Lipidomic analysis of human TANGO2-deficient cells suggests a lipid imbalance as a cause of TANGO2 deficiency disease.

TANGO2 deficiency disease (TDD) is a multisystem disease caused by variants in the TANGO2 gene. Symptoms include neurodevelopmental delays, seizures and potentially lethal metabolic crises and cardiac arrhythmias. While the function of TANGO2 remains elusive, vitamin B5/pantothenic acid supplementation has been shown to alleviate symptoms in a fruit fly model and has also been used with success to treat individuals suffering from TDD. Since vitamin B5 is the precursor to the lipid activator coenzyme A (CoA), we hypothesized that TANGO2-deficient cells would display changes in the lipid profile compared to control and that these changes would be rescued by vitamin B5 supplementation. In addition, the specific changes seen might point to a pathway in which TANGO2 functions. Indeed, we found profound changes in the lipid profile of human TANGO2-deficient cells as well as an increased pool of free fatty acids in both human cells devoid of TANGO2 and Drosophila harboring a previously described TANGO2 loss of function allele. All these changes were reversed upon vitamin B5 supplementation. Pathway analysis showed significant increases in triglyceride as well as in lysophospholipid levels as the top enriched pathways in the absence of TANGO2. Consistent with a defect in triglyceride metabolism, we found changes in lipid droplet numbers and sizes in the absence of TANGO2 compared to control. Our data will allow for comparison between other model systems of TDD and the homing in on critical lipid imbalances that lead to the disease state.

Vitamin B5, a coenzyme A precursor, rescues TANGO2 deficiency disease-associated defects in Drosophila and human cells.

Mutations in the Transport and Golgi Organization 2 (TANGO2) gene are associated with intellectual deficit, neurodevelopmental delay and regression. Individuals can also present with an acute metabolic crisis that includes rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, the latter of which are potentially lethal. While preventing metabolic crises has the potential to reduce mortality, no treatments currently exist for this condition. The function of TANGO2 remains unknown but is suspected to be involved in some aspect of lipid metabolism. Here, we describe a model of TANGO2-related disease in the fruit fly Drosophila melanogaster that recapitulates crucial disease traits. Pairing a new fly model with human cells, we examined the effects of vitamin B5, a coenzyme A (CoA) precursor, on alleviating the cellular and organismal defects associated with TANGO2 deficiency. We demonstrate that vitamin B5 specifically improves multiple defects associated with TANGO2 loss-of-function in Drosophila and rescues membrane trafficking defects in human cells. We also observed a partial rescue of one of the fly defects by vitamin B3, though to a lesser extent than vitamin B5. Our data suggest that a B complex supplement containing vitamin B5/pantothenate may have therapeutic benefits in individuals with TANGO2-deficiency disease. Possible mechanisms for the rescue are discussed that may include restoration of lipid homeostasis.

Drug discovery and chemical probing in Drosophila.

Flies are increasingly utilized in drug discovery and chemical probing in vivo, which are novel technologies complementary to genetic probing in fundamental biological studies. Excellent genetic conservation, small size, short generation time, and over one hundred years of genetics make Drosophila an attractive model for rapid assay readout and use of analytical amounts of compound, enabling the experimental iterations needed in early drug development at a fraction of time and costs. Here, we describe an effective drug-testing pipeline using adult flies that can be easily implemented to study several disease models and different genotypes to discover novel molecular insight, probes, quality lead compounds, and develop novel prototype drugs.

Modeling Neoplastic Growth in Renal Cell Carcinoma and Polycystic Kidney Disease.

Renal cell carcinoma (RCC) and autosomal dominant polycystic kidney disease (ADPKD) share several characteristics, including neoplastic cell growth, kidney cysts, and limited therapeutics. As well, both exhibit impaired vasculature and compensatory VEGF activation of angiogenesis. The PI3K/AKT/mTOR and Ras/Raf/ERK pathways play important roles in regulating cystic and tumor cell proliferation and growth. Both RCC and ADPKD result in hypoxia, where HIF-α signaling is activated in response to oxygen deprivation. Primary cilia and altered cell metabolism may play a role in disease progression. Non-coding RNAs may regulate RCC carcinogenesis and ADPKD through their varied effects. Drosophila exhibits remarkable conservation of the pathways involved in RCC and ADPKD. Here, we review the progress towards understanding disease mechanisms, partially overlapping cellular and molecular dysfunctions in RCC and ADPKD and reflect on the potential for the agile Drosophila genetic model to accelerate discovery science, address unresolved mechanistic aspects of these diseases, and perform rapid pharmacological screens.

Bicaudal C mutation causes myc and TOR pathway up-regulation and polycystic kidney disease-like phenotypes in Drosophila.

Progressive cystic kidney degeneration underlies diverse renal diseases, including the most common cause of kidney failure, autosomal dominant Polycystic Kidney Disease (PKD). Genetic analyses of patients and animal models have identified several key drivers of this disease. The precise molecular and cellular changes underlying cystogenesis remain, however, elusive. Drosophila mutants lacking the translational regulator Bicaudal C (BicC, the fly ortholog of vertebrate BICC1 implicated in renal cystogenesis) exhibited progressive cystic degeneration of the renal tubules (so called "Malpighian" tubules) and reduced renal function. The BicC protein was shown to bind to Drosophila (d-) myc mRNA in tubules. Elevation of d-Myc protein levels was a cause of tubular degeneration in BicC mutants. Activation of the Target of Rapamycin (TOR) kinase pathway, another common feature of PKD, was found in BicC mutant flies. Rapamycin administration substantially reduced the cystic phenotype in flies. We present new mechanistic insight on BicC function and propose that Drosophila may serve as a genetically tractable model for dissecting the evolutionarily-conserved molecular mechanisms of renal cystogenesis.

Cyst Reduction by Melatonin in a Novel Drosophila Model of Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) causes progressive cystic degeneration of the renal tubules, the nephrons, eventually severely compromising kidney function. ADPKD is incurable, with half of the patients eventually needing renal replacement. Treatments for ADPKD patients are limited and new effective therapeutics are needed. Melatonin, a central metabolic regulator conserved across all life kingdoms, exhibits oncostatic and oncoprotective activity and no detected toxicity. Here, we used the Bicaudal C (BicC) Drosophila model of polycystic kidney disease to test the cyst-reducing potential of melatonin. Significant cyst reduction was found in the renal (Malpighian) tubules upon melatonin administration and suggest mechanistic sophistication. Similar to vertebrate PKD, the BicC fly PKD model responds to the antiproliferative drugs rapamycin and mimics of the second mitochondria-derived activator of caspases (Smac). Melatonin appears to be a new cyst-reducing molecule with attractive properties as a potential candidate for PKD treatment.

Drosophila 4EHP is essential for the larval-pupal transition and required in the prothoracic gland for ecdysone biosynthesis.

Maternal expression of the translational regulator 4EHP (eIF4E-Homologous Protein) has an established role in generating protein gradients essential for specifying the Drosophila embryonic pattern. We generated a null mutation of 4EHP, which revealed for the first time that it is essential for viability and for completion of development. In fact, 4EHP null larvae, and larvae ubiquitously expressing RNAi targeting 4EHP, are developmentally delayed, fail to grow and eventually die. In addition, we found that expressing RNAi that targets 4EHP specifically in the prothoracic gland disrupted ecdysone biosynthesis, causing a block of the transition from the larval to pupal stages. This phenotype can be rescued by dietary administration of ecdysone. Consistent with this, 4EHP is highly expressed in the prothoracic gland and it is required for wild type expression levels of steroidogenic enzymes. Taken together, these results uncover a novel essential function for 4EHP in regulating ecdysone biosynthesis.

The CCR4-NOT complex mediates deadenylation and degradation of stem cell mRNAs and promotes planarian stem cell differentiation.

Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after Smed-not1 knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after Smed-not1 knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.

TANGO2 deficiency disease is predominantly caused by a lipid imbalance.

TANGO2 deficiency disease (TDD) is a rare genetic disorder estimated to affect ∼8000 individuals worldwide. It causes neurodegeneration often accompanied by potentially lethal metabolic crises that are triggered by diet or illness. Recent work has demonstrated distinct lipid imbalances in multiple model systems either depleted for or devoid of the TANGO2 protein, including human cells, fruit flies and zebrafish. Importantly, vitamin B5 supplementation has been shown to rescue TANGO2 deficiency-associated defects in flies and human cells. The notion that vitamin B5 is needed for synthesis of the lipid precursor coenzyme A (CoA) corroborates the hypothesis that key aspects of TDD pathology may be caused by lipid imbalance. A natural history study of 73 individuals with TDD reported that either multivitamin or vitamin B complex supplementation prevented the metabolic crises, suggesting this as a potentially life-saving treatment. Although recently published work supports this notion, much remains unknown about TANGO2 function, the pathological mechanism of TDD and the possible downsides of sustained vitamin supplementation in children and young adults. In this Perspective, we discuss these recent findings and highlight areas for immediate scientific attention.

Protocol to build a drug-testing pipeline using large populations of Drosophila melanogaster.

As a small animal that recapitulates many fundamental aspects of human disease, Drosophila lends itself to probing the biological activity of molecules and drug candidates. Here, we present a protocol to build a drug-testing pipeline in Drosophila. We describe steps for generating synchronous populations of Bicaudal C mutants by genetic crossing and wild-type fly culturing for controlled compound administration and exemplary phenotypic assays. For complete details on the use and execution of this protocol, please refer to Millet-Boureima et al.,1 Millet-Boureima et al.,2 and Gamberi et al.3.

Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression.

Bicaudal-C (Bic-C) encodes an RNA-binding protein required maternally for patterning the Drosophila embryo. We identified a set of mRNAs that associate with Bic-C in ovarian ribonucleoprotein complexes. These mRNAs are enriched for mRNAs that function in oogenesis and in cytoskeletal regulation, and include Bic-C RNA itself. Bic-C binds specific segments of the Bic-C 5' untranslated region and negatively regulates its own expression by binding directly to NOT3/5, a component of the CCR4 core deadenylase complex, thereby promoting deadenylation. Bic-C overexpression induces premature cytoplasmic-streaming, a posterior-group phenotype, defects in Oskar and Kinesin heavy chain:betaGal localization as well as dorsal-appendage defects. These phenotypes are largely reciprocal to those of Bic-C mutants, and they affect cellular processes that Bic-C-associated mRNAs are known, or predicted, to regulate. We conclude that Bic-C regulates expression of specific germline mRNAs by controlling their poly(A)-tail length.

The Bic-C family of developmental translational regulators.

Regulation of mRNA translation is especially important during cellular and developmental processes. Many evolutionarily conserved proteins act in the context of multiprotein complexes and modulate protein translation both at the spatial and the temporal levels. Among these, Bicaudal C constitutes a family of RNA binding proteins whose founding member was first identified in Drosophila and contains orthologs in vertebrates. We discuss recent advances towards understanding the functions of these proteins in the context of the cellular and developmental biology of many model organisms and their connection to human disease.

Empowering Melatonin Therapeutics with Drosophila Models.

Melatonin functions as a central regulator of cell and organismal function as well as a neurohormone involved in several processes, e.g., the regulation of the circadian rhythm, sleep, aging, oxidative response, and more. As such, it holds immense pharmacological potential. Receptor-mediated melatonin function mainly occurs through MT1 and MT2, conserved amongst mammals. Other melatonin-binding proteins exist. Non-receptor-mediated activities involve regulating the mitochondrial function and antioxidant cascade, which are frequently affected by normal aging as well as disease. Several pathologies display diseased or dysfunctional mitochondria, suggesting melatonin may be used therapeutically. Drosophila models have extensively been employed to study disease pathogenesis and discover new drugs. Here, we review the multiple functions of melatonin through the lens of functional conservation and model organism research to empower potential melatonin therapeutics to treat neurodegenerative and renal diseases.

Cap-dependent translational inhibition establishes two opposing morphogen gradients in Drosophila embryos.

In the early Drosophila embryo, asymmetric distribution of transcription factors, established as a consequence of translational control of their maternally derived mRNAs, initiates pattern formation . For instance, translation of the uniformly distributed maternal hunchback (hb) mRNA is inhibited at the posterior to form an anterior-to-posterior protein concentration gradient along the longitudinal axis . Inhibition of hb mRNA translation requires an mRNP complex (the NRE complex), which consists of Nanos (Nos), Pumilio (Pum), and Brain tumor (Brat) proteins, and the Nos responsive element (NRE) present in the 3' UTR of hb mRNA . The identity of the mRNA 5' effector protein that is responsible for this translational inhibition remained elusive. Here we show that d4EHP, a cap binding protein that represses caudal (cad) mRNA translation , also inhibits hb mRNA translation by interacting simultaneously with the mRNA 5' cap structure (m(7)GpppN, where N is any nucleotide) and Brat. Thus, by regulating Cad and Hb expression, d4EHP plays a key role in establishing anterior-posterior axis polarity in the Drosophila embryo.

The C elegans hunchback homolog, hbl-1, controls temporal patterning and is a probable microRNA target.

hunchback regulates the temporal identity of neuroblasts in Drosophila. Here we show that hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system.

Vasa protein is localized in the germ cells and in the oocyte-associated pyriform follicle cells during early oogenesis in the lizard Podarcis sicula.

The vasa gene, first identified in Drosophila, is a key determinant for germline formation in eukaryotes. Homologs of vasa have been identified and linked to germline development, in many invertebrates and vertebrates. Here, we analyze the distribution of Vasa in early germ cells (oogonia and oocytes) and previtellogenic ovarian follicles of the lizard Podarcis sicula. During most of its previtellogenic growth, the oocyte in this lizard species is structurally and functionally integrated through intercellular bridges with special follicle cells called pyriform cells. The pyriform cells function similarly to Drosophila nurse cells, but are somatic in origin. In the oogenesis of P. sicula, Vasa is initially highly detected in the oogonia, but its levels decrease in early stage oocytes before the onset of pyriform cell differentiation. In the later stages of oogenesis, the high level of Vasa is related with the nurse function of the pyriform follicle cells. These observations suggest that cells of somatic origin are engaged in the synthesis of Vasa in the oogenesis of this lizard.

Mapping the fly Malpighian tubule lipidome by imaging mass spectrometry.

Matrix-assisted laser/desorption ionization imaging mass spectrometry (MALDI IMS) is an analytical technique for understanding the spatial distribution of biomolecules across a sample surface. Originally employed for mammalian tissues, this technology has been adapted to study specimens as diverse as microbes and cell cultures, food such as strawberries, and invertebrates including the vinegar fly Drosophila melanogaster. As an ideal model organism, Drosophila has brought greater understanding about conserved biological processes, organism development, and diseased states and even informed management practices of agriculturally and environmentally important species. Drosophila displays anatomically separated renal (Malpighian) tubules that are the physiological equivalent to the vertebrate nephron. Insect Malpighian tubules are also responsible for pesticide detoxification. In this article, we first describe an effective workflow and sample preparation method to study the phospholipid distribution of the Malpighian tubules that initially involves the manual microdissection of the tubules in saline buffer followed by a series of washes to remove excess salt and enhances the phospholipid signals prior to matrix deposition and IMS at 25-µm spatial resolution. We also established a complementary methodology for lipid IMS analysis of whole-body fly sections using a dual-polarity data acquisition approach at the same spatial resolution after matrix deposition by sublimation. Both procedures yield rich signal profiles from the major phospholipid classes. The reproducibility and high-quality results offered by these methodologies enable cohort studies of Drosophila through MALDI IMS.

Cyst Reduction in a Polycystic Kidney Disease Drosophila Model Using Smac Mimics.

Autosomal dominant polycystic kidney disease (ADPKD) is an inherited malady affecting 12.5 million people worldwide. Therapeutic options to treat PKD are limited, due in part to lack of precise knowledge of underlying pathological mechanisms. Mimics of the second mitochondria-derived activator of caspases (Smac) have exhibited activity as antineoplastic agents and reported recently to ameliorate cysts in a murine ADPKD model, possibly by differentially targeting cystic cells and sparing the surrounding tissue. A first-in-kind Drosophila PKD model has now been employed to probe further the activity of novel Smac mimics. Substantial reduction of cystic defects was observed in the Malpighian (renal) tubules of treated flies, underscoring mechanistic conservation of the cystic pathways and potential for efficient testing of drug prototypes in this PKD model. Moreover, the observed differential rescue of the anterior and posterior tubules overall, and within their physiologically diverse intermediate and terminal regions implied a nuanced response in distinct tubular regions contingent upon the structure of the Smac mimic. Knowledge gained from studying Smac mimics reveals the capacity for the Drosophila model to precisely probe PKD pharmacology highlighting the value for such critical evaluation of factors implicated in renal function and pathology.

Modeling Renal Disease "On the Fly".

Detoxification is a fundamental function for all living organisms that need to excrete catabolites and toxins to maintain homeostasis. Kidneys are major organs of detoxification that maintain water and electrolyte balance to preserve physiological functions of vertebrates. In insects, the renal function is carried out by Malpighian tubules and nephrocytes. Due to differences in their circulation, the renal systems of mammalians and insects differ in their functional modalities, yet carry out similar biochemical and physiological functions and share extensive genetic and molecular similarities. Evolutionary conservation can be leveraged to model specific aspects of the complex mammalian kidney function in the genetic powerhouse Drosophila melanogaster to study how genes interact in diseased states. Here, we compare the human and Drosophila renal systems and present selected fly disease models.

Drosophila RNA binding proteins.

RNA binding proteins are fundamental mediators of gene expression. The use of the model organism Drosophila has helped to elucidate both tissue-specific and ubiquitous functions of RNA binding proteins. These proteins mediate all aspects of the mRNA lifespan including splicing, nucleocytoplasmic transport, localization, stability, translation, and degradation. Most RNA binding proteins fall into several major groups, based on their RNA binding domains. As well, experimental data have revealed several proteins that can bind RNA but lack canonical RNA binding motifs, suggesting the presence of as yet uncharacterized RNA binding domains. Here, we present the major classes of Drosophila RNA binding proteins with special focus on those with functional information.