Cachexia: A systemic consequence of progressive, unresolved disease.

Cachexia, a systemic wasting condition, is considered a late consequence of diseases, including cancer, organ failure, or infections, and contributes to significant morbidity and mortality. The induction process and mechanistic progression of cachexia are incompletely understood. Refocusing academic efforts away from advanced cachexia to the etiology of cachexia may enable discoveries of new therapeutic approaches. Here, we review drivers, mechanisms, organismal predispositions, evidence for multi-organ interaction, model systems, clinical research, trials, and care provision from early onset to late cachexia. Evidence is emerging that distinct inflammatory, metabolic, and neuro-modulatory drivers can initiate processes that ultimately converge on advanced cachexia.

"ISN't Thirst Sweet?" Says the Fly.

How food and water intake is reciprocally regulated to maintain homeostasis is unclear. New findings by Jourjine and colleagues identify four neurons in the Drosophila brain that receive both water and sugar abundance signals and oppositely regulate hunger and thirst.

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Cholinergic neurons trigger epithelial Ca2+ currents to heal the gut.

A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer1. We used the Drosophila midgut2 to investigate this and discovered that during regeneration a subpopulation of cholinergic3 neurons triggers Ca2+ currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. We found that downregulation of the conserved cholinergic enzyme acetylcholinesterase4 in the gut epithelium enables acetylcholine from specific Egr5 (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca2+, which spreads in the epithelium through Innexin2-Innexin7 gap junctions6, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation7, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel diseases8. Altogether, the conserved cholinergic pathway facilitates epithelial Ca2+ currents that heal the intestinal epithelium. Our findings demonstrate nerve- and bioelectric9-dependent intestinal regeneration and advance our current understanding of how a tissue returns to homeostasis after injury.

A phosphate-sensing organelle regulates phosphate and tissue homeostasis.

Inorganic phosphate (Pi) is one of the essential molecules for life. However, little is known about intracellular Pi metabolism and signalling in animal tissues1. Following the observation that chronic Pi starvation causes hyperproliferation in the digestive epithelium of Drosophila melanogaster, we determined that Pi starvation triggers the downregulation of the Pi transporter PXo. In line with Pi starvation, PXo deficiency caused midgut hyperproliferation. Interestingly, immunostaining and ultrastructural analyses showed that PXo specifically marks non-canonical multilamellar organelles (PXo bodies). Further, by Pi imaging with a Förster resonance energy transfer (FRET)-based Pi sensor2, we found that PXo restricts cytosolic Pi levels. PXo bodies require PXo for biogenesis and undergo degradation following Pi starvation. Proteomic and lipidomic characterization of PXo bodies unveiled their distinct feature as an intracellular Pi reserve. Therefore, Pi starvation triggers PXo downregulation and PXo body degradation as a compensatory mechanism to increase cytosolic Pi. Finally, we identified connector of kinase to AP-1 (Cka), a component of the STRIPAK complex and JNK signalling3, as the mediator of PXo knockdown- or Pi starvation-induced hyperproliferation. Altogether, our study uncovers PXo bodies as a critical regulator of cytosolic Pi levels and identifies a Pi-dependent PXo-Cka-JNK signalling cascade controlling tissue homeostasis.

Stress signaling between organs in metazoa.

Many organisms have developed a robust ability to adapt and survive in the face of environmental perturbations that threaten the integrity of their genome, proteome, or metabolome. Studies in multiple model organisms have shown that, in general, when exposed to stress, cells activate a complex prosurvival signaling network that includes immune and DNA damage response genes, chaperones, antioxidant enzymes, structural proteins, metabolic enzymes, and noncoding RNAs. The manner of activation runs the gamut from transcriptional induction of genes to increased stability of transcripts to posttranslational modification of important biosynthetic proteins within the stressed tissue. Superimposed on these largely autonomous effects are nonautonomous responses in which the stressed tissue secretes peptides and other factors that stimulate tissues in different organs to embark on processes that ultimately help the organism as a whole cope with stress. This review focuses on the mechanisms by which tissues in one organ adapt to environmental challenges by regulating stress responses in tissues of different organs.

mTORC1 promotes cell growth via m6A-dependent mRNA degradation.

Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.

Muscle mitohormesis promotes longevity via systemic repression of insulin signaling.

Mitochondrial dysfunction is usually associated with aging. To systematically characterize the compensatory stress signaling cascades triggered in response to muscle mitochondrial perturbation, we analyzed a Drosophila model of muscle mitochondrial injury. We find that mild muscle mitochondrial distress preserves mitochondrial function, impedes the age-dependent deterioration of muscle function and architecture, and prolongs lifespan. Strikingly, this effect is mediated by at least two prolongevity compensatory signaling modules: one involving a muscle-restricted redox-dependent induction of genes that regulate the mitochondrial unfolded protein response (UPR(mt)) and another involving the transcriptional induction of the Drosophila ortholog of insulin-like growth factor-binding protein 7, which systemically antagonizes insulin signaling and facilitates mitophagy. Given that several secreted IGF-binding proteins (IGFBPs) exist in mammals, our work raises the possibility that muscle mitochondrial injury in humans may similarly result in the secretion of IGFBPs, with important ramifications for diseases associated with aberrant insulin signaling.

Sestrin mediates detection of and adaptation to low-leucine diets in Drosophila.

Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine1. Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation2,3, but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn-/-) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (SesnL431E) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient.

CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.

Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.

Wnt signaling modulates the response to DNA damage in the Drosophila wing imaginal disc by regulating the EGFR pathway.

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.

Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion.

In Drosophila, the fat body (FB), a functional analog of the vertebrate adipose tissue, is the nutrient sensor that conveys the nutrient status to the insulin-producing cells (IPCs) in the fly brain to release Drosophila insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. We identify Unpaired 2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the fed state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, we find that human Leptin can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin.

RNAi screening comes of age: improved techniques and complementary approaches.

Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.

Control of the mitotic cleavage plane by local epithelial topology.

For nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g., Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage-plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long-axis orientations of their adjacent mitotic neighbors. Strikingly, analyses of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage-plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage-plane orientation, and that cleavage-plane bias may be a widespread property of polygonal cell sheets in plants and animals.

Expression Atlas update: insights from sequencing data at both bulk and single cell level.

Expression Atlas (www.ebi.ac.uk/gxa) and its newest counterpart the Single Cell Expression Atlas (www.ebi.ac.uk/gxa/sc) are EMBL-EBI's knowledgebases for gene and protein expression and localisation in bulk and at single cell level. These resources aim to allow users to investigate their expression in normal tissue (baseline) or in response to perturbations such as disease or changes to genotype (differential) across multiple species. Users are invited to search for genes or metadata terms across species or biological conditions in a standardised consistent interface. Alongside these data, new features in Single Cell Expression Atlas allow users to query metadata through our new cell type wheel search. At the experiment level data can be explored through two types of dimensionality reduction plots, t-distributed Stochastic Neighbor Embedding (tSNE) and Uniform Manifold Approximation and Projection (UMAP), overlaid with either clustering or metadata information to assist users' understanding. Data are also visualised as marker gene heatmaps identifying genes that help confer cluster identity. For some data, additional visualisations are available as interactive cell level anatomograms and cell type gene expression heatmaps.

split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80.

The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of "split-intein Gal4" in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type-specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm ("Two Against Background" or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.

Genomic screening with RNAi: results and challenges.

RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.

State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila.

For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens.

Protein phosphatase 1 regulates core PCP signaling.

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

FOXO/4E-BP signaling in Drosophila muscles regulates organism-wide proteostasis during aging.

The progressive loss of muscle strength during aging is a common degenerative event of unclear pathogenesis. Although muscle functional decline precedes age-related changes in other tissues, its contribution to systemic aging is unknown. Here, we show that muscle aging is characterized in Drosophila by the progressive accumulation of protein aggregates that associate with impaired muscle function. The transcription factor FOXO and its target 4E-BP remove damaged proteins at least in part via the autophagy/lysosome system, whereas foxo mutants have dysfunctional proteostasis. Both FOXO and 4E-BP delay muscle functional decay and extend life span. Moreover, FOXO/4E-BP signaling in muscles decreases feeding behavior and the release of insulin from producing cells, which in turn delays the age-related accumulation of protein aggregates in other tissues. These findings reveal an organism-wide regulation of proteostasis in response to muscle aging and a key role of FOXO/4E-BP signaling in the coordination of organismal and tissue aging.