotes longevity in a DAF-16-/FoxO-dependent manner. In contrast to AKT-1, which reduces resistance to multiple stresses, SGK-1 promotes resistance to oxidative stress and ultraviolet radiation but inhibits thermotolerance. Analysis of several DAF-16/FoxO target genes that are repressed by AKT-1 reveals that SGK-1 represses a subset of these genes while having little influence on the expression of others. Accordingly, unlike AKT-1, which promotes the cytoplasmic sequestration of DAF-16/FoxO, SGK-1 does not influence DAF-16/FoxO subcellular localization. Thus, in spite of their similar in vitro substrate specificities, Akt and Sgk influence longevity, stress resistance, and FoxO activity through distinct mechanisms in vivo. Our findings highlight the need for a re-evaluation of current paradigms of FoxO regulation by Sgk.

The AGC family serine-threonine kinases Akt and Sgk are similar in primary amino acid sequence and in vitro substrate specificity, and both kinases are thought to directly phosphorylate and inhibit FoxO transcription factors. In the nematode Caenorhabditis

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.

Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.

Serotonergic regulation of feeding behavior has been studied intensively, both for an understanding of the basic neurocircuitry of energy balance in various organisms and as a therapeutic target for human obesity. However, its underlying molecular mechanisms remain poorly understood. Here, we show that neural serotonin signaling in C. elegans modulates feeding behavior through inhibition of AMP-activated kinase (AMPK) in interneurons expressing the C. elegans counterpart of human SIM1, a transcription factor associated with obesity. In turn, glutamatergic signaling links these interneurons to pharyngeal neurons implicated in feeding behavior. We show that AMPK-mediated regulation of glutamatergic release is conserved in rat hippocampal neurons. These findings reveal cellular and molecular mediators of serotonergic signaling.

Bardet-Biedl syndrome, BBS, is a rare autosomal recessive disorder with clinical presentations including polydactyly, retinopathy, hyperphagia, obesity, short stature, cognitive impairment, and developmental delays. Disruptions of BBS proteins in a variety of organisms impair cilia formation and function and the multi-organ defects of BBS have been attributed to deficiencies in various cilia-associated signaling pathways. In C. elegans, bbs genes are expressed exclusively in the sixty ciliated sensory neurons of these animals and bbs mutants exhibit sensory defects as well as body size, feeding, and metabolic abnormalities. Here we show that in contrast to many other cilia-defective mutants, C. elegans bbs mutants exhibit increased release of dense-core vesicles and organism-wide phenotypes associated with enhanced activities of insulin, neuropeptide, and biogenic amine signaling pathways. We show that the altered body size, feeding, and metabolic abnormalities of bbs mutants can be corrected to wild-type levels by abrogating the enhanced secretion of dense-core vesicles without concomitant correction of ciliary defects. These findings expand the role of BBS proteins to the regulation of dense-core-vesicle exocytosis and suggest that some features of Bardet-Biedl Syndrome may be caused by excessive neuroendocrine secretion.

The regulation of energy homeostasis integrates diverse biological processes ranging from behavior to metabolism and is linked fundamentally to numerous disease states. To identify new molecules that can bypass homeostatic compensatory mechanisms of energy balance in intact animals, we screened for small-molecule modulators of Caenorhabditis elegans fat content. We report on several molecules that modulate fat storage without obvious deleterious effects on feeding, growth and reproduction. A subset of these compounds also altered fat storage in mammalian and insect cell culture. We found that one of the newly identified compounds exerts its effects in C. elegans through a pathway that requires previously undescribed functions of an AMP-activated kinase catalytic subunit and a transcription factor previously unassociated with fat regulation. Thus, our strategy identifies small molecules that are effective within the context of intact animals and reveals relationships between new pathways that operate across phyla to influence energy homeostasis.

The target of rapamycin (TOR) kinase coordinately regulates fundamental metabolic and cellular processes to support growth, proliferation, survival, and differentiation, and consequently it has been proposed as a therapeutic target for the treatment of cancer, metabolic disease, and aging. The TOR kinase is found in two biochemically and functionally distinct complexes, termed TORC1 and TORC2. Aided by the compound rapamycin, which specifically inhibits TORC1, the role of TORC1 in regulating translation and cellular growth has been extensively studied. The physiological roles of TORC2 have remained largely elusive due to the lack of pharmacological inhibitors and its genetic lethality in mammals. Among potential targets of TORC2, the pro-survival kinase AKT has garnered much attention. Within the context of intact animals, however, the physiological consequences of phosphorylation of AKT by TORC2 remain poorly understood. Here we describe viable loss-of-function mutants in the Caenorhabditis elegans homolog of the TORC2-specific component, Rictor (CeRictor). These mutants display a mild developmental delay and decreased body size, but have increased lipid storage. These functions of CeRictor are not mediated through the regulation of AKT kinases or their major downstream target, the insulin-regulated FOXO transcription factor DAF-16. We found that loss of sgk-1, a homolog of the serum- and glucocorticoid-induced kinase, mimics the developmental, growth, and metabolic phenotypes of CeRictor mutants, while a novel, gain-of-function mutation in sgk-1 suppresses these phenotypes, indicating that SGK-1 is a mediator of CeRictor activity. These findings identify new physiological roles for TORC2, mediated by SGK, in regulation of C. elegans lipid accumulation and growth, and they challenge the notion that AKT is the primary effector of TORC2 function.

For most organisms, food is only intermittently available; therefore, molecular mechanisms that couple sensation of nutrient availability to growth and development are critical for survival. These mechanisms, however, remain poorly defined. In the absence of nutrients, newly hatched first larval (L1) stage Caenorhabditis elegans halt development and survive in this state for several weeks. We isolated mutations in unc-31, encoding a calcium-activated regulator of neural dense-core vesicle release, which conferred enhanced starvation survival. This extended survival was reminiscent of that seen in daf-2 insulin-signaling deficient mutants and was ultimately dependent on daf-16, which encodes a FOXO transcription factor whose activity is inhibited by insulin signaling. While insulin signaling modulates metabolism, adult lifespan, and dauer formation, insulin-independent mechanisms that also regulate these processes did not promote starvation survival, indicating that regulation of starvation survival is a distinct program. Cell-specific rescue experiments identified a small subset of primary sensory neurons where unc-31 reconstitution modulated starvation survival, suggesting that these neurons mediate perception of food availability. We found that OCR-2, a transient receptor potential vanilloid (TRPV) channel that localizes to the cilia of this subset of neurons, regulates peptide-hormone secretion and L1 starvation survival. Moreover, inactivation of ocr-2 caused a significant extension in adult lifespan. These findings indicate that TRPV channels, which mediate sensation of diverse noxious, thermal, osmotic, and mechanical stimuli, couple nutrient availability to larval starvation survival and adult lifespan through modulation of neural dense-core vesicle secretion.

Author(s): Kaveh Ashrafi [1, 2]; Francesca Y. Chang [1]; Jennifer L. Watts [3]; Andrew G. Fraser [4]; Ravi S. Kamath [4]; Julie Ahringer [4]; Gary Ruvkun (corresponding author) [1, 2] [...]

Sip2p and Snf1p kinase affect aging in Saccharomyces cerevisiae. It has been shown that deletion of the N-myristoylprotein, Sip2p, reduces resistance to nutrient deprivation and shortens the lifespan in S. cerevisiae through accelerated aging. The fast aging is seen in nucleolar fragmentation, loss of silencing from telomeres and mating loci, and accumulation of extrachromosomal rDNA. Ability to endure nutrient deprivation periods correlates directly with lifespan in many organisms. The change seen in glucose metabolism in the recent study discussed here is a new correlate of aging in yeast. It happens more and at earlier generational ages in sip2(Delta) cells.