Identification and characterization of genes controlling energy homeostasis in Drosophila melanogaster

Energy homeostasis is a fundamental property of all organisms. It includes the ability to control storage and mobilization of fat, mainly triacylglycerols (TAG). A genome-wide comparative transcriptome analysis with fed and starved adult Drosophila flies was performed. Nutritionally regulated genes were identified and a subset was functionally characterized. The starvation-induced brummer (bmm) gene encodes the lipid droplet-associated TAG lipase Brummer, a homolog of the human ATGL protein. Overexpression of bmm depletes fat stores, whereas bmm loss-of-function causes obesity in the fly. The results indicate that bmm acts as an evolutionary conserved key regulator of Drosophila fat storage control. The only bmm paralogue in fly, doppelgänger von brummer (dob), is also upregulated under food-deprivation and exerts a Bmm-like TAG lipase activity in vivo. However, flies lacking both Bmm and Dob activities mobilize their TAG storage during food-deprivation. Thus, additional TAG lipases contribute to starvation-induced TAG mobilization in flies. Therefore, the Drosophila mutant of the homologue of the Hormone sensitive lipase, a key enzyme in mammalian lipolysis, was generated to further elucidate the lipolytic cascade in Drosophila. At the lipid droplet surface, Bmm antagonizes the function of the Drosophila PAT domain protein Lsd-2. Overexpression of Lsd-2 results in excessive TAG storage, whereas Lsd-2 mutants are lean. The lean phenotype of Lsd-2 mutant flies is reminiscent of mice deficient for the mammalian Lsd-2 homologue Perilipin, suggesting that Lsd-2 operates in a Perilipin-like manner by modulating the rate of lipolysis. The functional characterization of bmm and Lsd-2 suggests that the surface of lipid droplets represents an evolutionary conserved intracellular compartment boundary involved in the control of lipolysis. This observation emphasizes the value of Drosophila as a model organism to be used for research in energy homeostasis and metabolic disease.Energiehomöostase ist eine grundlegende Eigenschaft aller Organismen. Sie beinhaltet die kontrollierten Speicherung und Mobilisierung von Fett, hauptsächlich Triglyzeride (TAG). In einer genomweiten Transkriptomanalyse mit adulten Drosophila Fliegen wurden ernährungsabhängig-regulierte Gene identifiziert und funktionell untersucht. Das hungerinduzierte brummer Gen (bmm) kodiert für die Fetttröpfchen-assoziierte TAG Lipase Brummer, ein Homolog des humanen ATGL-Proteins. Überexpression von bmm verringert den Speicherfettgehalt, dagegen führt der Verlust der bmm Aktivität zu fettleibigen Fliegen. Diese Ergebnisse zeigen, daß bmm ein evolutionär konserviertes Schlüsselprotein der Fettspeichkontrolle von Drosophila ist. Das einzige bmm Paralog in der Fliege, doppelgänger von brummer (dob), ist ebenfalls hungerinduziert und zeigt Bmm-ähnliche TAG Lipase Aktivität in vivo. Allerdings mobilisieren Fliegen, denen sowohl Bmm und Dob Aktivität fehlt ihre Fettreserven unter Hungerbedingungen. Daher müssen weitere TAG Lipasen in die hungerinduzierte Fettmobilisierung der Fliege involviert sein. Um die lipolytische Kaskade weiter zu untersuchen, wurde eine Mutante für das Drosophila Homolog der Hormon Sensitiven Lipase, ein Schlüsselenzym der Säugerlipolyse, generiert. Auf der Oberflächen von Fettspeichertröpfchen wirkt Bmm antagonistisch zur Funktion des Drosophila PAT Domänen Proteins Lsd-2. Überexpression von Lsd-2 resultiert in erhöhter Fetteinlagerung, während Lsd-2 Mutanten dünn sind. Der Phänotyp von Lsd-2 Mutanten ähnelt dem Phänotyp von Mäusen defizient für das Lsd-2 Säugerhomolog Perilipin. Dies weißt darauf hin, daß Lsd-2 in einer Perilipin-ähnlichen Weise die Lipolyse moduliert. Die funktionelle Charakterisierung von bmm und Lsd-2 etabliert die Oberfläche der Fettspeichertröpfchen als eine evolutionär konservierte Kompartimentgrenze wichtig für die Kontrolle der Lipolyse und unterstreicht den Wert von Drosophila als ein Modellorganismus der Energiehomöostaseforschung

Dietary restriction mitigates the age-associated decline in mouse B cell receptor repertoire diversity

Aging impairs the capacity to respond to novel antigens, reducing immune protection against pathogens and vaccine efficacy. Dietary restriction (DR) extends life- and health span in diverse animals. However, little is known about the capacity of DR to combat the decline in immune function. Here, we study the changes in B cell receptor (BCR) repertoire during aging in DR and control mice. By sequencing the variable region of the BCR heavy chain in the spleen, we show that DR preserves diversity and attenuates the increase in clonal expansions throughout aging. Remarkably, mice starting DR in mid-life have repertoire diversity and clonal expansion rates indistinguishable from chronic DR mice. In contrast, in the intestine, these traits are unaffected by either age or DR. Reduced within-individual B cell repertoire diversity and increased clonal expansions are correlated with higher morbidity, suggesting a potential contribution of B cell repertoire dynamics to health during aging

Toxicity of C9orf72-associated dipeptide repeat peptides is modified by commonly used protein tags

Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. Here, we used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models

Hepatic gene body hypermethylation is a shared epigenetic signature of murine longevity.

Dietary, pharmacological and genetic interventions can extend health- and lifespan in diverse mammalian species. DNA methylation has been implicated in mediating the beneficial effects of these interventions; methylation patterns deteriorate during ageing, and this is prevented by lifespan-extending interventions. However, whether these interventions also actively shape the epigenome, and whether such epigenetic reprogramming contributes to improved health at old age, remains underexplored. We analysed published, whole-genome, BS-seq data sets from mouse liver to explore DNA methylation patterns in aged mice in response to three lifespan-extending interventions: dietary restriction (DR), reduced TOR signaling (rapamycin), and reduced growth (Ames dwarf mice). Dwarf mice show enhanced DNA hypermethylation in the body of key genes in lipid biosynthesis, cell proliferation and somatotropic signaling, which strongly correlates with the pattern of transcriptional repression. Remarkably, DR causes a similar hypermethylation in lipid biosynthesis genes, while rapamycin treatment increases methylation signatures in genes coding for growth factor and growth hormone receptors. Shared changes of DNA methylation were restricted to hypermethylated regions, and they were not merely a consequence of slowed ageing, thus suggesting an active mechanism driving their formation. By comparing the overlap in ageing-independent hypermethylated patterns between all three interventions, we identified four regions, which, independent of genetic background or gender, may serve as novel biomarkers for longevity-extending interventions. In summary, we identified gene body hypermethylation as a novel and partly conserved signature of lifespan-extending interventions in mouse, highlighting epigenetic reprogramming as a possible intervention to improve health at old age

Long-lasting geroprotection from brief rapamycin treatment in early adulthood by persistently increased intestinal autophagy

The licensed drug rapamycin has potential to be repurposed for geroprotection. A key challenge is to avoid adverse side effects from continuous dosing. Here we show that geroprotective effects of chronic rapamycin treatment can be obtained with a brief pulse of the drug in early adulthood in female Drosophila and mice. In Drosophila, a brief, early rapamycin treatment of adults extended lifespan and attenuated age-related decline in the intestine to the same degree as lifelong dosing. Lasting memory of earlier treatment was mediated by elevated autophagy in intestinal enterocytes, accompanied by increased levels of intestinal LManV and lysozyme. Brief elevation of autophagy in early adulthood itself induced a long-term increase in autophagy. In mice, a 3-month, early treatment also induced a memory effect, with maintenance similar to chronic treatment, of lysozyme distribution, Man2B1 level in intestinal crypts, Paneth cell architecture and gut barrier function, even 6 months after rapamycin was withdrawn

Quantitative Assessment of Eye Phenotypes for Functional Genetic Studies Using Drosophila melanogaster

About two-thirds of the vital genes in the Drosophila genome are involved in eye development, making the fly eye an excellent genetic system to study cellular function and development, neurodevelopment/degeneration, and complex diseases such as cancer and diabetes. We developed a novel computational method, implemented as Flynotyper software (http://flynotyper.sourceforge.net), to quantitatively assess the morphological defects in the Drosophila eye resulting from genetic alterations affecting basic cellular and developmental processes. Flynotyper utilizes a series of image processing operations to automatically detect the fly eye and the individual ommatidium, and calculates a phenotypic score as a measure of the disorderliness of ommatidial arrangement in the fly eye. As a proof of principle, we tested our method by analyzing the defects due to eye-specific knockdown of Drosophila orthologs of 12 neurodevelopmental genes to accurately document differential sensitivities of these genes to dosage alteration. We also evaluated eye images from six independent studies assessing the effect of overexpression of repeats, candidates from peptide library screens, and modifiers of neurotoxicity and developmental processes on eye morphology, and show strong concordance with the original assessment. We further demonstrate the utility of this method by analyzing 16 modifiers of sine oculis obtained from two genome-wide deficiency screens of Drosophila and accurately quantifying the effect of its enhancers and suppressors during eye development. Our method will complement existing assays for eye phenotypes and increase the accuracy of studies that use fly eyes for functional evaluation of genes and genetic interactions

Ageing-associated changes in transcriptional elongation influence longevity

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures

Enhancing autophagy by redox regulation extends lifespan in <i>Drosophila</i>

Redox signalling is an important modulator of diverse biological pathways and processes, and operates through specific post-translational modification of redox-sensitive thiols on cysteine residues 1–4. Critically, redox signalling is distinct from irreversible oxidative damage and functions as a reversible ‘redox switch’ to regulate target proteins. H2O2 acts as the major effector of redox signalling, both directly and through intracellular thiol redox relays 5,6. Dysregulation of redox homeostasis has long been implicated in the pathophysiology of many age-related diseases, as well as in the ageing process itself, however the underlying mechanisms remain largely unclear 7,8. To study redox signalling by H2O2in vivo and explore its involvement in metabolic health and longevity, we used the fruit fly Drosophila as a model organism, with its tractable lifespan and strong evolutionary conservation with mammals 9. Here we report that inducing an endogenous redox-shift, by manipulating levels of the H2O2-degrading enzyme catalase, improves health and robustly extends lifespan in flies, independently of oxidative stress resistance and dietary restriction. We find that the catalase redox-shifted flies are acutely sensitive to starvation stress, which relies on autophagy as a vital survival mechanism. Importantly, we show that autophagy is essential for the lifespan extension of the catalase flies. Furthermore, using redox-inactive knock-in mutants of Atg4a, a major effector of autophagy, we show that the lifespan extension in response to catalase requires a key redox-regulatory cysteine residue, Cys102 in Atg4a. These findings demonstrate that redox regulation of autophagy can extend lifespan, confirming the importance of redox signalling in ageing and as a potential pro-longevity target.</jats:p

Dual Lipolytic Control of Body Fat Storage and Mobilization in Drosophila

Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. We generated a Drosophila mutant lacking the receptor (AKHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ß-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. Our data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis

The Bicoid Stability Factor Controls Polyadenylation and Expression of Specific Mitochondrial mRNAs in Drosophila melanogaster

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation