ABSTRACT
Cell growth is positively controlled by the phosphoinositide 3-kinase (PI3K)-target of rapamycin (TOR) signaling pathway under conditions of abundant growth factors and nutrients. To discover additional mechanisms that regulate cell growth, here we performed RNAi-based mosaic analyses in the Drosophila fat body, the primary metabolic organ in the fly. Unexpectedly, the knockdown of the Drosophila von Hippel-Lindau (VHL) gene markedly decreased cell size and body size. These cell growth phenotypes induced by VHL loss of function were recovered by activation of TOR signaling in Drosophila Consistent with the genetic interactions between VHL and the signaling components of PI3K-TOR pathway in Drosophila, we observed that VHL loss of function in mammalian cells causes decreased phosphorylation of ribosomal protein S6 kinase and Akt, which represent the main activities of this pathway. We further demonstrate that VHL activates TOR signaling by directly interacting with the p110 catalytic subunit of PI3K. On the basis of the evolutionarily conserved regulation of PI3K-TOR signaling by VHL observed here, we propose that VHL plays an important role in the regulation and maintenance of proper cell growth in metazoans.
Subject(s)
Carrier Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Phosphatidylinositol 3-Kinase/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , Body Size , Cell Size , Drosophila melanogaster/cytology , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Protein BindingABSTRACT
Mutations in the cereblon (CRBN) gene cause human intellectual disability, one of the most common cognitive disorders. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. We investigated the role of CRBN in synaptic function and animal behavior using male mouse and Drosophila models. Crbn knock-out (KO) mice showed normal brain and spine morphology as well as intact synaptic plasticity; however, they also exhibited decreases in synaptic transmission and presynaptic release probability exclusively in excitatory synapses. Presynaptic function was impaired not only by loss of CRBN expression, but also by expression of pathogenic CRBN mutants (human R419X mutant and Drosophila G552X mutant). We found that the BK channel blockers paxilline and iberiotoxin reversed this decrease in presynaptic release probability in Crbn KO mice. In addition, paxilline treatment also restored normal cognitive behavior in Crbn KO mice. These results strongly suggest that increased BK channel activity is the pathological mechanism of intellectual disability in CRBN mutations.SIGNIFICANCE STATEMENTCereblon (CRBN), a well known target of the immunomodulatory drug thalidomide, was originally identified as a gene that causes human intellectual disability when mutated. However, the molecular mechanisms of CRBN-related intellectual disability remain poorly understood. Based on the idea that synaptic abnormalities are the most common factor in cognitive dysfunction, we monitored the synaptic structure and function of Crbn knock-out (KO) animals to identify the molecular mechanisms of intellectual disability. Here, we found that Crbn KO animals showed cognitive deficits caused by enhanced BK channel activity and reduced presynaptic glutamate release. Our findings suggest a physiological pathomechanism of the intellectual disability-related gene CRBN and will contribute to the development of therapeutic strategies for CRBN-related intellectual disability.
Subject(s)
Cognition , Intellectual Disability/genetics , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Nerve Tissue Proteins/metabolism , Synaptic Transmission , Adaptor Proteins, Signal Transducing , Animals , Brain/cytology , Brain/metabolism , Cells, Cultured , Drosophila , Glutamic Acid/metabolism , Indoles/pharmacology , Large-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Peptides/pharmacology , Potassium Channel Blockers/pharmacology , Synapses/drug effects , Synapses/metabolism , Synapses/physiologyABSTRACT
Wnt signaling pathway plays critical roles in body axes patterning, cell fate specification, cell proliferation, cell migration, stem cell maintenance, cancer development and etc. Deregulation of this pathway can be causative of cancer, metabolic disease and neurodegenerative disease such as Parkinson`s disease. Among the core components of Wnt signaling pathway, we discovered that Dishevelled (Dsh) interacts with ULK1 and is phosphorylated by ULK1. Unexpectedly, the knockdown of ULK1 elicited a marked increase in Wnt/ß-catenin signaling. Multiple ULK1 phosphorylation sites existed on Dsh and many of them were located on the PDZ-DEP region. By using evolutionarily well conserved Drosophila Dsh, we found that S239, S247 and S254 in the PDZ-DEP region are involved in phosphorylation of Dsh by ULK1. Among these, S247 and S254 were conserved in human Dsh. When phospho-mimetic mutants (2D and 2E Dsh mutants) of these conserved residues were generated and expressed in the eyes of the fruit flies, the activity of Dsh was significantly decreased compared to wild type Dsh. Through additional alanine scanning, we further identified that S239, S247, S254, S266, S376, S554 and S555 on full length Dsh were phosphorylated by ULK1. In regards to the S266A mutation located in the PDZ domain among these phosphorylated residues, our results suggested that Dsh forms an SDS-resistant high molecular weight complex with ß-catenin and TCF in the nucleus in an S266 phosphorylation-dependent manner. Based on these results, we propose that ULK1 plays a pivotal role in the regulation of Wnt/ß-catenin signaling pathway by phosphorylating Dsh.
Subject(s)
Autophagy-Related Protein-1 Homolog/metabolism , Dishevelled Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Wnt Signaling Pathway , Cells, Cultured , HEK293 Cells , Humans , PhosphorylationABSTRACT
Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. Here, we confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. We generated Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation. Basal metabolic activities were not significantly affected in these MCU mutants, as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. We also found that inositol 1,4,5-trisphosphate receptor genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for endoplasmic reticulum-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress.
Subject(s)
Apoptosis , Calcium Channels/metabolism , Calcium Signaling , Cation Transport Proteins/metabolism , Drosophila Proteins/metabolism , Endoplasmic Reticulum/metabolism , Mitochondria, Muscle/metabolism , Oxidative Stress , Animals , Animals, Genetically Modified , Apoptosis/drug effects , Autophagy/drug effects , Caffeine/pharmacology , Calcium Channels/chemistry , Calcium Channels/genetics , Calcium Signaling/drug effects , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cell Line , Central Nervous System Stimulants/pharmacology , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/drug effects , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Endoplasmic Reticulum/drug effects , Gene Silencing , Inositol 1,4,5-Trisphosphate Receptors/antagonists & inhibitors , Inositol 1,4,5-Trisphosphate Receptors/chemistry , Inositol 1,4,5-Trisphosphate Receptors/genetics , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Larva/drug effects , Larva/genetics , Larva/growth & development , Larva/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria, Muscle/drug effects , Mutation , Oxidative Stress/drug effects , Protein Sorting Signals/drug effects , RNA Interference , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.
Subject(s)
Mutation , Parkinson Disease/metabolism , Protein Kinases/physiology , Adenosine Triphosphate/metabolism , Animals , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Drosophila melanogaster , Fibroblasts/cytology , HEK293 Cells , HeLa Cells , Humans , Immunohistochemistry/methods , Male , Membrane Potential, Mitochondrial , Mice , Mitochondria/metabolism , Neurons/metabolism , Phenotype , Protein Kinases/metabolism , Transgenes , Ubiquitin-Protein Ligases/metabolismABSTRACT
Homoiotherms, for example mammals, regulate their body temperature with physiological responses such as a change of metabolic rate and sweating. In contrast, the body temperature of poikilotherms, for example Drosophila, is the result of heat exchange with the surrounding environment as a result of the large ratio of surface area to volume of their bodies. Accordingly, these animals must instinctively move to places with an environmental temperature as close as possible to their genetically determined desired temperature. The temperature that Drosophila instinctively prefers has a function equivalent to the 'set point' temperature in mammals. Although various temperature-gated TRP channels have been discovered, molecular and cellular components in Drosophila brain responsible for determining the desired temperature remain unknown. We identified these components by performing a large-scale genetic screen of temperature preference behaviour (TPB) in Drosophila. In parallel, we mapped areas of the Drosophila brain controlling TPB by targeted inactivation of neurons with tetanus toxin and a potassium channel (Kir2.1) driven with various brain-specific GAL4s. Here we show that mushroom bodies (MBs) and the cyclic AMP-cAMP-dependent protein kinase A (cAMP-PKA) pathway are essential for controlling TPB. Furthermore, targeted expression of cAMP-PKA pathway components in only the MB was sufficient to rescue abnormal TPB of the corresponding mutants. Preferred temperatures were affected by the level of cAMP and PKA activity in the MBs in various PKA pathway mutants.
Subject(s)
Body Temperature Regulation/physiology , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Drosophila melanogaster/physiology , Mushroom Bodies/metabolism , Signal Transduction , Temperature , Animals , Body Temperature/genetics , Body Temperature/physiology , Body Temperature Regulation/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Drosophila melanogaster/genetics , Motor Activity/genetics , Motor Activity/physiology , Mushroom Bodies/enzymologyABSTRACT
The ability to respond to environmental temperature variation is essential for survival in animals. Flies show robust temperature-preference behaviour (TPB) to find optimal temperatures. Recently, we have shown that Drosophila mushroom body (MB) functions as a center controlling TPB. However, neuromodulators that control the TPB in MB remain unknown. To identify the functions of dopamine in TPB, we have conducted various genetic studies in Drosophila. Inhibition of dopamine biosynthesis by genetic mutations or treatment with chemical inhibitors caused flies to prefer temperatures colder than normal. We also found that dopaminergic neurons are involved in TPB regulation, as the targeted inactivation of dopaminergic neurons by expression of a potassium channel (Kir2.1) induced flies with the loss of cold avoidance. Consistently, the mutant flies for dopamine receptor gene (DopR) also showed a cold temperature preference, which was rescued by MB-specific expression of DopR. Based on these results, we concluded that dopamine in MB is a key component in the homeostatic temperature control of Drosophila. The current findings will provide important bases to understand the logic of thermosensation and temperature preference decision in Drosophila.
Subject(s)
Behavior, Animal/physiology , Cold Temperature , Dopamine/metabolism , Drosophila/physiology , Signal Transduction , Animals , Body Temperature Regulation/genetics , Brain/metabolism , Drosophila/genetics , Drosophila/metabolism , Gene Expression Regulation/genetics , Mushroom Bodies/metabolism , Mutation/genetics , Neurons/metabolism , Receptors, Dopamine/genetics , Receptors, Dopamine/metabolismABSTRACT
Several transient receptor potential channels were recently found to be activated by temperature stimuli in vitro. Their physiological and behavioral roles are largely unknown. From a temperature-preference behavior screen of 27,000 Drosophila melanogaster P-insertion mutants, we isolated a gene, named pyrexia (pyx), encoding a new transient receptor potential channel. Pyx was opened by temperatures above 40 degrees C in Xenopus laevis oocytes and HEK293T cells. It was ubiquitously expressed along the dendrites of a subset of peripheral nervous system neurons and was more permeable to K(+) than to Na(+). Although some pyx alleles resulted in abnormal temperature preferences, pyx null flies did not have significantly different temperature preferences than wild-type flies. But 60% of pyx null flies were paralyzed within 3 min of exposure to 40 degrees C, whereas only 9% of wild-type flies were paralyzed by the same stimulus. From these findings, we propose that the primary in vivo role of Pyx is to protect flies from high-temperature stress.
Subject(s)
Calmodulin-Binding Proteins/physiology , Drosophila Proteins/physiology , Drosophila melanogaster/physiology , Fever/physiopathology , Hot Temperature , Membrane Proteins/physiology , Amino Acid Sequence , Animals , Calmodulin-Binding Proteins/chemistry , Cell Line , Cloning, Molecular , DNA, Complementary , Drosophila Proteins/chemistry , Humans , Immunohistochemistry , Membrane Proteins/chemistry , Molecular Sequence Data , Sequence Homology, Amino Acid , Transient Receptor Potential Channels , Xenopus laevisABSTRACT
The pigment-dispersing factor (PDF) is a neuropeptide controlling circadian behavioral rhythms in Drosophila, but its receptor is not yet known. From a large-scale temperature preference behavior screen in Drosophila, we isolated a P insertion mutant that preferred different temperatures during the day and night. This mutation, which we named han, reduced the transcript level of CG13758. We found that Han was expressed specifically in 13 pairs of circadian clock neurons in the adult brain. han null flies showed arrhythmic circadian behavior in constant darkness. The behavioral characteristics of han null mutants were similar to those of pdf null mutants. We also found that PDF binds specifically to S2 cells expressing Han, which results in the elevation of cAMP synthesis. Therefore, we herein propose that Han is a PDF receptor regulating circadian behavioral rhythm through coordination of activities of clock neurons.
Subject(s)
Circadian Rhythm/physiology , Drosophila Proteins/metabolism , Gene Expression Regulation/physiology , Neuropeptides/metabolism , Receptors, G-Protein-Coupled/metabolism , Amino Acid Sequence , Animals , Antibodies/metabolism , Behavior, Animal , Binding, Competitive , Blotting, Northern/methods , Brain/cytology , Cell Line , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/immunology , Drosophila Proteins/physiology , Gene Expression Regulation/drug effects , Immunohistochemistry/methods , Motor Activity/physiology , Mutation , Neurons/metabolism , Periodicity , Protein Binding/physiology , RNA, Messenger/biosynthesis , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/immunology , Receptors, G-Protein-Coupled/physiology , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Alignment , Thermosensing/genetics , Thermosensing/physiologyABSTRACT
Temperature profoundly influences various life phenomena, and most animals have developed mechanisms to respond properly to environmental temperature fluctuations. To identify genes involved in sensing ambient temperature and in responding to its change, >27,000 independent P-element insertion mutants of Drosophila were screened. As a result, we found that defects in the genes encoding for proteins involved in histamine signaling [histidine decarboxylase (hdc), histamine-gated chloride channel subunit 1 (hisCl1), ora transientless (ort)] cause abnormal temperature preferences. The abnormal preferences shown in these mutants were restored by genetic and pharmacological rescue and could be reproduced in wild type using the histamine receptor inhibitors cimetidine and hydroxyzine. Spatial expression of these genes was observed in various brain regions including pars intercerebralis, fan-shaped body, and circadian clock neurons but not in dTRPA1-expressing neurons, an essential element for thermotaxis. We also found that the histaminergic mutants showed reduced tolerance for high temperature and enhanced tolerance for cold temperature. Together, these results suggest that histamine signaling may have important roles in modulating temperature preference and in controlling tolerance of low and high temperature.
Subject(s)
Chloride Channels/metabolism , Drosophila Proteins/metabolism , Drosophila/physiology , Histamine/metabolism , Histidine Decarboxylase/metabolism , Receptors, Histamine/metabolism , Thermosensing/physiology , Animals , Behavior, Animal/physiology , Chloride Channels/genetics , Cold Temperature , Discrimination, Psychological/physiology , Drosophila/genetics , Drosophila Proteins/genetics , Histidine Decarboxylase/genetics , Hot Temperature , Mutagenesis , Receptors, Histamine/genetics , Signal Transduction/physiologyABSTRACT
BACKGROUND: Appropriate vertical movement is critical for the survival of flying animals. Although negative geotaxis (moving away from Earth) driven by gravity has been extensively studied, much less is understood concerning a static regulatory mechanism for inducing positive geotaxis (moving toward Earth). RESULTS: Using Drosophila melanogaster as a model organism, we showed that geomagnetic field (GMF) induces positive geotaxis and antagonizes negative gravitaxis. Remarkably, GMF acts as a sensory cue for an appetite-driven associative learning behavior through the GMF-induced positive geotaxis. This GMF-induced positive geotaxis requires the three geotaxis genes, such as cry, pyx and pdf, and the corresponding neurons residing in Johnston's organ of the fly's antennae. CONCLUSIONS: These findings provide a novel concept with the neurogenetic basis on the regulation of vertical movement by GMF in the flying animals.
Subject(s)
Behavior, Animal/physiology , Drosophila melanogaster/physiology , Gravitation , Locomotion/physiology , Magnetic Fields , Animals , Neurons/metabolism , Organ Specificity , Signal TransductionABSTRACT
Hereditary spastic paraplegias (HSPs) are human genetic disorders causing increased stiffness and overactive muscle reflexes in the lower extremities. atlastin (atl) is one of the major genes in which mutations result in HSP. We generated a Drosophila model of HSP that has a null mutation in atl. As they aged, atl null flies were paralyzed by mechanical shock such as bumping or vortexing. Furthermore, the flies showed age-dependent degeneration of dopaminergic neurons. These phenotypes were rescued by targeted expression of atl in dopaminergic neurons or feeding L-DOPA or SK&F 38393, an agonist of dopamine receptor. Our data raised the possibility that one of the causes of HSP disease symptoms in human patients with alt mutations is malfunction or degeneration of dopaminergic neurons.