Inducible progenitor-derived Wingless regulates adult midgut regeneration in Drosophila.

The ability to regenerate following stress is a hallmark of self-renewing tissues. However, little is known about how regeneration differs from homeostatic tissue maintenance. Here, we study the role and regulation of Wingless (Wg)/Wnt signalling during intestinal regeneration using the Drosophila adult midgut. We show that Wg is produced by the intestinal epithelial compartment upon damage or stress and it is exclusively required for intestinal stem cell (ISC) proliferation during tissue regeneration. Reducing Wg or downstream signalling components from the intestinal epithelium blocked tissue regeneration. Importantly, we demonstrate that Wg from the undifferentiated progenitor cell, the enteroblast, is required for Myc-dependent ISC proliferation during regeneration. Similar to young regenerating tissues, ageing intestines required Wg and Myc for ISC hyperproliferation. Unexpectedly, our results demonstrate that epithelial but not mesenchymal Wg is essential for ISC proliferation in response to damage, while neither source of the ligand is solely responsible for ISC maintenance and tissue self-renewal in unchallenged tissues. Therefore, fine-tuning Wnt results in optimal balance between the ability to respond to stress without negatively affecting organismal viability.

Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut.

Inactivating mutations within adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, are responsible for most sporadic and hereditary forms of colorectal cancer (CRC). Here, we use the adult Drosophila midgut as a model system to investigate the molecular events that mediate intestinal hyperplasia following loss of Apc in the intestine. Our results indicate that the conserved Wnt target Myc and its binding partner Max are required for the initiation and maintenance of intestinal stem cell (ISC) hyperproliferation following Apc1 loss. Importantly, we find that loss of Apc1 leads to the production of the interleukin-like ligands Upd2/3 and the EGF-like Spitz in a Myc-dependent manner. Loss of Apc1 or high Wg in ISCs results in non-cell-autonomous upregulation of upd3 in enterocytes and subsequent activation of Jak/Stat signaling in ISCs. Crucially, knocking down Jak/Stat or Spitz/Egfr signaling suppresses Apc1-dependent ISC hyperproliferation. In summary, our results uncover a novel non-cell-autonomous interplay between Wnt/Myc, Egfr and Jak/Stat signaling in the regulation of intestinal hyperproliferation. Furthermore, we present evidence suggesting potential conservation in mouse models and human CRC. Therefore, the Drosophila adult midgut proves to be a powerful genetic system to identify novel mediators of APC phenotypes in the intestine.

Reduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferation.

OBJECTIVE: Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development. DESIGN: LIMK2 expression and activity were measured by immunostaining tumours from CRC-prone mice, human CRC cell lines and 650 human tumours. LIMK knockdown in Drosophila or Limk2 deletion in mice allowed for assessment of their contributions to gastrointestinal stem cell homeostasis and tumour development. RESULTS: LIMK2 expression was reduced in intestinal tumours of cancer-prone mice, as well as in human CRC cell lines and tumours. Reduced LIMK2 expression and substrate phosphorylation were associated with shorter patient survival. Genetic analysis in Drosophila midgut and intestinal epithelial cells isolated from genetically modified mice revealed a conserved role for LIMK2 in constraining gastrointestinal stem cell proliferation. Limk2 deletion increased colon tumour size in a colitis-associated colorectal mouse cancer model. CONCLUSIONS: This study revealed that LIMK2 expression and activity progressively decrease with advancing stage, and supports the hypothesis that there is selective pressure for reduced LIMK2 expression in CRC to relieve negative constraints imposed upon gastrointestinal stem cells.

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction.

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.

Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients.

Myotonic dystrophy type 1 (DM1) is one of the most variable inherited human disorders. It is characterized by the involvement of multiple tissues and is caused by the expansion of a highly unstable CTG repeat. Variation in disease severity is partially accounted for by the number of CTG repeats inherited. However, the basis of the variable tissue-specific symptoms is unknown. We have determined that an unusual Dutch family co-segregating DM1, Charcot-Marie-Tooth neuropathy, encephalopathic attacks and early hearing loss, carries a complex variant repeat at the DM1 locus. The mutation comprises an expanded CTG tract at the 5'-end and a complex array of CTG repeats interspersed with multiple GGC and CCG repeats at the 3'-end. The complex variant repeat tract at the 3'-end of the array is relatively stable in both blood DNA and the maternal germ line, although the 5'-CTG tract remains genetically unstable and prone to expansion. Surprisingly though, even the pure 5'-CTG tract is more stable in blood DNA and the maternal germ line than archetypal DM1 alleles of a similar size. Complex variant repeats were also identified at the 3'-end of the CTG array of approximately 3-4% of unrelated DM1 patients. The observed polarity and the stabilizing effect of the variant repeats implicate a cis-acting modifier of mutational dynamics in the 3'-flanking DNA. The presence of such variant repeats very likely contributes toward the unusual symptoms in the Dutch family and additional symptomatic variation in DM1 via affects on both RNA toxicity and somatic instability.

Mitochondrial ROS signalling requires uninterrupted electron flow and is lost during ageing in flies.

Mitochondrial reactive oxygen species (mtROS) are cellular messengers essential for cellular homeostasis. In response to stress, reverse electron transport (RET) through respiratory complex I generates high levels of mtROS. Suppression of ROS production via RET (ROS-RET) reduces survival under stress, while activation of ROS-RET extends lifespan in basal conditions. Here, we demonstrate that ROS-RET signalling requires increased electron entry and uninterrupted electron flow through the electron transport chain (ETC). We find that in old fruit flies, ROS-RET is abolished when electron flux is decreased and that their mitochondria produce consistently high levels of mtROS. Finally, we demonstrate that in young flies, limiting electron exit, but not entry, from the ETC phenocopies mtROS generation observed in old individuals. Our results elucidate the mechanism by which ROS signalling is lost during ageing.

Autophagy promotes cell survival by maintaining NAD levels.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Production of reactive oxygen species by the mitochondrial electron transport chain in Drosophila melanogaster.

Mitochondrial free radicals and in particular mitochondrial Reactive Oxygen Species (mtROS) are considered to be totally or partially responsible for several different diseases including Parkinson, diabetes or cancer. Even more importantly, mtROS have also been proposed as the main driving force behind the aging process. Thus, in the last decade, there has been a growing interest in the role of free radicals as signalling molecules. Collectively this makes understanding mechanisms controlling free radical production extremely important. There is extensive published literature on mammalian models (essentially rat, mouse and guinea pig) however; this is not the case in Drosophila melanogaster. Drosophila is an excellent model to study different physiological and pathological processes. Additionally a robust method to study mtROS is extremely useful. In the present article, we describe a simple--but extremely sensitive--method to study mtROS production in Drosophila. We have performed various experiments to determine which specific respiratory complexes produce free radicals in the electron transport chain of Drosophila melanogaster. Complex I is the main generator of ROS in Drosophila mitochondria, leaking electrons either in the forward or reverse direction. The production of ROS during reverse electron transport can be prevented either by rotenone or by the oxidation of NADH by complex I. These results clearly show that Drosophila mitochondria function in a very similar way to mammalian mitochondria, and therefore are a very relevant experimental model for biochemical studies related to ageing.

Mitochondrial complex I derived ROS regulate stress adaptation in Drosophila melanogaster.

Reactive Oxygen Species (ROS) are essential cellular messengers required for cellular homeostasis and regulate the lifespan of several animal species. The main site of ROS production is the mitochondrion, and within it, respiratory complex I (CI) is the main ROS generator. ROS produced by CI trigger several physiological responses that are essential for the survival of neurons, cardiomyocytes and macrophages. Here, we show that CI produces ROS when electrons flow in either the forward (Forward Electron Transport, FET) or reverse direction (Reverse Electron Transport, RET). We demonstrate that ROS production via RET (ROS-RET) is activated under thermal stress conditions and that interruption of ROS-RET production, through ectopic expression of the alternative oxidase AOX, attenuates the activation of pro-survival pathways in response to stress. Accordingly, we find that both suppressing ROS-RET signalling or decreasing levels of mitochondrial H2O2 by overexpressing mitochondrial catalase (mtCAT), reduces survival dramatically in flies under stress. Our results uncover a specific ROS signalling pathway where hydrogen peroxide (H2O2) generated by CI via RET is required to activate adaptive mechanisms, maximising survival under stress conditions.

Essential Physiological Differences Characterize Short- and Long-Lived Strains of Drosophila melanogaster.

Aging is a multifactorial process which affects all animals. Aging as a result of damage accumulation is the most accepted explanation but the proximal causes remain to be elucidated. There is also evidence indicating that aging has an important genetic component. Animal species age at different rates and specific signaling pathways, such as insulin/insulin-like growth factor, can regulate life span of individuals within a species by reprogramming cells in response to environmental changes. Here, we use an unbiased approach to identify novel factors that regulate life span in Drosophila melanogaster. We compare the transcriptome and metabolome of two wild-type strains used widely in aging research: short-lived Dahomey and long-lived Oregon R flies. We found that Dahomey flies carry several traits associated with short-lived individuals and species such as increased lipoxidative stress, decreased mitochondrial gene expression, and increased Target of Rapamycin signaling. Dahomey flies also have upregulated octopamine signaling known to stimulate foraging behavior. Accordingly, we present evidence that increased foraging behavior, under laboratory conditions where nutrients are in excess increases damage generation and accelerates aging. In summary, we have identified several new pathways, which influence longevity highlighting the contribution and importance of the genetic component of aging.

The role of mitochondrial ROS in the aging brain.

The brain is the most complex human organ, consuming more energy than any other tissue in proportion to its size. It relies heavily on mitochondria to produce energy and is made up of mitotic and postmitotic cells that need to closely coordinate their metabolism to maintain essential bodily functions. During aging, damaged mitochondria that produce less ATP and more reactive oxygen species (ROS) accumulate. The current consensus is that ROS cause oxidative stress, damaging mitochondria and resulting in an energetic crisis that triggers neurodegenerative diseases and accelerates aging. However, in model organisms, increasing mitochondrial ROS (mtROS) in the brain extends lifespan, suggesting that ROS may participate in signaling that protects the brain. Here, we summarize the mechanisms by which mtROS are produced at the molecular level, how different brain cells and regions produce different amounts of mtROS, and how mtROS levels change during aging. Finally, we critically discuss the possible roles of ROS in aging as signaling molecules and damaging agents, addressing whether age-associated increases in mtROS are a cause or a consequence of aging.

Oxidation of p62 as an evolutionary adaptation to promote autophagy in stress conditions.

Ageing and age-related diseases are characterised by increased oxidative and proteotoxic stress, which results in negative effects on cell function and survival. The cell possesses several mechanisms to deal with damaged proteins, including degradation via macroautophagy (hereafter called autophagy). This essential cellular pathway is conserved from yeast to humans and it is well established that its impairment reduces lifespan in multiple model organisms, including worms, flies and mice. In our study, recently published in Nature Communications, we asked if longer lifespan characteristic of higher organisms is the result of evolutionary adaptations to the autophagy machinery. We found that the autophagy receptor p62 can be oxidised leading to its oligomerisation which ultimately promotes autophagy. However this mechanism, present in vertebrates, has been acquired late in evolution. We propose that the ability of p62 to sense reactive oxygen species (ROS) via oxidation, and potentially other similar modifications, may have evolved in higher organisms and contributed to their increased lifespan. Indeed, impairment of this process could result in age-related neurodegeneration in humans.

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis.

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.

Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in Drosophila melanogaster.

Drosophila melanogaster is a popular research model organism thanks to its' powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates "the background advantage", but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail.

Transformed epithelia trigger non-tissue-autonomous tumor suppressor response by adipocytes via activation of Toll and Eiger/TNF signaling.

High tumor burden is associated with increased levels of circulating inflammatory cytokines that influence the pathophysiology of the tumor and its environment. The cellular and molecular events mediating the organismal response to a growing tumor are poorly understood. Here, we report a bidirectional crosstalk between epithelial tumors and the fat body-a peripheral immune tissue-in Drosophila. Tumors trigger a systemic immune response through activation of Eiger/TNF signaling, which leads to Toll pathway upregulation in adipocytes. Reciprocally, Toll elicits a non-tissue-autonomous program in adipocytes, which drives tumor cell death. Hemocytes play a critical role in this system by producing the ligands Spätzle and Eiger, which are required for Toll activation in the fat body and tumor cell death. Altogether, our results provide a paradigm for a long-range tumor suppression function of adipocytes in Drosophila, which may represent an evolutionarily conserved mechanism in the organismal response to solid tumors.

p120 catenin is required for the stress response in Drosophila.

p120ctn is a ubiquitously expressed core component of cadherin junctions and essential for vertebrate development. Surprisingly, Drosophila p120ctn (dp120ctn) is dispensable for adherens junctions and development, which has discouraged Drosophila researchers from further pursuing the biological role of dp120ctn. Here we demonstrate that dp120ctn loss results in increased heat shock sensitivity and reduced animal lifespan, which are completely rescued by ectopic expression of a dp120ctn-GFP transgene. Transcriptomic analysis revealed multiple relish/NF-κB target genes differentially expressed upon loss of dp120ctn. Importantly, this aberrant gene expression was rescued by overexpression of dp120ctn-GFP or heterozygosity for relish. Our results uncover a novel role for dp120ctn in the regulation of animal stress response and immune signalling. This may represent an ancient role of p120ctn and can influence further studies in Drosophila and mammals.

Regulation of lifespan by the mitochondrial electron transport chain: reactive oxygen species-dependent and reactive oxygen species-independent mechanisms.

SIGNIFICANCE: Aging is a consequence of the accumulation of cellular damage that impairs the capacity of an aging organism to adapt to stress. The Mitochondrial Free Radical Theory of Aging (MFRTA) has been one of the most influential ideas over the past 50 years. The MFRTA is supported by the accumulation of oxidative damage during aging along with comparative studies demonstrating that long-lived species or individuals produce fewer mitochondrial reactive oxygen species and have lower levels of oxidative damage. RECENT ADVANCES: Recently, however, species that combine high oxidative damage with a longer lifespan (i.e., naked mole rats) have been described. Moreover, most of the interventions based on antioxidant supplementation do not increase longevity, as would be predicted by the MFRTA. Studies to date provide a clear understanding that mitochondrial function regulates the rate of aging, but the underlying mechanisms remain unclear. CRITICAL ISSUES: Here, we review the reactive oxygen species (ROS)-dependent and ROS-independent mechanisms by which mitochondria can affect longevity. We discuss the role of different ROS (superoxide, hydrogen peroxide, and hydroxyl radical), both as oxidants as well as signaling molecules. We also describe how mitochondria can regulate longevity by ROS-independent mechanisms. We discuss alterations in mitochondrial DNA, accumulation of cellular waste as a consequence of glyco- and lipoxidative damage, and the regulation of DNA maintenance enzymes as mechanisms that can determine longevity without involving ROS. FUTURE DIRECTIONS: We also show how the regulation of longevity is a complex process whereby ROS-dependent and ROS-independent mechanisms interact to determine the maximum lifespan of species and individuals.

dj-1ß regulates oxidative stress, insulin-like signaling and development in Drosophila melanogaster.

DJ-1 (or PARK-7) is a multifunctional protein implicated in numerous pathologies including cancer, sterility and Parkinson disease (PD). The popular genetic model Drosophila melanogaster has two orthologs, dj-1: α and ß. Dysfunction of dj-1ß strongly impairs fly mobility in an age-dependent manner. In this study, we analyze in detail the molecular mechanism underlying the dj-1ß mutant phenotype. Mitochondrial hydrogen peroxide production, but not superoxide production, was increased in mutant flies. An increase in peroxide leak from mitochondria causes oxidative damage elsewhere and explains the strong reduction in mobility caused by dj-1ß mutation. However, at the same time, increased levels of hydrogen peroxide activated a pro-survival program characterized by (1) an alteration in insulin-like signaling, (2) an increase in mitochondrial biogenesis and (3) an increase in the de-acetylase activity of sirtuins. The activation of this pro-survival program was associated with increased longevity under conditions of moderate oxidative stress. Additionally, the dj-1ß mutation unexpectedly accelerated development, a phenotype not previously associated with this mutation. Our results reveal an important role of dj-1ß in oxidative stress handling, insulin-like signaling and development in Drosophila melanogaster.

Mitochondrial complex I: a central regulator of the aging process.

Mitochondria are considered major regulators of longevity, although their exact role in aging is not fully understood. Data from different laboratories show a negative correlation between reactive oxygen species (ROS) generated by complex I and lifespan. This suggests that complex I has a central role in the regulation of longevity. Here, we review data that both support and refute the role of complex I as a pacemaker of aging. We include data from our laboratory, where we have manipulated ROS production by the electron transport chain (ETC) in Drosophila melanogaster. The bypass of complex I increases the lifespan of the fruit fly, but it is not clear if this is caused by a reduction in ROS or by a change in the NAD+ to NADH ratio. We propose that complex I regulates aging through at least two mechanisms: (1) an ROS-dependent mechanism that leads to mitochondrial DNA damage and (2) an ROS-independent mechanism through the control of the NAD+ to NADH ratio. Control of the relative levels of NAD+ and NADH would allow the regulation of (1) glyco- and (2) lipoxidative-damage and (3) the activation of sirtuins.

Tumor invasion and metastasis in Drosophila: a bold past, a bright future.

Invasion and metastasis are the most deadly hallmarks of cancer. Once a cancer has acquired the ability to colonize new sites in the body it becomes dramatically more difficult to treat. This has made it a focus of much of cancer research. The humble fruit fly, Drosophila melanogaster, has despite its relative simplicity, made significant contributions to the understanding of tumor progression. In this review we outline and highlight those with an emphasis on modeling the genetic and epigenetic changes required for invasion and metastasis. We will revisit the early years of cancer modeling in Drosophila where the first parallels were drawn between Drosophila and vertebrate neoplasms and highlight recent advances using genetic screens and interactions with the epithelial microenvironment and innate immune system. We focus on the power and limitations of current fly models of metastasis.