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Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme whose function is important for the biosynthesis of purines, pyrimidines, and pyridines. Importantly, while missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, how they contribute to neuropathogenesis is still unclear. We identified the Drosophila ortholog of PRPS (dPRPS) as a direct target of RB/E2F in Drosophila, a vital cell cycle regulator, and engineered dPRPS alleles carrying patient-derived mutations. Interestingly, while they are able to develop normally, dPRPS mutant flies have a shortened lifespan and locomotive defects, common phenotypes associated with neurodegeneration. Careful analysis of the fat body revealed that patient-derived PRPS mutations result in profound defects in lipolysis, macroautophagy, and lysosome function. Significantly, we show evidence that the nervous system of dPRPS mutant flies is affected by these defects. Overall, we uncovered an unexpected link between nucleotide metabolism and autophagy/lysosome function, providing a possible mechanism by which PRPS-dysfunction contributes to neurological disorders. [ABSTRACT FROM AUTHOR]

Ageing populations pose one of the main public health crises of our time. Reprogramming gene expression by altering the activities of sequence-specific transcription factors (TFs) can ameliorate deleterious effects of age. Here we explore how a circuit of TFs coordinates pro-longevity transcriptional outcomes, which reveals a multi-tissue and multi-species role for an entire protein family: the E-twenty-six (ETS) TFs. In Drosophila, reduced insulin/IGF signalling (IIS) extends lifespan by coordinating activation of Aop, an ETS transcriptional repressor, and Foxo, a Forkhead transcriptional activator. Aop and Foxo bind the same genomic loci, and we show that, individually, they effect similar transcriptional programmes in vivo. In combination, Aop can both moderate or synergise with Foxo, dependent on promoter context. Moreover, Foxo and Aop oppose the gene-regulatory activity of Pnt, an ETS transcriptional activator. Directly knocking down Pnt recapitulates aspects of the Aop/Foxo transcriptional programme and is sufficient to extend lifespan. The lifespan-limiting role of Pnt appears to be balanced by a requirement for metabolic regulation in young flies, in which the Aop-Pnt-Foxo circuit determines expression of metabolic genes, and Pnt regulates lipolysis and responses to nutrient stress. Molecular functions are often conserved amongst ETS TFs, prompting us to examine whether other Drosophila ETS-coding genes may also affect ageing. We show that five out of eight Drosophila ETS TFs play a role in fly ageing, acting from a range of organs and cells including the intestine, adipose and neurons. We expand the repertoire of lifespan-limiting ETS TFs in C. elegans, confirming their conserved function in ageing and revealing that the roles of ETS TFs in physiology and lifespan are conserved throughout the family, both within and between species. [ABSTRACT FROM AUTHOR]

Schnyder corneal dystrophy (SCD) is a rare genetic eye disease characterized by corneal opacification resulted from deposition of excess free cholesterol. UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is an enzyme catalyzing biosynthesis of coenzyme Q10 and vitamin K2. More than 20 UBIAD1 mutations have been found to associate with human SCD. How these mutants contribute to SCD development is not fully understood. Here, we identified HMGCR as a binding partner of UBIAD1 using mass spectrometry. In contrast to the Golgi localization of wild-type UBIAD1, SCD-associated mutants mainly resided in the endoplasmic reticulum (ER) and competed with Insig-1 for HMGCR binding, thereby preventing HMGCR from degradation and increasing cholesterol biosynthesis. The heterozygous Ubiad1 G184R knock-in (Ubiad1G184R/+) mice expressed elevated levels of HMGCR protein in various tissues. The aged Ubiad1G184R/+ mice exhibited corneal opacification and free cholesterol accumulation, phenocopying clinical manifestations of SCD patients. In summary, these results demonstrate that SCD-associated mutations of UBIAD1 impair its ER-to-Golgi transportation and enhance its interaction with HMGCR. The stabilization of HMGCR by UBIAD1 increases cholesterol biosynthesis and eventually causes cholesterol accumulation in the cornea. [ABSTRACT FROM AUTHOR]

Berardinelli-Seip congenital generalized lipodystrophy is associated with increased bone mass suggesting that fat tissue regulates the skeleton. Because there is little mechanistic information regarding this issue, we generated "fat-free" (FF) mice completely lacking visible visceral, subcutaneous and brown fat. Due to robust osteoblastic activity, trabecular and cortical bone volume is markedly enhanced in these animals. FF mice, like Berardinelli-Seip patients, are diabetic but normalization of glucose tolerance and significant reduction in circulating insulin fails to alter their skeletal phenotype. Importantly, the skeletal phenotype of FF mice is completely rescued by transplantation of adipocyte precursors or white or brown fat depots, indicating that adipocyte derived products regulate bone mass. Confirming such is the case, transplantation of fat derived from adiponectin and leptin double knockout mice, unlike that obtained from their WT counterparts, fails to normalize FF bone. These observations suggest a paucity of leptin and adiponectin may contribute to the increased bone mass of Berardinelli-Seip patients. [ABSTRACT FROM AUTHOR]

Dipteran or “true” flies occupy nearly every terrestrial habitat, and have evolved to feed upon a wide variety of sources including fruit, pollen, decomposing animal matter, and even vertebrate blood. Here we analyze the molecular, genetic and cellular basis of odor response in the tsetse fly Glossina morsitans, which feeds on the blood of humans and their livestock, and is a vector of deadly trypanosomes. The G. morsitans antenna contains specialized subtypes of sensilla, some of which line a sensory pit not found in the fruit fly Drosophila. We characterize distinct patterns of G. morsitans Odor receptor (GmmOr) gene expression in the antenna. We devise a new version of the “empty neuron” heterologous expression system, and use it to functionally express several GmmOrs in a mutant olfactory receptor neuron (ORN) of Drosophila. GmmOr35 responds to 1-hexen-3-ol, an odorant found in human emanations, and also alpha-pinene, a compound produced by malarial parasites. Another receptor, GmmOr9, which is expressed in the sensory pit, responds to acetone, 2-butanone and 2-propanol. We confirm by electrophysiological recording that neurons of the sensory pit respond to these odorants. Acetone and 2-butanone are strong attractants long used in the field to trap tsetse. We find that 2-propanol is also an attractant for both G. morsitans and the related species G. fuscipes, a major vector of African sleeping sickness. The results identify 2-propanol as a candidate for an environmentally friendly and practical tsetse attractant. Taken together, this work characterizes the olfactory system of a highly distinct kind of fly, and it provides an approach to identifying new agents for controlling the fly and the devastating diseases that it carries. [ABSTRACT FROM AUTHOR]

The Drosophila protocadherins Dachsous and Fat regulate growth and tissue polarity by modulating the levels, membrane localization and polarity of the atypical myosin Dachs. Localization to the apical junctional membrane is critical for Dachs function, and the adapter protein Vamana/Dlish and palmitoyl transferase Approximated are required for Dachs membrane localization. However, how Dachs levels are regulated is poorly understood. Here we identify the early girl gene as playing an essential role in Fat signaling by limiting the levels of Dachs protein. early girl mutants display overgrowth of the wings and reduced cross vein spacing, hallmark features of mutations affecting Fat signaling. Genetic experiments reveal that it functions in parallel with Fat to regulate Dachs. early girl encodes an E3 ubiquitin ligase, physically interacts with Dachs, and regulates its protein stability. Concomitant loss of early girl and approximated results in accumulation of Dachs and Vamana in cytoplasmic punctae, suggesting that it also regulates their trafficking to the apical membrane. Our findings establish a crucial role for early girl in Fat signaling, involving regulation of Dachs and Vamana, two key downstream effectors of this pathway. [ABSTRACT FROM AUTHOR]

Signaling pathways that control the activities in non-photosynthetic plastids, important sites of plant metabolism, are largely unknown. Previously, we demonstrated that WRKY2 and WRKY34 transcription factors play an essential role in pollen development downstream of mitogen-activated protein kinase 3 (MPK3) and MPK6 in Arabidopsis. Here, we report that GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR 1 (GPT1) is a key target gene of WRKY2/WRKY34. GPT1 transports glucose-6-phosphate (Glc6P) into plastids for starch and/or fatty acid biosynthesis depending on the plant species. Loss of function of WRKY2/WRKY34 results in reduced GPT1 expression, and concomitantly, reduced accumulation of lipid bodies in mature pollen, which leads to compromised pollen viability, germination, pollen tube growth, and male transmission in Arabidopsis. Pollen-specific overexpression of GPT1 rescues the pollen defects of wrky2 wrky34 double mutant. Furthermore, gain-of-function activation of MPK3/MPK6 enhances GPT1 expression; whereas GPT1 expression is reduced in mkk4 mkk5 double mutant. Together, this study revealed a cytoplasmic/nuclear signaling pathway capable of coordinating the metabolic activities in plastids. High-level expression of GPT1 at late stages of pollen development drives Glc6P from cytosol into plastids, where Glc6P is used for fatty acid biosynthesis, an important step of lipid body biogenesis. The accumulation of lipid bodies during pollen maturation is essential to pollen fitness and successful reproduction. [ABSTRACT FROM AUTHOR]

The human 1q21.1 deletion of ten genes is associated with increased risk of schizophrenia. This deletion involves the β-subunit of the AMP-activated protein kinase (AMPK) complex, a key energy sensor in the cell. Although neurons have a high demand for energy and low capacity to store nutrients, the role of AMPK in neuronal physiology is poorly defined. Here we show that AMPK is important in the nervous system for maintaining neuronal integrity and for stress survival and longevity in Drosophila. To understand the impact of this signaling system on behavior and its potential contribution to the 1q21.1 deletion syndrome, we focused on sleep, an important role of which is proposed to be the reestablishment of neuronal energy levels that are diminished during energy-demanding wakefulness. Sleep disturbances are one of the most common problems affecting individuals with psychiatric disorders. We show that AMPK is required for maintenance of proper sleep architecture and for sleep recovery following sleep deprivation. Neuronal AMPKβ loss specifically leads to sleep fragmentation and causes dysregulation of genes believed to play a role in sleep homeostasis. Our data also suggest that AMPKβ loss may contribute to the increased risk of developing mental disorders and sleep disturbances associated with the human 1q21.1 deletion. [ABSTRACT FROM AUTHOR]

The control of the size of internal tubular organs, such as the lungs or vascular system, is critical for proper physiological activity and to prevent disease or malformations. This control incorporates the intrinsic physical anisotropy of tubes to generate proportionate organs that match their function. The exact mechanisms underlying tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. Here we investigate these mechanisms using the Drosophila tracheal system. We show that the apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. We provide evidence indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. We find that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. Our results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, our results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process. [ABSTRACT FROM AUTHOR]

Basement membranes (BMs) are thin sheet-like specialized extracellular matrices found at the basal surface of epithelia and endothelial tissues. They have been conserved across evolution and are required for proper tissue growth, organization, differentiation and maintenance. The major constituents of BMs are two independent networks of Laminin and Type IV Collagen in addition to the proteoglycan Perlecan and the glycoprotein Nidogen/entactin (Ndg). The ability of Ndg to bind in vitro Collagen IV and Laminin, both with key functions during embryogenesis, anticipated an essential role for Ndg in morphogenesis linking the Laminin and Collagen IV networks. This was supported by results from in vitro and cultured embryonic tissues experiments. However, the fact that elimination of Ndg in C. elegans and mice did not affect survival strongly questioned this proposed linking role. Here, we have isolated mutations in the only Ndg gene present in Drosophila. We find that while, similar to C.elegans and mice, Ndg is not essential for overall organogenesis or viability, it is required for appropriate fertility. We also find, alike in mice, tissue-specific requirements of Ndg for proper assembly and maintenance of certain BMs, namely those of the adipose tissue and flight muscles. In addition, we have performed a thorough functional analysis of the different Ndg domains in vivo. Our results support an essential requirement of the G3 domain for Ndg function and unravel a new key role for the Rod domain in regulating Ndg incorporation into BMs. Furthermore, uncoupling of the laminin and Collagen IV networks is clearly observed in the larval adipose tissue in the absence of Ndg, indeed supporting a linking role. In light of our findings, we propose that BM assembly and/or maintenance is tissue-specific, which could explain the diverse requirements of a ubiquitous conserved BM component like Nidogen. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer’s and Parkinson’s diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types in response to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient for expansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic and promoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-metFB Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions. [ABSTRACT FROM AUTHOR]

It is uncertain which β4-galactosyltransferase (β4GalT; gene name, B4galt), β4GalT-5 and/or β4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation. [ABSTRACT FROM AUTHOR]

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) in wheat and barley. Autophagy is a highly conserved vacuolar degradation pathway essential for cellular homeostasis in which Atg9 serves as a multispanning membrane protein important for generating membranes for the formation of phagophore assembly site. However, the mechanism of autophagy or autophagosome formation in phytopathogens awaits further clarifications. In this study, we identified and characterized the Atg9 homolog (FgAtg9) in F. graminearum by live cell imaging, biochemical and genetic analyses. We find that GFP-FgAtg9 localizes to late endosomes and trans-Golgi network under both nutrient-rich and nitrogen starvation conditions and also show its dynamic actin-dependent trafficking in the cell. Further targeted gene deletion of FgATG9 demonstrates that it is important for growth, aerial hyphae development, and pathogenicity in F. graminearum. Furthermore, the deletion mutant (ΔFgatg9) shows severe defects in autophagy and lipid metabolism in response to carbon starvation. Interestingly, small GTPase FgRab7 is found to be required for the dynamic trafficking of FgAtg9, and co-immunoprecipitation (Co-IP) assays show that FgAtg9 associates with FgRab7 in vivo. Finally, heterologous complementation assay shows that Atg9 is functionally conserved in F. graminearum and Magnaporthe oryzae. Taken together, we conclude that FgAtg9 is essential for autophagy-dependent development and pathogenicity of F. graminearum, which may be regulated by the small GTPase FgRab7. [ABSTRACT FROM AUTHOR]

Tri-methylation on histone H3 lysine 4 (H3K4me3) is associated with active gene expression but its regulatory role in transcriptional activation is unclear. Here we used Caenorhabditis elegans to investigate the connection between H3K4me3 and gene expression regulation during aging. We uncovered around 30% of H3K4me3 enriched regions to show significant and reproducible changes with age. We further showed that these age-dynamic H3K4me3 regions largely mark gene-bodies and are acquired during adult stages. We found that these adult-specific age-dynamic H3K4me3 regions are correlated with gene expression changes with age. In contrast, H3K4me3 marking established during developmental stages remained largely stable with age, even when the H3K4me3 associated genes exhibited RNA expression changes during aging. Importantly, the genes associated with changes in H3K4me3 and RNA levels with age are enriched for functional groups commonly implicated in aging biology. Therefore, our findings suggested divergent roles of H3K4me3 in gene expression regulation during aging, with important implications on aging-dependent pathophysiologies. [ABSTRACT FROM AUTHOR]

Polymyxin is the last line of defense against severe infections caused by carbapenem-resistant gram-negative pathogens. The emergence of transferable MCR-1/2 polymyxin resistance greatly challenges the renewed interest in colistin (polymyxin E) for clinical treatments. Recent studies have suggested that Moraxella species are a putative reservoir for MCR-1/2 genetic determinants. Here, we report the functional definition of ICR-Mo from M. osloensis, a chromosomally encoded determinant of colistin resistance, in close relation to current MCR-1/2 family. ICR-Mo transmembrane protein was prepared and purified to homogeneity. Taken along with an in vitro enzymatic detection, MALDI-TOF mass spectrometry of bacterial lipid A pools determined that the ICR-Mo enzyme might exploit a possible “ping-pong” mechanism to accept the hosphothanolmine (PEA) moiety from its donor hosphatidylthanolamine (PE) and then transfer it to the 1(or 4’)-phosphate position of lipid A via an ICR-Mo-bound PEA adduct. Structural decoration of LPS-lipid A by ICR-Mo renders the recipient strain of E. coli resistant to polymyxin. Domain swapping assays indicate that the two domains of ICR-Mo cannot be functionally-exchanged with its counterparts in MCR-1/2 and EptA, validating its phylogenetic position in a distinct set of MCR-like genes. Structure-guided functional mapping of ICR-Mo reveals a PE lipid substrate recognizing cavity having a role in enzymatic catalysis and the resultant conference of antibiotic resistance. Expression of icr-Mo in E. coli significantly prevents the formation of reactive oxygen species (ROS) induced by colistin. Taken together, our results define a member of a group of intrinsic colistin resistance genes phylogenetically close to the MCR-1/2 family, highlighting the evolution of transferable colistin resistance. [ABSTRACT FROM AUTHOR]

Human GWAS of obesity have been successful in identifying loci associated with adiposity, but for the most part, these are non-coding SNPs whose function, or even whose gene of action, is unknown. To help identify the genes on which these human BMI loci may be operating, we conducted a high throughput screen in Drosophila Melanogaster. Starting with 78 BMI loci from two recently published GWAS meta-analyses, we identified fly orthologs of all nearby genes (± 250KB). We crossed RNAi knockdown lines of each gene with flies containing tissue-specific drivers to knock down (KD) the expression of the genes only in the brain and the fat body. We then raised the flies on a control diet and compared the amount of fat/triglyceride in the tissue-specific KD group compared to the driver-only control flies. 16 of the 78 BMI GWAS loci could not be screened with this approach, as no gene in the 500-kb region had a fly ortholog. Of the remaining 62 GWAS loci testable in the fly, we found a significant fat phenotype in the KD flies for at least one gene for 26 loci (42%) even after correcting for multiple comparisons. By contrast, the rate of significant fat phenotypes in RNAi KD found in a recent genome-wide Drosophila screen (Pospisilik et al. (2010) is ~5%. More interestingly, for 10 of the 26 positive regions, we found that the nearest gene the one that showed a significant phenotype in the fly. Specifically, our screen suggests that for the 10 human BMI SNPs rs11057405, rs205262, rs9925964, rs9914578, rs2287019, rs11688816, rs13107325, rs7164727, rs17724992, and rs299412, the functional genes may NOT be the nearest ones (CLIP1, C6orf106, KAT8, SMG6, QPCTL, EHBP1, SLC39A8, ADPGK /ADPGK-AS1, PGPEP1, KCTD15, respectively), but instead, the specific nearby cis genes are the functional target (namely: ZCCHC8, VPS33A, RSRC2; SPDEF, NUDT3; PAGR1; SETD1, VKORC1; SGSM2, SRR; VASP, SIX5; OTX1; BANK1; ARIH1; ELL; CHST8, respectively). The study also suggests further functional experiments to elucidate mechanism of action for genes evolutionarily conserved for fat storage. [ABSTRACT FROM AUTHOR]

PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes. [ABSTRACT FROM AUTHOR]

The mechanisms by which the sensory environment influences metabolic homeostasis remains poorly understood. In this report, we show that oxygen, a potent environmental signal, is an important regulator of whole body lipid metabolism. C. elegans oxygen-sensing neurons reciprocally regulate peripheral lipid metabolism under normoxia in the following way: under high oxygen and food absence, URX sensory neurons are activated, and stimulate fat loss in the intestine, the major metabolic organ for C. elegans. Under lower oxygen conditions or when food is present, the BAG sensory neurons respond by repressing the resting properties of the URX neurons. A genetic screen to identify modulators of this effect led to the identification of a BAG-neuron-specific neuropeptide called FLP-17, whose cognate receptor EGL-6 functions in URX neurons. Thus, BAG sensory neurons counterbalance the metabolic effect of tonically active URX neurons via neuropeptide communication. The combined regulatory actions of these neurons serve to precisely tune the rate and extent of fat loss to the availability of food and oxygen, and provides an interesting example of the myriad mechanisms underlying homeostatic control. [ABSTRACT FROM AUTHOR]

Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson’s disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. Here, we investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. We show that dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. We further show that dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD. [ABSTRACT FROM AUTHOR]

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation. [ABSTRACT FROM AUTHOR]

Animals use taste to sample and ingest essential nutrients for survival. Free fatty acids (FAs) are energy-rich nutrients that contribute to various cellular functions. Recent evidence suggests FAs are detected through the gustatory system to promote feeding. In Drosophila, phospholipase C (PLC) signaling in sweet-sensing cells is required for FA detection but other signaling molecules are unknown. Here, we show Gr64e is required for the behavioral and electrophysiological responses to FAs. GR64e and TRPA1 are interchangeable when they act downstream of PLC: TRPA1 can substitute for GR64e in FA but not glycerol sensing, and GR64e can substitute for TRPA1 in aristolochic acid but not N-methylmaleimide sensing. In contrast to its role in FA sensing, GR64e functions as a ligand-gated ion channel for glycerol detection. Our results identify a novel FA transduction molecule and reveal that Drosophila Grs can act via distinct molecular mechanisms depending on context. [ABSTRACT FROM AUTHOR]

Fatty liver is a major health problem worldwide. People with hereditary deficiency of hormone-sensitive lipase (HSL) are reported to develop fatty liver. In this study, systemic and tissue-specific HSL-deficient mice were used as models to explore the underlying mechanism of this association. We found that systemic HSL deficient mice developed fatty liver in an age-dependent fashion between 3 and 8 months of age. To further explore the mechanism of fatty liver in HSL deficiency, liver-specific HSL knockout mice were created. Surprisingly, liver HSL deficiency did not influence liver fat content, suggesting that fatty liver in HSL deficiency is not liver autonomous. Given the importance of adipose tissue in systemic triglyceride metabolism, we created adipose-specific HSL knockout mice and found that adipose HSL deficiency, to a similar extent as systemic HSL deficiency, causes age-dependent fatty liver in mice. Mechanistic study revealed that deficiency of HSL in adipose tissue caused inflammatory macrophage infiltrates, progressive lipodystrophy, abnormal adipokine secretion and systemic insulin resistance. These changes in adipose tissue were associated with a constellation of changes in liver: low levels of fatty acid oxidation, of very low density lipoprotein secretion and of triglyceride hydrolase activity, each favoring the development of hepatic steatosis. In conclusion, HSL-deficient mice revealed a complex interorgan interaction between adipose tissue and liver: the role of HSL in the liver is minimal but adipose tissue deficiency of HSL can cause age-dependent hepatic steatosis. Adipose tissue is a potential target for treating the hepatic steatosis of HSL deficiency. [ABSTRACT FROM AUTHOR]

Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of free fatty acids (FAs), one of the building blocks of fat, promotes food consumption, activates reward circuitry, and is thought to contribute to hedonic feeding underlying many metabolism-related disorders. Despite a role in the etiology of metabolic diseases, little is known about how dietary fats are detected by the gustatory system to promote feeding. Previously, we showed that a broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (Gr64f) is required for reflexive feeding responses to both FAs and sugars. Here, we report a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid (FA) taste. We find neurons identified by expression of Ionotropic Receptor 56d (IR56d) are necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes FA taste, and their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding response to FAs. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in Drosophila. Taken together, these findings localize FA taste within the Drosophila gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants. [ABSTRACT FROM AUTHOR]

Dietary fatty acids can be incorporated directly into phospholipids. This poses a specific challenge to cellular membranes since their composition, hence properties, could greatly vary with different diets. That vast variations in diets are tolerated therefore implies the existence of regulatory mechanisms that monitor and regulate membrane compositions. Here we show that the adiponectin receptor AdipoR2, and its C. elegans homolog PAQR-2, are essential to counter the membrane rigidifying effects of exogenously provided saturated fatty acids. In particular, we use dietary supplements or mutated E. coli as food, together with direct measurements of membrane fluidity and composition, to show that diets containing a high ratio of saturated to monounsaturated fatty acids cause membrane rigidity and lethality in the paqr-2 mutant. We also show that mammalian cells in which AdipoR2 has been knocked-down by siRNA are unable to prevent the membrane-rigidifying effects of palmitic acid. We conclude that the PAQR-2 and AdipoR2 proteins share an evolutionarily conserved function that maintains membrane fluidity in the presence of exogenous saturated fatty acids. [ABSTRACT FROM AUTHOR]

Individuals choose their mates so as to maximize reproductive success, and one important component of this choice is assessment of traits reflecting mate quality. Little is known about why specific traits are used for mate quality assessment nor about how they reflect it. We have previously shown that global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in the fruit fly, Drosophila melanogaster. Here we demonstrate that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, here we describe a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells. [ABSTRACT FROM AUTHOR]

Preventing obesity requires a precise balance between deposition into and mobilization from fat stores, but regulatory mechanisms are incompletely understood. Drosophila Split ends (Spen) is the founding member of a conserved family of RNA-binding proteins involved in transcriptional regulation and frequently mutated in human cancers. We find that manipulating Spen expression alters larval fat levels in a cell-autonomous manner. Spen-depleted larvae had defects in energy liberation from stores, including starvation sensitivity and major changes in the levels of metabolic enzymes and metabolites, particularly those involved in β-oxidation. Spenito, a small Spen family member, counteracted Spen function in fat regulation. Finally, mouse Spen and Spenito transcript levels scaled directly with body fat in vivo, suggesting a conserved role in fat liberation and catabolism. This study demonstrates that Spen is a key regulator of energy balance and provides a molecular context to understand the metabolic defects that arise from Spen dysfunction. [ABSTRACT FROM AUTHOR]

Phenotypic variance heterogeneity across genotypes at a single nucleotide polymorphism (SNP) may reflect underlying gene-environment (G×E) or gene-gene interactions. We modeled variance heterogeneity for blood lipids and BMI in up to 44,211 participants and investigated relationships between variance effects (Pv), G×E interaction effects (with smoking and physical activity), and marginal genetic effects (Pm). Correlations between Pv and Pm were stronger for SNPs with established marginal effects (Spearman’s ρ = 0.401 for triglycerides, and ρ = 0.236 for BMI) compared to all SNPs. When Pv and Pm were compared for all pruned SNPs, only BMI was statistically significant (Spearman’s ρ = 0.010). Overall, SNPs with established marginal effects were overrepresented in the nominally significant part of the Pv distribution (Pbinomial <0.05). SNPs from the top 1% of the Pm distribution for BMI had more significant Pv values (PMann–Whitney = 1.46×10−5), and the odds ratio of SNPs with nominally significant (<0.05) Pm and Pv was 1.33 (95% CI: 1.12, 1.57) for BMI. Moreover, BMI SNPs with nominally significant G×E interaction P-values (Pint<0.05) were enriched with nominally significant Pv values (Pbinomial = 8.63×10−9 and 8.52×10−7 for SNP × smoking and SNP × physical activity, respectively). We conclude that some loci with strong marginal effects may be good candidates for G×E, and variance-based prioritization can be used to identify them. [ABSTRACT FROM AUTHOR]

It is now established that the central nervous system plays an important role in regulating whole body metabolism and energy balance. However, the extent to which sensory systems relay environmental information to modulate metabolic events in peripheral tissues has remained poorly understood. In addition, it has been challenging to map the molecular mechanisms underlying discrete sensory modalities with respect to their role in lipid metabolism. In previous work our lab has identified instructive roles for serotonin signaling as a surrogate for food availability, as well as oxygen sensing, in the control of whole body metabolism. In this study, we now identify a role for a pair of pheromone-sensing neurons in regulating fat metabolism in C. elegans, which has emerged as a tractable and highly informative model to study the neurobiology of metabolism. A genetic screen revealed that GPA-3, a member of the Gα family of G proteins, regulates body fat content in the intestine, the major metabolic organ for C. elegans. Genetic and reconstitution studies revealed that the potent body fat phenotype of gpa-3 null mutants is controlled from a pair of neurons called ADL(L/R). We show that cAMP functions as the second messenger in the ADL neurons, and regulates body fat stores via the neurotransmitter acetylcholine, from downstream neurons. We find that the pheromone ascr#3, which is detected by the ADL neurons, regulates body fat stores in a GPA-3-dependent manner. We define here a third sensory modality, pheromone sensing, as a major regulator of body fat metabolism. The pheromone ascr#3 is an indicator of population density, thus we hypothesize that pheromone sensing provides a salient 'denominator' to evaluate the amount of food available within a population and to accordingly adjust metabolic rate and body fat levels. [ABSTRACT FROM AUTHOR]

Joint genetic models for multiple traits have helped to enhance association analyses. Most existing multi-trait models have been designed to increase power for detecting associations, whereas the analysis of interactions has received considerably less attention. Here, we propose iSet, a method based on linear mixed models to test for interactions between sets of variants and environmental states or other contexts. Our model generalizes previous interaction tests and in particular provides a test for local differences in the genetic architecture between contexts. We first use simulations to validate iSet before applying the model to the analysis of genotype-environment interactions in an eQTL study. Our model retrieves a larger number of interactions than alternative methods and reveals that up to 20% of cases show context-specific configurations of causal variants. Finally, we apply iSet to test for sub-group specific genetic effects in human lipid levels in a large human cohort, where we identify a gene-sex interaction for C-reactive protein that is missed by alternative methods. [ABSTRACT FROM AUTHOR]

The general transcription factor TBP (TATA-box binding protein) and its associated factors (TAFs) together form the TFIID complex, which directs transcription initiation. Through RNAi and mutant analysis, we identified a specific TBP family protein, TRF2, and a set of TAFs that regulate lipid droplet (LD) size in the Drosophila larval fat body. Among the three Drosophila TBP genes, trf2, tbp and trf1, only loss of function of trf2 results in increased LD size. Moreover, TRF2 and TAF9 regulate fatty acid composition of several classes of phospholipids. Through RNA profiling, we found that TRF2 and TAF9 affects the transcription of a common set of genes, including peroxisomal fatty acid β-oxidation-related genes that affect phospholipid fatty acid composition. We also found that knockdown of several TRF2 and TAF9 target genes results in large LDs, a phenotype which is similar to that of trf2 mutants. Together, these findings provide new insights into the specific role of the general transcription machinery in lipid homeostasis. [ABSTRACT FROM AUTHOR]

The heart has emerged as an important organ in the regulation of systemic lipid homeostasis; however, the underlying mechanism remains poorly understood. Here, we show that Drosophila cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body. In a Drosophila genetic screen, we discovered that when haplo-insufficient, microsomal triglyceride transfer protein (mtp), required for the biosynthesis of apoB-lipoproteins, suppressed the development of diet-induced obesity. Tissue-specific inhibition of Mtp revealed that whereas knockdown of mtp only in the fat body decreases systemic triglyceride (TG) content on normal food diet (NFD) as expected, knockdown of mtp only in the cardiomyocytes also equally decreases systemic TG content on NFD, suggesting that the cardiomyocyte- and fat body-derived apoB-lipoproteins serve similarly important roles in regulating whole-body lipid metabolism. Unexpectedly, on high fat diet (HFD), knockdown of mtp in the cardiomyocytes, but not in fat body, protects against the gain in systemic TG levels. We further showed that inhibition of the Drosophila apoB homologue, apolipophorin or apoLpp, another gene essential for apoB-lipoprotein biosynthesis, affects systemic TG levels similarly to that of Mtp inhibition in the cardiomyocytes on NFD or HFD. Finally, we determined that HFD differentially alters Mtp and apoLpp expression in the cardiomyocytes versus the fat body, culminating in higher Mtp and apoLpp levels in the cardiomyocytes than in fat body and possibly underlying the predominant role of cardiomyocyte-derived apoB-lipoproteins in lipid metabolic regulation. Our findings reveal a novel and significant function of heart-mediated apoB-lipoproteins in controlling lipid homeostasis. [ABSTRACT FROM AUTHOR]

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point. [ABSTRACT FROM AUTHOR]

Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. [ABSTRACT FROM AUTHOR]

While most yeast enzymes for the biosynthesis of glycerophospholipids, sphingolipids and ergosterol are known, genes for several postulated transporters allowing the flopping of biosynthetic intermediates and newly made lipids from the cytosolic to the lumenal side of the membrane are still not identified. An E-MAP measuring the growth of 142'108 double mutants generated by systematically crossing 543 hypomorphic or deletion alleles in genes encoding multispan membrane proteins, both on media with or without an inhibitor of fatty acid synthesis, was generated. Flc proteins, represented by 4 homologous genes encoding presumed FAD or calcium transporters of the ER, have a severe depression of sphingolipid biosynthesis and elevated detergent sensitivity of the ER. FLC1, FLC2 and FLC3 are redundant in granting a common function, which remains essential even when the severe cell wall defect of flc mutants is compensated by osmotic support. Biochemical characterization of some other genetic interactions shows that Cst26 is the enzyme mainly responsible for the introduction of saturated very long chain fatty acids into phosphatidylinositol and that the GPI lipid remodelase Cwh43, responsible for introducing ceramides into GPI anchors having a C26:0 fatty acid in sn-2 of the glycerol moiety can also use lyso-GPI protein anchors and various base resistant lipids as substrates. Furthermore, we observe that adjacent deletions in several chromosomal regions show strong negative genetic interactions with a single gene on another chromosome suggesting the presence of undeclared suppressor mutations in certain chromosomal regions that need to be identified in order to yield meaningful E-map data. [ABSTRACT FROM AUTHOR]

Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds. [ABSTRACT FROM AUTHOR]

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival. [ABSTRACT FROM AUTHOR]

Familial combined hyperlipidemia (FCH) is a complex and common familial dyslipidemia characterized by elevated total cholesterol and/or triglyceride levels with over five-fold risk of coronary heart disease. The genetic architecture and contribution of rare Mendelian and common variants to FCH susceptibility is unknown. In 53 Finnish FCH families, we genotyped and imputed nine million variants in 715 family members with DNA available. We studied the enrichment of variants previously implicated with monogenic dyslipidemias and/or lipid levels in the general population by comparing allele frequencies between the FCH families and population samples. We also constructed weighted polygenic scores using 212 lipid-associated SNPs and estimated the relative contributions of Mendelian variants and polygenic scores to the risk of FCH in the families. We identified, across the whole allele frequency spectrum, an enrichment of variants known to elevate, and a deficiency of variants known to lower LDL-C and/or TG levels among both probands and affected FCH individuals. The score based on TG associated SNPs was particularly high among affected individuals compared to non-affected family members. Out of 234 affected FCH individuals across the families, seven (3%) carried Mendelian variants and 83 (35%) showed high accumulation of either known LDL-C or TG elevating variants by having either polygenic score over the 90th percentile in the population. The positive predictive value of high score was much higher for affected FCH individuals than for similar sporadic cases in the population. FCH is highly polygenic, supporting the hypothesis that variants across the whole allele frequency spectrum contribute to this complex familial trait. Polygenic SNP panels improve identification of individuals affected with FCH, but their clinical utility remains to be defined. [ABSTRACT FROM AUTHOR]

TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes. [ABSTRACT FROM AUTHOR]

In spite of the worldwide impact of diabetes on human health, the mechanisms behind glucose toxicity remain elusive. Here we show that C. elegans mutants lacking paqr-2, the worm homolog of the adiponectin receptors AdipoR1/2, or its newly identified functional partner iglr-2, are glucose intolerant and die in the presence of as little as 20 mM glucose. Using FRAP (Fluorescence Recovery After Photobleaching) on living worms, we found that cultivation in the presence of glucose causes a decrease in membrane fluidity in paqr-2 and iglr-2 mutants and that genetic suppressors of this sensitivity act to restore membrane fluidity by promoting fatty acid desaturation. The essential roles of paqr-2 and iglr-2 in the presence of glucose are completely independent from daf-2 and daf-16, the C. elegans homologs of the insulin receptor and its downstream target FoxO, respectively. Using bimolecular fluorescence complementation, we also show that PAQR-2 and IGLR-2 interact on plasma membranes and thus may act together as a fluidity sensor that controls membrane lipid composition. [ABSTRACT FROM AUTHOR]

SIRT1 is a member of the sirtuin family of NAD+-dependent deacetylases, which couple cellular metabolism to systemic physiology. Although studies in mouse models have defined a central role for SIRT1 in maintaining metabolic health, the molecular mechanisms remain unclear. Here we show that loss of the Drosophila SIRT1 homolog sir2 leads to the age-progressive onset of hyperglycemia, obesity, glucose intolerance, and insulin resistance. Tissue-specific functional studies show that Sir2 is both necessary and sufficient in the fat body (analogous to the mammalian liver) to maintain glucose homeostasis and peripheral insulin sensitivity. Transcriptional profiling of sir2 mutants by RNA-seq revealed a major overlap with genes regulated by the nuclear receptor Hepatocyte Nuclear Factor 4 (HNF4). Consistent with this, Drosophila HNF4 mutants display diabetic phenotypes similar to those of sir2 mutants, and protein levels for dHNF4 are reduced in sir2 mutant animals. We show that Sir2 exerts these effects by deacetylating and stabilizing dHNF4 through protein interactions. Increasing dHNF4 expression in sir2 mutants is sufficient to rescue their insulin signaling defects, defining this nuclear receptor as an important downstream effector of Sir2 signaling. This study demonstrates that the key metabolic activities of SIRT1 have been conserved through evolution, provides a genetic model for functional studies of phenotypes related to type 2 diabetes, and establishes HNF4 as a critical downstream target by which Sir2 maintains metabolic health. [ABSTRACT FROM AUTHOR]

The accurate diagnosis and clinical management of the growth restriction disorder Silver Russell Syndrome (SRS) has confounded researchers and clinicians for many years due to the myriad of genetic and epigenetic alterations reported in these patients and the lack of suitable animal models to test the contribution of specific gene alterations. Some genetic alterations suggest a role for increased dosage of the imprinted CYCLIN DEPENDENT KINASE INHIBITOR 1C (CDKN1C) gene, often mutated in IMAGe Syndrome and Beckwith-Wiedemann Syndrome (BWS). Cdkn1c encodes a potent negative regulator of fetal growth that also regulates placental development, consistent with a proposed role for CDKN1C in these complex childhood growth disorders. Here, we report that a mouse modelling the rare microduplications present in some SRS patients exhibited phenotypes including low birth weight with relative head sparing, neonatal hypoglycemia, absence of catch-up growth and significantly reduced adiposity as adults, all defining features of SRS. Further investigation revealed the presence of substantially more brown adipose tissue in very young mice, of both the classical or canonical type exemplified by interscapular-type brown fat depot in mice (iBAT) and a second type of non-classic BAT that develops postnatally within white adipose tissue (WAT), genetically attributable to a double dose of Cdkn1c in vivo and ex-vivo. Conversely, loss-of-function of Cdkn1c resulted in the complete developmental failure of the brown adipocyte lineage with a loss of markers of both brown adipose fate and function. We further show that Cdkn1c is required for post-transcriptional accumulation of the brown fat determinant PR domain containing 16 (PRDM16) and that CDKN1C and PRDM16 co-localise to the nucleus of rare label-retaining cell within iBAT. This study reveals a key requirement for Cdkn1c in the early development of the brown adipose lineages. Importantly, active BAT consumes high amounts of energy to generate body heat, providing a valid explanation for the persistence of thinness in our model and supporting a major role for elevated CDKN1C in SRS. [ABSTRACT FROM AUTHOR]

Although genome-wide association studies (GWASs) have discovered numerous novel genetic variants associated with many complex traits and diseases, those genetic variants typically explain only a small fraction of phenotypic variance. Factors that account for phenotypic variance include environmental factors and gene-by-environment interactions (GEIs). Recently, several studies have conducted genome-wide gene-by-environment association analyses and demonstrated important roles of GEIs in complex traits. One of the main challenges in these association studies is to control effects of population structure that may cause spurious associations. Many studies have analyzed how population structure influences statistics of genetic variants and developed several statistical approaches to correct for population structure. However, the impact of population structure on GEI statistics in GWASs has not been extensively studied and nor have there been methods designed to correct for population structure on GEI statistics. In this paper, we show both analytically and empirically that population structure may cause spurious GEIs and use both simulation and two GWAS datasets to support our finding. We propose a statistical approach based on mixed models to account for population structure on GEI statistics. We find that our approach effectively controls population structure on statistics for GEIs as well as for genetic variants. [ABSTRACT FROM AUTHOR]

Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity. [ABSTRACT FROM AUTHOR]

Intracellular triacylglycerol (TAG) is a ubiquitous energy storage lipid also involved in lipid homeostasis and signaling. Comparatively, little is known about TAG’s role in other cellular functions. Here we show a pro-longevity function of TAG in the budding yeast Saccharomyces cerevisiae. In yeast strains derived from natural and laboratory environments a correlation between high levels of TAG and longer chronological lifespan was observed. Increased TAG abundance through the deletion of TAG lipases prolonged chronological lifespan of laboratory strains, while diminishing TAG biosynthesis shortened lifespan without apparently affecting vegetative growth. TAG-mediated lifespan extension was independent of several other known stress response factors involved in chronological aging. Because both lifespan regulation and TAG metabolism are conserved, this cellular pro-longevity function of TAG may extend to other organisms. [ABSTRACT FROM AUTHOR]

We report the first systems biology investigation of regulators controlling arterial plaque macrophage transcriptional changes in response to lipid lowering in vivo in two distinct mouse models of atherosclerosis regression. Transcriptome measurements from plaque macrophages from the Reversa mouse were integrated with measurements from an aortic transplant-based mouse model of plaque regression. Functional relevance of the genes detected as differentially expressed in plaque macrophages in response to lipid lowering in vivo was assessed through analysis of gene functional annotations, overlap with in vitro foam cell studies, and overlap of associated eQTLs with human atherosclerosis/CAD risk SNPs. To identify transcription factors that control plaque macrophage responses to lipid lowering in vivo, we used an integrative strategy – leveraging macrophage epigenomic measurements – to detect enrichment of transcription factor binding sites upstream of genes that are differentially expressed in plaque macrophages during regression. The integrated analysis uncovered eight transcription factor binding site elements that were statistically overrepresented within the 5′ regulatory regions of genes that were upregulated in plaque macrophages in the Reversa model under maximal regression conditions and within the 5′ regulatory regions of genes that were upregulated in the aortic transplant model during regression. Of these, the TCF/LEF binding site was present in promoters of upregulated genes related to cell motility, suggesting that the canonical Wnt signaling pathway may be activated in plaque macrophages during regression. We validated this network-based prediction by demonstrating that β-catenin expression is higher in regressing (vs. control group) plaques in both regression models, and we further demonstrated that stimulation of canonical Wnt signaling increases macrophage migration in vitro. These results suggest involvement of canonical Wnt signaling in macrophage emigration from the plaque during lipid lowering-induced regression, and they illustrate the discovery potential of an epigenome-guided, systems approach to understanding atherosclerosis regression. [ABSTRACT FROM AUTHOR]