RESUMO
BACKGROUND: Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. RESULTS: Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of ß-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. CONCLUSIONS: These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Metabolismo dos Lipídeos , Animais , Humanos , Trifosfato de Adenosina/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Corpo Adiposo/metabolismo , Flavoproteínas/metabolismo , Larva , Lipase/genética , Lipase/metabolismo , Metabolismo dos Lipídeos/genética , Lipídeos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by â¼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF) set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP) signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Ritmo Circadiano/fisiologia , Transdução de Sinais/fisiologia , Animais , Encéfalo/fisiologia , Proteínas CLOCK/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Atividade Motora/fisiologia , Neurônios/fisiologiaRESUMO
Neurodegenerative diseases encompass a broad variety of motor and cognitive disorders that are accompanied by death of specific neuronal populations or brain regions. Cellular and molecular mechanisms underlying these complex disorders remain largely unknown. In a previous work we searched for novel Drosophila genes relevant for neurodegeneration and singled out enabled (ena), which encodes a protein involved in cytoskeleton remodeling. To extend our understanding on the mechanisms of ENA-triggered degeneration we now investigated the effect of silencing ena ortholog genes in mouse hippocampal neurons. We found that ENA/VASP downregulation led to neurite retraction and concomitant neuronal cell death through an apoptotic pathway. Remarkably, this retraction initially affected the axonal structure, showing no effect on dendrites. Reduction in ENA/VASP levels blocked the neuritogenic effect of a specific RhoA kinase (ROCK) inhibitor, thus suggesting that these proteins could participate in the Rho-signaling pathway. Altogether these observations demonstrate that ENA/VASP proteins are implicated in the establishment and maintenance of the axonal structure and that a change on their expression levels triggers neuronal degeneration.
Assuntos
Apoptose/genética , Axônios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hipocampo/metabolismo , Degeneração Neural/metabolismo , Animais , Axônios/patologia , Células Cultivadas , Proteínas do Citoesqueleto/genética , Regulação para Baixo/genética , Inativação Gênica/fisiologia , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos , Proteínas dos Microfilamentos , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismoRESUMO
Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.
Assuntos
Drosophila/genética , Expressão Gênica , Doenças Neurodegenerativas/genética , Envelhecimento/patologia , Animais , Apoptose , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Atividade MotoraRESUMO
The GAL4/UAS system has been extensively employed in Drosophila to control gene expression in defined spatial patterns. More recently this system has been successfully applied to express genes involved in neurodegeneration to model various diseases in the fruit fly. We used transgenic lines expressing different levels of GAL4 in a particular subset of neurons involved in the control of rhythmic behaviour, so that its impact on neuronal physiology would result in altered locomotor activity, which could be readily assessed. We observed a striking correlation between gal4 dosage and behavioural defects associated with apoptotic neuronal loss in the specific GAL4-expressing neurons. Increased gal4 dosage correlated with accumulation of insoluble GAL4, suggesting that the cascade of events leading to apoptosis might be triggered by protein deposits of either GAL4 or protein intermediates. Behavioural defects were rescued by expression of hsp70, a classic chaperone that also interferes with cell death pathways. In agreement with the latter, the viral caspase inhibitor p35 also rescued GAL4-induced behavioural defects. Our observations demonstrate the intrinsic effects of GAL4 deregulation on neuronal viability and suggest that an excess of GAL4 might enhance neuronal deficits observed in models of neurodegeneration.
Assuntos
Apoptose/fisiologia , Degeneração Neural/fisiopatologia , Neurônios/patologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transgenes/fisiologia , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/fisiologia , Proteínas de Ligação a DNA , Drosophila , Dosagem de Genes/fisiologia , Regulação da Expressão Gênica , Marcação In Situ das Extremidades Cortadas , Larva/fisiologia , Masculino , Microscopia Eletrônica de Varredura , Atividade Motora/fisiologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/fisiologia , Fenótipo , Células Fotorreceptoras de Invertebrados/fisiologia , Células Fotorreceptoras de Invertebrados/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, delta-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. RESULTS: We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.