A hormone-dependent module regulating energy balance.

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.

Crtc modulates fasting programs associated with 1-C metabolism and inhibition of insulin signaling.

Fasting in mammals promotes increases in circulating glucagon and decreases in circulating insulin that stimulate catabolic programs and facilitate a transition from glucose to lipid burning. The second messenger cAMP mediates effects of glucagon on fasting metabolism, in part by promoting the phosphorylation of CREB and the dephosphorylation of the cAMP-regulated transcriptional coactivators (CRTCs) in hepatocytes. In Drosophila, fasting also triggers activation of the single Crtc homolog in neurons, via the PKA-mediated phosphorylation and inhibition of salt-inducible kinases. Crtc mutant flies are more sensitive to starvation and oxidative stress, although the underlying mechanism remains unclear. Here we use RNA sequencing to identify Crtc target genes that are up-regulated in response to starvation. We found that Crtc stimulates a subset of fasting-inducible genes that have conserved CREB binding sites. In keeping with its role in the starvation response, Crtc was found to induce the expression of genes that inhibit insulin secretion (Lst) and insulin signaling (Impl2). In parallel, Crtc also promoted the expression of genes involved in one-carbon (1-C) metabolism. Within the 1-C pathway, Crtc stimulated the expression of enzymes that encode modulators of S-adenosyl-methionine metabolism (Gnmt and Sardh) and purine synthesis (ade2 and AdSl) Collectively, our results point to an important role for the CREB/CRTC pathway in promoting energy balance in the context of nutrient stress.

Sustainable Lithium Recovery from Hypersaline Salt-Lakes by Selective Electrodialysis: Transport and Thermodynamics.

Evaporative technology for lithium mining from salt-lakes exacerbates freshwater scarcity and wetland destruction, and suffers from protracted production cycles. Electrodialysis (ED) offers an environmentally benign alternative for continuous lithium extraction and is amenable to renewable energy usage. Salt-lake brines, however, are hypersaline multicomponent mixtures, and the impact of the complex brine-membrane interactions remains poorly understood. Here, we quantify the influence of the solution composition, salinity, and acidity on the counterion selectivity and thermodynamic efficiency of electrodialysis, leveraging 1250 original measurements with salt-lake brines that span four feed salinities, three pH levels, and five current densities. Our experiments reveal that commonly used binary cation solutions, which neglect Na+ and K+ transport, may overestimate the Li+/Mg2+ selectivity by 250% and underpredict the specific energy consumption (SEC) by a factor of 54.8. As a result of the hypersaline conditions, exposure to salt-lake brine weakens the efficacy of Donnan exclusion, amplifying Mg2+ leakage. Higher current densities enhance the Donnan potential across the solution-membrane interface and ameliorate the selectivity degradation with hypersaline brines. However, a steep trade-off between counterion selectivity and thermodynamic efficiency governs ED's performance: a 6.25 times enhancement in Li+/Mg2+ selectivity is accompanied by a 71.6% increase in the SEC. Lastly, our analysis suggests that an industrial-scale ED module can meet existing salt-lake production capacities, while being powered by a photovoltaic farm that utilizes <1% of the salt-flat area.

Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance.

The starvation-inducible coactivator cAMP response element binding protein (CREB)-cAMP-regulated transcription coactivator (Crtc) has been shown to promote starvation resistance in Drosophila by up-regulating CREB target gene expression in neurons, although the underlying mechanism is unclear. We found that Crtc and its binding partner CREB enhance energy homeostasis by stimulating the expression of short neuropeptide F (sNPF), an ortholog of mammalian neuropeptide Y, which we show here is a direct target of CREB and Crtc. Neuronal sNPF was found to promote energy homeostasis via gut enterocyte sNPF receptors, which appear to maintain gut epithelial integrity. Loss of Crtc-sNPF signaling disrupted epithelial tight junctions, allowing resident gut flora to promote chronic increases in antimicrobial peptide (AMP) gene expression that compromised energy balance. Growth on germ-free food reduced AMP gene expression and rescued starvation sensitivity in Crtc mutant flies. Overexpression of Crtc or sNPF in neurons of wild-type flies dampens the gut immune response and enhances starvation resistance. Our results reveal a previously unidentified tolerance defense strategy involving a brain-gut pathway that maintains homeostasis through its effects on epithelial integrity.

Equivalent Activities of Repulsive Axon Guidance Receptors.

Receptors on the growth cone at the leading edge of elongating axons play critical guidance roles by recognizing cues via their extracellular domains and transducing signals via their intracellular domains, resulting in changes in direction of growth. An important concept to have emerged in the axon guidance field is the importance of repulsion as a major guidance mechanism. Given the number and variety of different repulsive receptors, it is generally thought that there are likely to be qualitative differences in the signals they transduce. However, the nature of these possible differences is unknown. By creating chimeras using the extracellular and intracellular domains of three different Drosophila repulsive receptors, Unc5, Roundabout (Robo), and Derailed (Drl) and expressing them in defined cells within the embryonic nervous system, we examined the responses elicited by their intracellular domains systematically. Surprisingly, we found no qualitative differences in growth cone response or axon growth, suggesting that, despite their highly diverged sequences, each intracellular domain elicits repulsion via a common pathway. In terms of the signaling pathway(s) used by the repulsive receptors, mutations in the guanine nucleotide exchange factor Trio strongly enhance the repulsive activity of all three intracellular domains, suggesting that repulsion by Unc5, Robo, and Drl, and perhaps repulsion in general, involves Trio activity. SIGNIFICANCE STATEMENT: A prevailing concept that has emerged in the axon guidance field is the importance of repulsion as a guidance mechanism for steering axons to their appropriate targets. Given the number and variety of different repulsive receptors, it is generally thought that there are differences in the signals that they transduce. However, this has never been tested directly. We have used the advanced genetics of Drosophila to compare directly the outputs of different repulsive receptors. Surprisingly, we found no qualitative differences in receptor-mediated repulsion, suggesting that, despite their highly diverged domain structure, each receptor couples to a common repulsive pathway. We went on to show that this common pathway involves Trio, a guanine nucleotide exchange factor known to promote cytoskeletal remodeling.

A subset of interneurons required for Drosophila larval locomotion.

Efforts to define the neural circuits generating locomotor behavior have produced an initial understanding of some of the components within the spinal cord, as well as a basic understanding of several invertebrate motor pattern generators. However, how these circuits are assembled during development is poorly understood. We are defining the neural circuit that generates larval locomotion in the genetically tractable fruit fly Drosophila melanogaster to study locomotor circuit development. Forward larval locomotion involves a stereotyped posterior-to-anterior segmental translocation of body wall muscle contraction and is generated by a relatively small number of identified muscles, motor and sensory neurons, plus an unknown number of the ~270 bilaterally-paired interneurons per segment of the 1st instar larva. To begin identifying the relevant interneurons, we have conditionally inactivated synaptic transmission of interneuron subsets and assayed for the effects on locomotion. From this screen we have identified a subset of 25 interneurons per hemisegment, called the lateral locomotor neurons (LLNs), that are required for locomotion. Both inactivation and constitutive activation of the LLNs disrupt locomotion, indicating that patterned output of the LLNs is required. By expressing a calcium indicator in the LLNs, we found that they display a posterior-to-anterior wave of activity within the CNS corresponding to the segmental translocation of the muscle contraction wave. Identification of the LLNs represents the first step toward elucidating the circuit generating larval locomotion.

A kinome-wide RNAi screen in Drosophila Glia reveals that the RIO kinases mediate cell proliferation and survival through TORC2-Akt signaling in glioblastoma.

Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK) and Pi-3 kinase (PI3K) signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

The nutrient sensor CRTC and Sarcalumenin/thinman represent an alternate pathway in cardiac hypertrophy.

CREB-regulated transcription co-activator (CRTC) is activated by Calcineurin (CaN) to regulate gluconeogenic genes. CaN also has roles in cardiac hypertrophy. Here, we explore a cardiac-autonomous role for CRTC in cardiac hypertrophy. In Drosophila, CRTC mutants exhibit severe cardiac restriction, myofibrillar disorganization, fibrosis, and tachycardia. Cardiac-specific CRTC knockdown (KD) phenocopies mutants, and cardiac overexpression causes hypertrophy. CaN-induced hypertrophy in Drosophila is reduced in CRTC mutants, suggesting that CRTC mediates the effects. RNA sequencing (RNA-seq) of CRTC-KD and -overexpressing hearts reveals contraregulation of metabolic genes. Genes with conserved CREB sites include the fly ortholog of Sarcalumenin, a Ca2+-binding protein. Cardiac manipulation of this gene recapitulates the CRTC-KD and -overexpression phenotypes. CRTC KD in zebrafish also causes cardiac restriction, and CRTC KD in human induced cardiomyocytes causes a reduction in Srl expression and increased action potential duration. Our data from three model systems suggest that CaN-CRTC-Sarcalumenin signaling represents an alternate, conserved pathway underlying cardiac function and hypertrophy.

The nutrient sensor CRTC & Sarcalumenin / Thinman represent a new pathway in cardiac hypertrophy.

Obesity and type 2 diabetes are at epidemic levels and a significant proportion of these patients are diagnosed with left ventricular hypertrophy. CREB R egulated T ranscription C o-activator ( CRTC ) is a key regulator of metabolism in mammalian hepatocytes, where it is activated by calcineurin (CaN) to increase expression of gluconeogenic genes. CaN is known its role in pathological cardiac hypertrophy, however, a role for CRTC in the heart has not been identified. In Drosophila , CRTC null mutants have little body fat and exhibit severe cardiac restriction, myofibrillar disorganization, cardiac fibrosis and tachycardia, all hallmarks of heart disease. Cardiac-specific knockdown of CRTC , or its coactivator CREBb , mimicked the reduced body fat and heart defects of CRTC null mutants. Comparative gene expression in CRTC loss- or gain-of-function fly hearts revealed contra-regulation of genes involved in glucose, fatty acid, and amino acid metabolism, suggesting that CRTC also acts as a metabolic switch in the heart. Among the contra-regulated genes with conserved CREB binding sites, we identified the fly ortholog of Sarcalumenin, which is a Ca 2+ -binding protein in the sarcoplasmic reticulum. Cardiac knockdown recapitulated the loss of CRTC cardiac restriction and fibrotic phenotypes, suggesting it is a downstream effector of CRTC we named thinman ( tmn ). Importantly, cardiac overexpression of either CaN or CRTC in flies caused hypertrophy that was reversed in a CRTC mutant background, suggesting CRTC mediates hypertrophy downstream of CaN, perhaps as an alternative to NFAT. CRTC novel role in the heart is likely conserved in vertebrates as knockdown in zebrafish also caused cardiac restriction, as in fl ies. These data suggest that CRTC is involved in myocardial cell maintenance and that CaN-CRTC- Sarcalumenin/ tmn signaling represents a novel and conserved pathway underlying cardiac hypertrophy.

A drosophila model for EGFR-Ras and PI3K-dependent human glioma.

Gliomas, the most common malignant tumors of the nervous system, frequently harbor mutations that activate the epidermal growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) signaling pathways. To investigate the genetic basis of this disease, we developed a glioma model in Drosophila. We found that constitutive coactivation of EGFR-Ras and PI3K pathways in Drosophila glia and glial precursors gives rise to neoplastic, invasive glial cells that create transplantable tumor-like growths, mimicking human glioma. Our model represents a robust organotypic and cell-type-specific Drosophila cancer model in which malignant cells are created by mutations in signature genes and pathways thought to be driving forces in a homologous human cancer. Genetic analyses demonstrated that EGFR and PI3K initiate malignant neoplastic transformation via a combinatorial genetic network composed primarily of other pathways commonly mutated or activated in human glioma, including the Tor, Myc, G1 Cyclins-Cdks, and Rb-E2F pathways. This network acts synergistically to coordinately stimulate cell cycle entry and progression, protein translation, and inappropriate cellular growth and migration. In particular, we found that the fly orthologs of CyclinE, Cdc25, and Myc are key rate-limiting genes required for glial neoplasia. Moreover, orthologs of Sin1, Rictor, and Cdk4 are genes required only for abnormal neoplastic glial proliferation but not for glial development. These and other genes within this network may represent important therapeutic targets in human glioma.

Differentially expressed Drl and Drl-2 play opposing roles in Wnt5 signaling during Drosophila olfactory system development.

In Drosophila, odor information received by olfactory receptor neurons (ORNs) is processed by glomeruli, which are organized in a stereotypic manner in the antennal lobe (AL). This glomerular organization is regulated by Wnt5 signaling. In the embryonic CNS, Wnt5 signaling is transduced by the Drl receptor, a member of the Ryk family. During development of the olfactory system, however, it is antagonized by Drl. Here, we identify Drl-2 as a receptor mediating Wnt5 signaling. Drl is found in the neurites of brain cells in the AL and specific glia, whereas Drl-2 is predominantly found in subsets of growing ORN axons. A drl-2 mutation produces only mild deficits in glomerular patterning, but when it is combined with a drl mutation, the phenotype is exacerbated and more closely resembles the Wnt5 phenotype. Wnt5 overexpression in ORNs induces aberrant glomeruli positioning. This phenotype is ameliorated in the drl-2 mutant background, indicating that Drl-2 mediates Wnt5 signaling. In contrast, forced expression of Drl-2 in the glia of drl mutants rescues the glomerular phenotype caused by the loss of antagonistic Drl function. Therefore, Drl-2 can also antagonize Wnt5 signaling. Additionally, our genetic data suggest that Drl localized to developing glomeruli mediates Wnt5 signaling. Thus, these two members of the Ryk family are capable of carrying out a similar molecular function, but they can play opposing roles in Wnt5 signaling, depending on the type of cells in which they are expressed. These molecules work cooperatively to establish the olfactory circuitry in Drosophila.

Wnt4 is a local repulsive cue that determines synaptic target specificity.

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. We conducted single-cell microarray analysis of two neighboring muscles (M12 and M13) in Drosophila, which are innervated by distinct motor neurons, by directly isolating them from dissected embryos. We identified a number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity.

The homeobox transcription factor even-skipped regulates netrin-receptor expression to control dorsal motor-axon projections in Drosophila.

Homeobox transcription-factor codes control motor-neuron subtype identity and dorsal versus ventral axon guidance in both vertebrate and invertebrate nervous systems; however, the specific axon guidance-receptors that are regulated by these transcription factors to control pathfinding are poorly defined. In Drosophila, the Even-skipped (Eve) transcription factor specifies dorsal motor-axon projection through the regulation of unidentified guidance molecules. The Netrins and their attractive and repulsive receptors DCC and Unc-5, respectively, define important conserved cue and receptor families that control growth-cone guidance. In Drosophila, the Netrins and frazzled (the fly homolog of DCC) contribute to motor-axon guidance. Here, using genetics and single-cell mRNA-expression analysis, we show that expression and requirement of different Netrin receptor combinations correlate with distinct dorsal and ventral motor-axon projections in Drosophila. Mis-expression of eve dorsalizes ventral axons in part through the upregulation of Unc-5, whereas loss of eve function in two dorsally projecting motor neurons results in aberrant axon projections and a failure to express Unc-5. Our results support a functional link between the expression of distinct Netrin receptor combinations and the transcriptional control of dorsal motor-axon guidance.

Motor neuron specification in worms, flies and mice: conserved and 'lost' mechanisms.

Motor neuron differentiation has been studied intensively in both invertebrates and vertebrates in recent years. These studies have led to the identification of several key regulatory genes acting to generate motor neurons and to specify their subclass identities. By comparing findings from Caenorhabditis elegans, Drosophila and vertebrate model systems, it is apparent that both evolutionarily conserved and non-conserved mechanisms are used.

Secreted cell signaling molecules in axon guidance.

The growth cones of developing neurons respond to specific guidance cues in their extracellular environment. Recent studies have shown that secreted signaling molecules from protein families that are best known for their roles as morphogens in specifying cell fate can function as axon guidance molecules. These signaling molecules seem to act directly on the growth cone and thus are likely to activate non-canonical signaling pathways that are coupled to the cytoskeleton.

Mutation of Drosophila homer disrupts control of locomotor activity and behavioral plasticity.

Homer proteins have been proposed to play a role in synaptogenesis, synapse function, receptor trafficking, and axon pathfinding. Here we report the isolation and characterization of the Drosophila gene homer, the single Homer-related gene in fly. Using anti-Homer antibody we show that Homer is expressed in a broad range of tissues but is highly enriched in the CNS. Similarly to its mammalian counterpart, the Drosophila Homer localizes to the dendrites and the endoplasmic reticulum (ER). This subcellular distribution is dependent on an intact Enabled/Vasp homology 1 domain, suggesting that Homer must bind to one or more of its partners for proper localization. We have created a mutation of homer and show that flies homozygous for this mutation are viable and show coordinated locomotion, suggesting that Homer is not essential for basic neurotransmission. However, we found that homer mutants display defects in behavioral plasticity and the control of locomotor activity. Our results argue that in the CNS, Homer-related proteins operate in the ER and in dendrites to regulate the development and function of neural networks underlying locomotor control and behavioral plasticity.

Twenty-one cases of aortoenteric fistula: lessons for the general surgeon.

We retrospectively reviewed our experience from 1984 to 2001 with 21 cases of aortoenteric fistula (AEF) in 19 patients. The majority of cases were in men (13 of 19, 68%). One AEF was spontaneous, the other 20 developed after prior vascular reconstruction (95%). The majority of AEF were duodenal (48%) followed by small bowel (38%), colon (10%), and esophageal AEF (5%). The proximal anastomosis of the prior vascular repair was the site of AEF origin in 62 per cent of cases, the distal anastomosis accounted for 19 per cent, and the body of the graft for 14 per cent. The intestinal repair was chosen on a case-by-case basis by the general surgeon and consisted of a simple primary repair in 48 per cent, resection with primary anastomosis in 38 per cent, and patching with pleura or omentum in individual cases. Colostomies were created in the two cases with colonic AEF. The duodenum was excluded in one of 10 duodenal AEF. Six patients (32%) died in the 90 days following surgery. The biggest risk of postoperative death was presentation with sepsis (P = 0.069); interestingly, women were more likely to present with sepsis (P = 0.019) and experienced a disproportionate rate of postoperative death (male 23%, female 50%, P = 0.24). The method used to repair the bowel was linked to a higher rate of postoperative death, and patients that required bowel resection died more frequently (66%) than those who had a simple repair (10%, P = 0.07). Overall mortality with AEF remains high despite routine SICU care. The biggest risk for death is preoperative sepsis. Women presented with sepsis more frequently than men. The method of bowel repair appears to be related to overall survival and along with sepsis is, perhaps, a surrogate for the degree of erosion present at the site of the AEF. Simple bowel repairs were sufficient when technically possible. Duodenal exclusion is not an obligatory adjunct to duodenal repairs.

The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila.

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance.

A sensory feedback circuit coordinates muscle activity in Drosophila.

Drosophila larval crawling is a simple behavior that allows us to dissect the functions of specific neurons in the intact animal and explore the roles of genes in the specification of those neurons. By inhibiting subsets of neurons in the PNS, we have found that two classes of multidendritic neurons play a major role in larval crawling. The bipolar dendrites and class I mds send a feedback signal to the CNS that keeps the contraction wave progressing quickly, allowing smooth forward movement. Genetic manipulation of the sensory neurons suggests that this feedback depends on proper dendritic morphology and axon pathfinding to appropriate synaptic target areas in the CNS. Our data suggest that coordination of muscle activity in larval crawling requires feedback from neurons acting as proprioceptors, sending a "mission accomplished" signal in response to segment contraction, and resulting in rapid relaxation of the segment and propagation of the wave.