The Interactive Fly

Zygotically transcribed genes

Apoptosis and Autophagy

  • Cell death regulation in Drosophila: Conservation of mechanism and unique insights
  • JAK/STAT autocontrol of ligand-producing cell number through apoptosis
  • A steroid-controlled global switch in sensitivity to apoptosis during Drosophila development
  • CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid
  • The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB
  • The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis
  • Low levels of p53 protein and chromatin silencing of p53 target genes repress apoptosis in Drosophila endocycling cells
  • Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila
  • Dying cells protect survivors from radiation-induced cell death in Drosophila
  • Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster
  • Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila
  • Tumor suppressor gene OSCP1/NOR1 regulates apoptosis, proliferation, differentiation, and ROS generation during eye development of Drosophila melanogaster
  • Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye
  • Analysis of the function of apoptosis during imaginal wing disc regeneration in Drosophila melanogaster
  • Different cell cycle modifications repress apoptosis at different steps independent of developmental signaling in Drosophila
  • The human Bcl-2 family member Bcl-rambo localizes to mitochondria and induces apoptosis and morphological aberrations in Drosophila
  • Necrotic pyknosis is a morphologically and biochemically distinct event from apoptotic pyknosis
  • The defender against apoptotic cell death 1 gene is required for tissue growth and efficient N-glycosylation in Drosophila melanogaster
  • Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster
  • In vivo biosensor tracks non-apoptotic caspase activity in Drosophila
  • Cellular aspects of gonadal atrophy in Drosophila P-M hybrid dysgenesis
  • Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium

  • Autophagy in neurodegeneration: two sides of the same coin
  • Genes involved in autophagic cell death
  • Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila
  • Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy
  • Atg9 Interacts with dTRAF2/TRAF6 to Regulate Oxidative Stress-Induced JNK Activation and Autophagy Induction
  • Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila
  • Aging and Autophagic Function Influences the Progressive Decline of Adult Drosophila Behaviors
  • Uba1 functions in Atg7- and Atg3-independent autophagy
  • The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila
  • Retromer ensures the degradation of autophagic cargo via maintaining lysosome function in Drosophila
  • UTX coordinates steroid hormone-mediated autophagy and cell death
  • Hox proteins mediate developmental and environmental control of autophagy
  • Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster
  • Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila
  • An ancient defense system eliminates unfit cells from developing tissues during cell competition
  • Elimination of unfit cells maintains tissue health and prolongs lifespan
  • The metabolite alpha-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR
  • Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced neurodegeneration
  • Age-induced reduction of autophagy-related gene expression is associated with onset of Alzheimer's disease
  • P62 plays a protective role in the autophagic degradation of polyglutamine protein oligomers in polyglutamine disease model flies
  • Huntingtin functions as a scaffold for selective macroautophagy
  • Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model
  • p62/Sequstosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB
  • Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis
  • β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy
  • Tousled-like kinase mediated a new type of cell death pathway in Drosophila
  • Ceramides and stress signalling intersect with autophagic defects in neurodegenerative Drosophila blue cheese (bchs) mutants
  • deubiquitinating enzyme UBPY is required for lysosomal biogenesis and productive autophagy in Drosophila
  • Drosophila Mitf regulates the V-ATPase and the lysosomal-autophagic pathway
  • Selective endosomal microautophagy is starvation-inducible in Drosophila
  • Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila
  • CCT complex restricts neuropathogenic protein aggregation via autophagy
  • Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy
  • Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy
  • Microenvironmental autophagy promotes tumour growth
  • EndoA/Endophilin-A creates docking stations for autophagic proteins at synapses
  • Heparan sulfate proteoglycans regulate autophagy in Drosophila
  • Rab2 promotes autophagic and endocytic lysosomal degradation
  • Complement-related regulates autophagy in neighboring cells
  • Epigenetic regulation of starvation-induced autophagy in Drosophila by histone methyltransferase G9a
  • Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification
  • Small chaperons and autophagy protected neurons from necrotic cell death
  • Zonda is a novel early component of the autophagy pathway in Drosophila

    Genes functioning in Apoptosis

    Genes functioning in Autophagy

    Cell death regulation in Drosophila: Conservation of mechanism and unique insights

    The caspase family of cysteine proteases is central to apoptotic signaling and cell execution in all animals that have been studied, including worms, flies, and vertebrates. As with many proteases, caspases are synthesized as inactive zymogens, known as procaspases, and are generally thought to be present in all cells at levels sufficient to induce apoptosis when activated. Death stimuli lead to one or more cleavages COOH-terminal to specific aspartate residues. These cleavage events separate the large and small subunits that make up the active caspase. Two sets of these subunits assemble to form the active caspase heterotetramer, which has two active sites. Frequently an NH2-terminal prodomain is also removed during caspase processing. An important point is that the sites cleaved to produce an active caspase often correspond to caspase target sites. Thus, once activated, caspases can participate in proteolytic cascades (Vernooy, 2000 and references therein).

    Caspases play two roles in bringing about the death of the cell. They transduce death signals that are generated in specific cellular compartments, and they cleave a number of cellular proteins, resulting in the activation of some and the inactivation of others. These latter cleavage events are thought to lead, through a number of mechanisms, to many of the biochemical and morphological changes associated with apoptosis. Caspases that act as signal transducers (known as apical or upstream caspases) have long prodomains. These regions contain specific sequence motifs (known as death effector domains [DEDs] or caspase recruitment domains [CARDs]) that are thought to mediate procaspase recruitment into complexes in which caspase activation occurs in response to forced oligomerization. Some caspases may also become activated as a consequence of prodomain-dependent homodimerization. Once activated, long prodomain caspases are thought to cleave and activate short prodomain caspases (known as downstream or executioner caspases) that rely on cleavage by other caspases for activation. It is important to note that, in mammals and flies, mutant phenotypes suggest caspases can also play important nonapoptotic roles, and the functions of a number of caspases are still unclear (Vernooy, 2000 and references therein).

    Drosophila encodes three long prodomain caspases: dcp-2/dredd, dronc (Dorstyn, 1999a), and dream, as well as four caspases with short prodomains: dcp-1, drICE (Fraser, 1997), decay (Dorstyn, 1999b), and daydream. An eighth Drosophila caspase, a head-to-head partial duplication of daydream, is likely to be nonfunctional because of numerous mutations (including premature stop codons and deletions). The Caenorhabditis elegans genome encodes three caspases, the known apoptosis inducer ced-3, and csp-1 and csp-2, all of which have long prodomains. 14 caspases have been identified in mammals, 10 of which have long prodomains (Vernooy, 2000 and references therein).

    All long prodomain caspases that have been identified to date in mammals contain either CARD or DED sequences. In contrast, both Drosophila and C. elegans encode caspases that have long prodomains with unique sequences, as well as a single caspase with a CARD. The unique prodomain sequences in these caspases may promote death-inducing caspase activation in response to unknown stimuli. Alternatively, they may regulate caspase activation in contexts other than cell death. Several Drosophila and C. elegans caspases, Dronc and Csp-1a and Csp-2a, respectively, are unique in a second way as well. Caspases are described as being specific for cleavage after aspartate and typically have an active site that conforms to the consensus QAC(R/Q/G)(G/E) (catalytic cysteine is underlined). Dronc, Csp-1a, and Csp-2a have active sites that differ in the first two positions. Because the glutamine at the first position of the active site pentapeptide QACRG is part of the substrate binding pocket, it is likely that caspases with different amino acids at this position will have unique cleavage preferences. In support of this hypothesis, Dronc, which has the active site sequence PFCRG, cleaves itself after glutamate rather than aspartate, and cleaves tetrapeptide substrates after glutamate as well as aspartate (Hawkins, 2000). Cleavage specificity data for Csp-1 and Csp-2 have not been reported. Why might these caspases have altered cleavage specificity? All are long prodomain caspases, suggesting that they act to transduce signals. One possibility is simply that these proteins have unique substrates (which may or may not be death related) that require an altered cleavage specificity. The altered cleavage specificity may also have evolved to be able to efficiently cleave the sequences present between their large and small caspase subunits, which contain sequences predicted to be very poor target sites for traditional caspases. An altered cleavage specificity, in conjunction with an absence of good target sites for other caspases in the linker region, may also serve as a way of making the activation of these caspases more strictly dependent on oligomerization rather than activation by other caspases (Vernooy, 2000 and references therein).

    In mammals, three pathways have been described that lead to caspase activation. In one pathway a serine protease, granzyme B, is delivered directly into the cytoplasm of target cells from cytotoxic T cells, where it activates executioner caspases. In the other two pathways, cytoplasmic adaptor proteins link a cell death signal transducer to a long prodomain caspase through homophilic receptor-adaptor and adaptor-caspase interactions leading to caspase activation. In one pathway, initiating at the plasma membrane, caspase recruitment is initiated by the binding of ligands to receptors of the tumor necrosis factor/nerve growth factor receptor superfamily. The cytoplasmic region of these receptors contains a region known as the death domain (DD). Ligand-dependent receptor multimerization results in the recruitment of DD-containing cytoplasmic adaptors such as Fas-associated death domain (FADD) through homophilic DD interactions. FADD and related adaptors also contain a second motif known as DED, copies of which are also present in the prodomains of caspase-8 and caspase-10. Homophilic interactions between the DEDs present in receptor-bound adaptors and procaspases leads to caspase oligomerization and subsequent autoactivation. Other adaptors that include DD and CARD domains may also couple activated receptors to CARD domain-containing caspases (Vernooy, 2000 and references therein).

    Database searches were used to find candidate death receptors (predicted type 1 transmembrane proteins containing intracellular DDs) in the fly genome. A number of proteins or predicted proteins with DD homology were found, including the kinase Pelle, a Drosophila netrin receptor, a protein with a number of ankyrin repeats (CG7462), and three other proteins that lack significant similarity to other proteins (CG2031, AF22205, and AF22206). However, none of these also shows DED or CARD homology. The prodomain of Dcp-2/Dredd does share weak homology with that of caspase-8, but the Dcp-2/Dredd prodomain is not itself identified in searches for Drosophila proteins. In fact, no Drosophila proteins with significant DED homology were identified in similar searches. These observations suggest several possibilities. One is that Drosophila lacks death receptor signaling pathways. A second possibility is that Drosophila has a death receptor pathway analogous to that found in mammals, but that the level of homology of these proteins with their mammalian counterparts is very low. Finally, Drosophila death receptors may incorporate a distinct set of oligomerization motifs. In the context of this possibility, it will be interesting to identify proteins that interact with the Dream and Dcp-2/Dredd prodomains (Vernooy, 2000 and references therein).

    In a second major pathway of apical caspase activation in mammals, cellular stress of various sorts leads to the release of mitochondrial cytochrome c, which in conjunction with the cytosolic adapter protein Apaf-1, promotes caspase-9 activation. Apaf-1 shows large regions of homology with the C. elegans apoptosis inducer, Ced-4. In both organisms, caspase-activating adapter-caspase interactions are dependent on homophilic interactions between the two proteins, mediated at least, in part, by CARDs present at the NH2 terminus of Ced-4/Apaf-1 and in the caspase prodomain. In the case of worms, caspase activation by Ced-4 requires disruption of an association between Ced-4 and the apoptosis inhibitor and Bcl-2 family member Ced-9 by Egl-1, which is a second Bcl-2 family member that acts as an apoptosis inducer. Activation of Apaf-1 in mammals in vitro requires cytochrome c, which stably interacts with WD-40 repeats present at the COOH terminus of Apaf-1 but which are absent in Ced-4. The Apaf-1 WD-40 repeats inhibit its function, and this inhibition is relieved after cytochrome c binding in the presence of ATP/dATP, allowing the formation of a multimeric Apaf-1/cytochrome c complex. Procaspase-9 is recruited to this complex and activated through autocatalysis. Recently, several Apaf-1-like genes have been identified in vertebrates. The proteins encoded by these genes contain distinct NH2- and COOH-terminal sequences, suggesting that they may activate other caspases through different upstream signaling pathways (Vernooy, 2000 and references therein).

    The Drosophila genome has one Ced-4/Apaf-1 homolog, variously known as dapaf-1(Kanuka, 1999), dark (Rodriguez, 1999), or hac-1 (Zhou, 1999). Here, this gene is referred to as apaf-1-related killer (ark), its designation in the FlyBase. This gene encodes two splice forms. The long form most closely resembles Apaf-1, in that it contains a series of COOH-terminal WD-40 repeats that presumably mediate regulation by cytochrome c. The short form most closely resembles CED-4, which lacks these repeats, and would thus be predicted to be constitutively active. Genetic evidence indicates that Ark is important for cell death induction in the fly (as well as other processes such as specification of photoreceptor number), and biochemical data point toward interactions between Ark, cytochrome c, and Drosophila caspases. Mitochondrial cytochrome c is at least shifted in localization (Varkey, 1999), and perhaps released into the cytoplasm during apoptosis (Kanuka, 1999). Thus, the weight of evidence suggests that in Drosophila , as in vertebrates, cytochrome c functions to transduce apoptotic signals through Apaf-1 (Vernooy, 2000 and references therein).

    Since proteolysis is irreversible, and caspases have the potential to engage in amplifying cascades of proteolysis, caspase activation and activity must be carefully regulated in cells that normally live. The only known cellular caspase inhibitors are members of the inhibitor of apoptosis (IAP) family. Genetic and biochemical evidence from Drosophila argues that IAP-dependent inhibition of caspase activity is essential for cell survival, and that one mechanism for cell death activation involves inhibition of IAP function (Wang, 1999; Goyal, 2000; Lisi, 2000; Vernooy, 2000 and references therein).

    IAPs were first identified as baculovirus-encoded cell death inhibitors. These proteins contain several NH2-terminal repeats of an ~70-amino acid motif known as a baculovirus IAP repeat (BIR) as well as a COOH-terminal RING finger domain. RING fingers have since been found in proteins that function in a number of different contexts. For a number of proteins this domain confers E3 ubiquitin protein ligase activity. A number of cellular proteins that share homology with the viral IAPs, based on the presence of one or more BIR repeats (referred to as BIR repeat-containing proteins, or BIRPs) have now been identified in organisms ranging from yeast to humans. The Drosophila genome encodes four BIRPs, including DIAP1, the product of thread locus, Inhibitor of apoptosis 2, deterin, a homolog of Survivin (Jones, 2000), and Bruce, a homolog of BRUCE. A number of the cellular BIRPs, including XIAP, cIAP-1, cIAP-2, NAIP, and Survivin in mammals, and DIAP1, DIAP2, and Deterin in Drosophila, have been tested and shown to act as cell death inhibitors. Notable exceptions are BIRPs from C. elegans and yeast, which regulate cell division. Thus, whereas all IAPs contain BIR repeats by definition, not all proteins with BIRs are IAPs. Many of the death-inhibiting BIRPs, including XIAP, cIAP-1, cIAP-2, Survivin, and DIAP1, have been shown to directly inhibit caspase activation or activity. However, IAPs have been found to associate with a number of different proteins, and may have multiple mechanisms of action. This is particularly suggested in the case of those proteins that contain domains associated with ubiquitin conjugation (Vernooy, 2000 and references therein).

    Mitochondria are necessary for cellular energy production, and, thus, are essential for cell survival. In vertebrates (and probably also in Drosophila ) the mitochondria are an important site of integration for cell death and survival signals. The decision to release cytochrome c constitutes one proapoptotic output of this calculation. A second proapoptotic protein released from mitochondria is apoptosis-inducing factor (AIF), which in mammals translocates from the mitochondria to the nucleus upon receipt of a death signal and causes large-scale fragmentation of the DNA. Drosophila , but not C. elegans, encodes a clear AIF homolog (CG7263) (Vernooy, 2000 and references therein).

    In some cells undergoing apoptosis, caspase inhibitors are unable to prevent cell death. One cause of this caspase-independent death is thought to be due to mitochondrial damage that occurs upstream of caspase activation. The Bcl-2 family of proteins constitutes a major family of cell death regulators, and many of their pro- and anti-apoptotic functions in vertebrates can be traced to their effects on mitochondrial function. Currently 19 distinct vertebrate Bcl-2 family members have been identified that share up to four Bcl-2 homology domains (BH1-4). Some also have a hydrophobic COOH terminus that targets them to membranes. An important aspect of Bcl-2 family member function is that pro- and anti-apoptotic proteins can heterodimerize (though this is not always required for function), and a large body of evidence argues that they titrate each other's function. However, exactly how these proteins regulate cell death is still unclear. Drosophila encodes two clear Bcl-2 family members. The first is known variously as debcl, drob-1, dBorg-1, or dbok. The second gene is known as buffy (Colussi, 2000) or dBorg-2 (Brachmann, 2000). Both proteins have BH1, BH2, and BH3 domains. Weak BH4 domain homology may also be present. They show the greatest overall homology to the mammalian proapoptotic protein Bok/Mtd, and have proapoptotic function. Genes encoding candidate prosurvival Bcl-2 proteins are not apparent in the fly genome. One possibility is that prosurvival Bcl-2 proteins do not exist. Alternatively, prosurvival members may exist, but have such low homology that it was not possible to identify them. Finally, prosurvival Bcl-2 function may be obtained from posttranslational conversion of one or both of these proteins into an antiapoptotic form (Brachmann, 2000; Vernooy, 2000 and references therein).

    A common feature of apoptotic cell death is nuclear condensation and extensive DNA degradation. Apoptotic DNA degradation involves at least several steps. In vertebrates, the initial degradation of DNA is triggered by the caspase-dependent activation of a 40-kD nuclease known as CPAN/CAD/DFF. This protein is synthesized in a form that is complexed to a specific chaperone/inhibitor known as DFF45/ICAD. Caspase cleavage of DFF45/ICAD by caspase-3, releases CPAN/DFF40/CAD, which moves to the nucleus and cleaves DNA. Both DFF45/ICAD and CPAN/DFF40/CAD, as well as several other vertebrate proteins, contain a motif known as a CIDE domain. Experimental observations suggest that CIDE-CIDE interactions are important for regulation of CPAN/DFF40/CAD activity. Degradation of DNA after cell death also occurs in Drosophila and C. elegans. The fly genome encodes functional homologs of caspase-activated DNase (CAD) and CAD inhibitor (ICAD), as well as several other predicted proteins that have CIDE domains (Inohara, 1998; Inohara, 1999; Yokoyama, 2000). CAD-like DNases or other proteins with CIDE domains have not been identified in the C. elegans genome. However, DNA fragmentation occurs cell autonomously in a CED-3-dependent manner in dying cells, suggesting that a CAD-like activity is present. In a second step in apoptotic DNA degradation, which involves the participation of cells that engulf the dying cell, DNA is further processed by an acidic endonuclease. In mammals, this activity is probably an acid lysosomal DNase, either DNase II or a DNase II-like enzyme, and in C. elegans it is the product of the nuc-1 gene. Drosophila also encodes a DNase II-like protein (CG7780), and it seems likely that this form of DNA degradation occurs in flies as well (Vernooy, 2000 and references therein).

    Two other mammalian proteins that promote nuclear apoptotic events are AIF and acinus. AIF translocates from the mitochondria to cause chromatin condensation and large-scale DNA fragmentation. Acinus, a DNA-condensing factor with no nuclease activity, localizes to the nucleus, and is activated during apoptosis by combined caspase and serine protease cleavage. Drosophila, but not C. elegans, encodes clear homologs of both these proteins Acinus and AIF) (Vernooy, 2000 and references therein).

    One of the reasons for working with a model system such as the fly is the hope of finding a different perspective that will afford unique insight into a conserved, but complex process such as apoptosis. Drosophila has arguably been in this position for some time. An early genetic screen identified a genomic region at 75C that contained genes required for essentially all normally occurring cell deaths during Drosophila embryogenesis. Three genes within this region, reaper, head involution defective, and grim, mediate this proapoptotic requirement. A large body of evidence argues that they act to integrate and transduce many different cell death signals that, ultimately, lead to the activation of caspase-dependent cell death. Rpr, Hid, and Grim have only very limited homology with each other (a short stretch of roughly 14 amino acids near their NH2 termini), and sequence homologs have not been identified in other organisms. However, recent observations argue that the mechanisms of action defined by these genes are likely to be conserved: (1) each of these proteins induces apoptosis in mammalian cells, strongly suggesting that some aspect of their function is evolutionarily conserved; (2) despite their very low level of homology with each other, they each interact with several different conserved death regulators. This suggests that putative mammalian homologs may also be quite divergent in sequence. For example, they each bind the Drosophila caspase inhibitor DIAP1 through interactions that require their NH2 termini, and genetic and biochemical data argue that one way they promote apoptosis is by inhibiting DIAP1's ability to prevent death-inducing caspase activity. Since IAPs and caspases also function to regulate death in vertebrates, it seems reasonable that Rpr, Hid, and Grim orthologs exist that perform a similar death-promoting function. A mammalian protein called Smac/DIABLO, which appears to play such a role has recently been described (Du, 2000). Rpr, Hid, and Grim also bind a Xenopus protein, Scythe, in an interaction that does not require their NH2 termini. In the case of at least Rpr this interaction leads to release of a Scythe-bound proapoptotic factor that promotes cytochrome c release. Drosophila encodes a Scythe homolog (CG7546), suggesting that a similar pathway may exist in flies as well (Vernooy, 2000 and references therein).

    JAK/STAT autocontrol of ligand-producing cell number through apoptosis

    During development, specific cells are eliminated by apoptosis to ensure that the correct number of cells is integrated in a given tissue or structure. How the apoptosis machinery is activated selectively in vivo in the context of a developing tissue is still poorly understood. In the Drosophila ovary, specialised follicle cells [polar cells (PCs)] are produced in excess during early oogenesis and reduced by apoptosis to exactly two cells per follicle extremity. PCs act as an organising centre during follicle maturation as they are the only source of the JAK/STAT pathway ligand Unpaired (Upd), the morphogen activity of which instructs distinct follicle cell fates. This study shows that reduction of Upd levels leads to prolonged survival of supernumerary PCs, downregulation of the pro-apoptotic factor Hid, upregulation of the anti-apoptotic factor Diap1 and inhibition of caspase activity. Upd-mediated activation of the JAK/STAT pathway occurs in PCs themselves, as well as in adjacent terminal follicle and interfollicular stalk cells, and inhibition of JAK/STAT signalling in any one of these cell populations protects PCs from apoptosis. Thus, a Stat-dependent unidentified relay signal is necessary for inducing supernumerary PC death. Finally, blocking apoptosis of PCs leads to specification of excess adjacent border cells via excessive Upd signalling. These results therefore show that Upd and JAK/STAT signalling induce apoptosis of supernumerary PCs to control the size of the PC organising centre and thereby produce appropriate levels of Upd. This is the first example linking this highly conserved signalling pathway with developmental apoptosis in Drosophila (Borensztejn, 2013).

    A role for STAT in cell death and survival has been clearly documented in mammals, and depending on which of the seven mammalian Stat genes is considered and on the cellular context, both pro- and anti-apoptotic functions have been characterised. In the Drosophila developing wing, phosphorylated Stat92E has been shown to be necessary for protection against stress-induced apoptosis, but not for wing developmental apoptosis. This study provides evidence that Upd and the JAK/STAT pathway control developmental apoptosis during Drosophila oogenesis (Borensztejn, 2013).

    This study demonstrated that the JAK/STAT pathway ligand, Upd, and all components of the JAK/STAT transduction cascade (the receptor Dome, JAK/Hop and Stat92E) are involved in promoting apoptosis of supernumerary PCs produced during early oogenesis. It is argued that The JAK/STAT pathway is essential for this event for several reasons. Indeed, in the strongest mutant context tested, follicle poles containing large TFC and PC clones homozygous for Stat92E amorphic alleles, almost all of these (95%) maintained more than two PCs through oogenesis. Also, RNAi-mediated reduction of upd, dome and hop blocked PC number reduction and deregulated several apoptosis markers, inhibiting Hid accumulation, Diap1 downregulation and caspase activation in supernumerary PCs. Altogether, these data, along with what has already been shown for JAK/STAT signalling in this system, fit the following model. Upd is secreted from PCs and diffuses in the local environment. Signal transduction via Dome/Hop/Stat92E occurs in nearby TFCs, interfollicular stalks and PCs themselves, leading to specific target gene transcription in these cells, as revealed by a number of pathway reporters. An as-yet-unidentified Stat92E-dependent pro-apoptotic relay signal (X) is produced in TFCs, interfollicular stalks and possibly PCs, which promotes supernumerary PC elimination via specific expression of hid in these cells, consequent downregulation of Diap1 and finally caspase activation. An additional cell-autonomous role for JAK/STAT signal transduction in supernumerary PC apoptosis of these cells is also consistent with, though not demonstrated by, the results (Borensztejn, 2013).

    Relay signalling allows for spatial and temporal positioning of multiple signals in a tissue and thus exquisite control of differentiation and morphogenetic programmes. In the Drosophila developing eye, the role of Upd and the JAK/STAT pathway in instructing planar polarity has been shown to require an as-yet-uncharacterised secondary signal. In the ovary, the fact that JAK/STAT-mediated PC apoptosis depends on a relay signal may provide a mechanism by which PC apoptosis and earlier JAK/STAT-dependent stalk-cell specification can be separated temporally (Borensztejn, 2013).

    Although neither the identity, nor the nature, of the relay signal are known, it is possible to propose that the signal is not likely to be contact-dependent, and could be diffusible at only a short range. Indeed, Stat92E homozygous mutant TFC clones in contact with PCs, as well as those positioned up to three cell diameters away from PCs, are both associated with prolonged survival of supernumerary PCs, whereas clones further than three cell diameters away from PCs are not. In addition, fully efficient apoptosis of supernumerary PCs may require participation of all surrounding TFCs, stalk cells and possibly PCs, for production of a threshold level of relay signal. In support of this, large stat mutant TFC clones are more frequently associated with prolonged survival of supernumerary PCs, and the effects of removing JAK/STAT signal transduction in several cell populations at the same time are additive. Interestingly, the characterisation of two other Drosophila models of developmental apoptosis, interommatidial cells of the eye and glial cells at the midline of the embryonic central nervous system, also indicates that the level and relative position of signals (EGFR and Notch pathways) is determinant in selection of specific cells to be eliminated by apoptosis (Borensztejn, 2013).

    The results indicate that only the supernumerary PCs respond to the JAK/STAT-mediated pro-apoptotic relay signal, whereas two PCs per pole are always protected. Indeed, this study found that overexpression of Upd did not lead to apoptosis of the mature PC pairs and delayed rather than accelerated elimination of supernumerary PCs. Recently, it was reported that selection of the two surviving PCs requires high Notch activation in one of the two cells and an as-yet-unknown Notch-independent mechanism for the second cell. Intriguingly, expression of both Notch and Stat reporters is dynamic in PC clusters and PC survival and death fates are associated with respective activation of the Notch and JAK/STAT pathways. However, this study found that RNAi-mediated downregulation of upd did not affect either expression of Notch or that of two Notch activity reporters. Therefore, JAK/STAT does not promote supernumerary PC apoptosis by downregulating Notch activity in these cells. Identification of the relay signal and/or of Stat target genes should help further elucidate the mechanism underlying the induction of apoptosis in selected PCs (Borensztejn, 2013).

    Interfollicular stalk formation during early oogenesis has been shown to depend on activation of the JAK/STAT pathway. The presence of more than two PCs during these stages may be important to produce the appropriate level of Upd ligand to induce specification of the correct number of stalk cells. Later, at stages 7-8 of oogenesis, correct specification of anterior follicle cell fates (border, stretch and centripetal cells) depends on a decreasing gradient of Upd signal emanating from two PCs positioned centrally in this field of cells. Attaining the correct number of PCs per follicle pole has been shown to be relevant to this process and border cells (BC) specification seems to be particularly sensitive to the number of PCs present. Previously work has shown apoptosis of supernumerary PCs is physiological necessary for PC organiser function, as blocking caspase activity in PCs such that more than two PCs are present from stage 7 leads to defects in PC/BC migration and stretch cell morphogenesis. This study now shows that the excess PCs produced by blocking apoptosis lead to increased levels of secreted Upd and induce specification of excess BCs compared with the control, and these exhibit inefficient migration. These results indicate that reduction of PC number to two is necessary to limit the amount of Upd signal such that the correct numbers of BCs are specified for efficient migration to occur. Taken together with the role shown for Upd and JAK/STAT signalling in promoting PC apoptosis, it is possible to propose a model whereby Upd itself controls the size of the Upd-producing organising centre composed of PCs by inducing apoptosis of supernumerary PCs. Interestingly, in the polarising region in the vertebrate limb bud, which secretes the morphogen Sonic Hedgehog (Shh), Shh-induced apoptosis counteracts Fgf4-stimulated proliferation to maintain the size of the polarising region and thus stabilise levels of Shh. It is likely that signal autocontrol via apoptosis of signal-producing cells will prove to be a more widespread mechanism as knowledge of apoptosis control during development advances (Borensztejn, 2013).

    A steroid-controlled global switch in sensitivity to apoptosis during Drosophila development

    Precise control over activation of the apoptotic machinery is critical for development, tissue homeostasis and disease. In Drosophila, the decision to trigger apoptosis-whether in response to developmental cues or to DNA damage-converges on transcription of inhibitor of apoptosis protein (IAP) antagonists Reaper, Hid and Grim. This study describes a parallel process that regulates the sensitivity to, rather than the execution of, apoptosis. This process establishes developmental windows that are permissive or restrictive for triggering apoptosis, where the status of cells determines their capacity to die. One switch was characterized in the sensitivity to apoptotic triggers, from restrictive to permissive, that occurs during third-instar larval (L3) development. Early L3 animals are highly resistant to induction of apoptosis by expression of IAP-antagonists, DNA-damaging agents and even knockdown of the IAP diap1. This resistance to apoptosis, however, is lost in wandering L3 animals after acquiring a heightened sensitivity to apoptotic triggers. This switch in sensitivity to death activators is mediated by a change in mechanisms available for activating endogenous caspases, from an apoptosome-independent to an apoptosome-dependent pathway. This switch in apoptotic pathways is regulated in a cell-autonomous manner by the steroid hormone ecdysone, through changes in expression of critical pro-, but not anti-, apoptotic genes. This steroid-controlled switch defines a novel, physiologically-regulated, mechanism for controlling sensitivity to apoptosis and provides new insights into the control of apoptosis during development (Kang, 2013).

    CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid

    The mitochondrial outer membrane is a major site of apoptosis regulation across phyla. Human and C. elegans Bcl-2 family proteins and Drosophila Hid require the C-terminal tail-anchored (TA) sequence in order to insert into the mitochondrial membrane, but it remains unclear whether cytosolic proteins actively regulate the mitochondrial localization of these proteins. This study reports that the cdk7 complex regulates the mitochondrial localization of Hid and its ability to induce apoptosis. cdk7 was identified through an in vivo RNAi screen of genes required for cell death. Although CDK7 is best known for its role in transcription and cell-cycle progression, a hypomorphic cdk7 mutant suppresses apoptosis without impairing these other known functions. In this cdk7 mutant background, Hid fails to localize to the mitochondria and fails to bind to recombinant inhibitors of apoptosis (IAPs). These findings indicate that apoptosis is promoted by a newly identified function of CDK7, which couples the mitochondrial localization and IAP binding of Hid (Morishita, 2013).

    This study reports a mechanism of cell death regulation in Drosophila in which the mitochondrial localization of a proapoptotic TA protein is regulated by CDK7. Moreover, the mitochondrial localization of Hid is coupled with its ability to bind to DIAP1. These finding provides an explanation for the mitochondrial requirement of IAP antagonists (Morishita, 2013).

    Future studies are required to elucidate the structural nature of these Hid subspecies, and how they can be generated in a CDK7-dependent manner. Since only the faster-migrating form binds to DIAP1, the idea is favored that the two isoforms differ in their N terminus. In one speculative model, the faster-migrating form represents the proteolytically processed form that exposes the critical N-terminal alanine, which is responsible for DIAP1 binding. Alternatively, it is also possible that the slower-migrating form undergoes a modification that inhibits DIAP1 binding (Morishita, 2013).

    Recent studies indicated that dedicated trafficking machinery exists for other TA proteins destined for the endoplasmic reticulum. However, the equivalent trafficking factors for mitochondria-destined TA proteins have not yet been found, and it is widely assumed that these TA proteins insert into the mitochondrial outer membrane without active assistance. By contrast, the finding of this study indicates that Hid's mitochondrial localization can be regulated in cells, suggesting the existence of an active trafficking machinery for the mitochondrial TA protein (Morishita, 2013).

    The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

    Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin-proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. This study reports the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which was named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation (Yang, 2013).

    Low levels of p53 protein and chromatin silencing of p53 target genes repress apoptosis in Drosophila endocycling cells

    Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. This study shows that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. It was also found that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, the data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, these results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals (Zhang, 2014).

    This study used Drosophila as a model system to define the molecular mechanisms for tissue-specific apoptotic responses to genotoxic stress. The data suggest that Drosophila endocycling cells repress the apoptotic response in two ways: low level expression of the p53 transcription factor and epigenetic silencing of the p53 target genes at the H99 locus (see Model for tissue-specific apoptotic responses in Drosophila). In mitotic cycling B-D cells, the major p53 protein isoform is p53A, and no expression was detected of the other predicted p53 protein isoforms. In endocycling salivary glands (SG) and fat body (FB) cells, all of the p53 protein isoforms, including p53A, were below the level of detection. The data suggest that, similar to human p53, Drosophila p53A is ubiquitinated and degraded by the proteasome in endocycling cells. Over-riding this proteolysis by forced expression of p53A did not activate H99 gene transcription or apoptosis in endocycling cells. These results suggest that downstream chromatin silencing of the H99 locus represses apoptosis in endocycling cells even when p53A protein is abundant. In contrast, over-expression of the longer p53B isoform was found to induced H99 gene expression and apoptosis in endocycling cells. However, the normal physiological expression of p53B protein and binding to the H99 locus was undetectable in endocycling cells, suggesting that the low level of expression of this isoform also contributes to the repression of apoptosis. In the absence of genotoxic stress, a paused RNA Pol II was found at the H99 gene promoters in both mitotic cycling and endocycling cells. In endocycling cells, this paused RNA Pol II complex is activated only when the longer p53B isoform is highly over-expressed. This result implicates polymerase activation as one step that is blocked after DNA damage or p53A over-expression. In mitotic cycling cells, both paused RNA pol II and p53A protein are bound to H99 promoters in the absence of stress, which may prepare cells for a rapid apoptotic response to DNA damage. In addition, the data suggest that p53A protein levels are regulated in mitotic cycling cells, which likely ensures that apoptosis occurs only in response to stress. Together, these results have revealed new mechanisms by which different cells in development modulate their apoptotic response (Zhang, 2014).

    Previous evidence suggested that Drosophila p53 is regulated primarily by Chk2 phosphorylation and not protein stability. Consistent with this, it was found that in mitotic cycling cells p53A protein levels do not increase during the early response to radiation, a time when H99 genes are highly induced. At later times after irradiation, p53A protein levels increased only 2-3 fold, a magnitude that is proportional to the increase in p53 mRNA levels, as has been previously reported. Therefore, there is no evidence that the protein stability of p53A or other p53 isoforms changes in response to genotoxic stress. Both with and without genotoxic stress, the cellular levels of p53A protein were relatively low in mitotic cycling cells, and it was observed that the epitope tag on p53-Ch increased the abundance of p53A protein in p53 mutant but not p53 wild type cells. A cogent model is that the epitope-tag on p53-Ch partially interferes with p53A proteolysis in mitotic cycling cells, and that untagged p53 can promote the degradation of tagged p53-Ch in the same tetramer. Dampening of p53 protein levels may be critically important to prevent inappropriate apoptosis in the absence of stress. Consistent with this idea, it was found that elevated levels of p53A or p53B protein were sufficient to induce apoptosis in mitotic cycling cells even in Chk2 null animals. It is proposed that regulation of p53 protein levels in mitotic cycling cells tunes a threshold level of p53 protein that is poised to rapidly activate H99 gene expression when phosphorylated by activated Chk2 in response to DNA damage (Zhang, 2014).

    In endocycling cells, however, no p53 protein isoforms were detected using a variety of methods. This tissue-specific regulation of p53 protein abundance is post-transcriptional because mRNA levels were similar between mitotic cycling and endocycling cells. This low level of p53 protein suggests that either its translation is repressed and/or that it is more efficiently proteolyzed in endocycling cells. A model is favored wherein it is p53 proteolysis that is regulated in endocycling cells (see Model for tissue-specific apoptotic responses in Drosophila). In support of this model, compromising proteasome function elevated p53A protein levels in salivary glands. Moreover, p53A is ubiquitinated in endocycling cells, and these modified forms increase when proteasome function is compromised, which is consistent with previous data that p53 turnover is regulated by ubiquitination in Drosophila S2 cells (Chen, 2011). In contrast, the longer p53B isoform remained undetectable when the proteasome function was reduced. Given that proteasome function was only partially compromised, the inability to detect p53B may reflect a more efficient degradation of this longer isoform. This idea is consistent with the known correlation between transactivation domains and ubiquitin-mediated proteolysis for mammalian p53 and other proteins (Zhang, 2014).

    Although the results suggest that at least the p53A isoform is modified and targeted for degradation by a ubiquitin ligase, the identity of this ligase is unknown. The Drosophila genome does not have an obvious ortholog of the ubiquitin ligase MDM2, which targets p53 for degradation in mammalian cells. It remains possible that another family of ubiquitin ligases mediate p53 degradation in endocycling cells. Nonetheless, the results indicate that regulation of p53 is more similar between flies and humans than previously suspected, a finding that is interesting in the context of growing evidence for conserved p53 functions in flies and humans, including the response to hyperplasia (Zhang, 2014).

    The data suggest that apoptosis in endocycling cells is repressed in part through chromatin silencing of the pro-apoptotic genes at the H99 locus. The evidence for silent chromatin marks H3K9me3 and H3K27me3 at H99 are consistent with cytogenetic observations that the H99 chromosome region (75C) is a highly-condensed constriction on salivary gland polytene chromosomes, and genome-wide studies that showed that H3K27me3 is enriched at H99 relative to other loci in salivary glands. Although genetic data indicate that knockdown of the writers and readers of H3K9me3 and H3K27me3 results in salivary gland apoptosis, it remains possible that knockdown of these regulators causes other types of stress that triggers apoptosis. It is important to note, however, that the results in endocycling cells are also consistent with a previous analysis that indicated that chromatin silencing at H99 dampens the apoptotic response during late embryogenesis (Zhang, 2014).

    It was previously shown that the chromatin organization at the H99 locus impedes its DNA replication in endocycling cells. As a result, DNA at this locus is not duplicated every endocycle S phase, resulting in a final lower DNA copy number relative to euchromatic loci. This 'under-replication' is not the cause of apoptotic repression because it was found that in Suppressor of Underreplication (Su(UR)) mutants, in which the H99 locus is almost fully replicated, endocycling SG cells still did not apoptose in response to DNA damage (Zhang, 2014).

    The data suggest that the apoptotic response to genotoxic stress is repressed in endocycling cells because paused RNA Pol II is not activated at rpr and hid genes. One possibility is that chromatin silencing in endocycling cells restricts recruitment of transcription elongation factors to H99 promoters. This study found that over-expressed p53A and p53B were similar in binding and recruitment of acetylation to rpr and hid promoters, but only p53B activated transcription and apoptosis in endocycling cells. This difference between p53A and p53B isoform activity is attributable to an additional 110 AA amino- terminal transactivation domain in p53B that is somewhat conserved with human p53. The N-terminus of over-expressed p53B, therefore, may bypass silencing of the H99 genes in endocycling cells by activating this paused RNA polymerase to promote transcriptional elongation. The normal biological function of these paused RNA pol II complexes may be to coordinate a rapid response to developmental signals that trigger apoptosis and autophagy of endocycling larval tissues during metamorphosis (Zhang, 2014).

    It is proposed that low levels of p53 protein and downstream silencing of its target genes both prevent endocycling cell apoptosis. It has been proposed that the apoptotic response to genotoxic stress must be tightly repressed in polyploid endocycling cells because they have constitutive genotoxic stress caused by under-replication of heterochromatic DNA. Consistent with a possible linkage between the endocycle program and apoptotic repression, it was recently found that experimentally-induced endocycling cells (iECs) repress apoptosis independent of cell differentiation. It is clear that low levels of p53 protein is not the only mechanism of repression because over-expression of p53A resulted in abundant protein in endocycling cells, but failed to induce H99 transcription or apoptosis. Notably, over-expressed p53 had lower occupancy at H99 promoters in SG than B-D cells, another possible mechanism by which chromatin organization represses apoptosis downstream of p53. Moreover, the complete absence of endocycling cell apoptosis in response to IR suggests that both p53-dependent and p53-independent apoptotic pathways are repressed through silencing of the H99 locus, a point where these pathways intersect. These data, however, do not rule out the possibility that endocycling cells may use other mechanisms to repress the apoptotic response to DNA damage to ensure their survival despite the continuous genotoxic stress caused by under-replication (Zhang, 2014).

    In mitotic cycling cells, the p53 protein and paused RNA Pol II were bound to rpr and hid gene promoters in the absence of stress. This suggests that Chk2 phosphorylation of p53 pre-bound to these promoters activates the paused RNA Pol II to elicit a coordinated and rapid transcriptional response to genotoxic stress. This is consistent with previous evidence that p53-dependent activation of rpr and hid transcription is readily detectable within 15 minutes of ionizing radiation. This strategy to rapidly respond to stress appears to be conserved to humans where it has been shown that p53 activates paused RNA Pol II at some of its target genes, by indirect or direct physical interaction of p53 with elongation factors. Together, these results suggest that mitotic cycling cells in Drosophila are poised to respond to stress by tuning a threshold level of p53 protein that is bound to H99 promoters with a stalled RNA Pol II (Zhang, 2014).

    The data raise the question as to whether similar mechanisms repress apoptosis in mammalian polyploid cells. The transcriptome signatures of fly endocycles is very similar to that of polyploid cycles of mouse liver, megakaryocytes, and placental Trophoblast Giant Cells (TGCs), suggesting a conservation of cell cycle regulation. It is also known that mouse TGCs do not apoptose in response to UV. Moreover, evidence suggests that p53 protein levels decline when trophoblast stem cells switch into the endocycle and differentiate into TGCs, suggesting that the endocycle repression of apoptosis may be a theme conserved to mammals. The ubiquitin ligase that targets p53 for degradation in TGCs has not been identified, and it is possible that in both Drosophila and mouse the same family of ubiquitin ligases targets p53 for degradation in endocycling cells. In addition to developmentally-programmed endocycles, recent evidence suggests that cells can inappropriately switch from mitotic cycles into endocycles, and that this cell cycle switch contributes to genome instability and oncogenesis. Similar to developmental endocycles, apoptosis may be repressed in these endocycling cancer cells. In support of this idea, recent evidence showed that pro-apoptotic p53 target genes are epigenetically silenced in polyploid cancer cells. Therefore, the mechanisms that repress apoptosis in Drosophila endocycling cells may be conserved to humans and relevant to tissue-specific radiation therapy response and oncogenesis (Zhang, 2014).

    Structure of the apoptosome: mechanistic insights into activation of an initiator caspase from Drosophila

    Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog,Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. This study reports the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome (Pang, 2015).

    This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosom (Pang, 2015).

    This study presents the cryo-EM structures of the Dark apoptosome and the multimeric Dronc-Dark complex at overall resolutions of 4.0 and 4.1 Å, respectively. Notably, the EM density in the central region of the structures exhibits considerably higher resolutions, which allow assignment of specific side chains and atomic interactions. Because the overall domain organization of Dark is identical to that of Apaf-1, the structures reveal for the first time conserved atomic features of an apoptosome from a higher organism. The observed structural features of the Dark apoptosome, most of which are likely preserved in the Apaf-1 apoptosome, reveal the underpinnings of initiator caspase activation. Supporting this analysis, structure of the Dark protomer can be very well aligned with that of the activated Apaf-1 protomer from the Apaf-1 apoptosome (Pang, 2015).

    Dying cells protect survivors from radiation-induced cell death in Drosophila

    Induction of cell death by a variety of means in wing imaginal discs of Drosophila larvae resulted in the activation of an anti-apoptotic microRNA, bantam. Cells in the vicinity of dying cells also become harder to kill by ionizing radiation (IR)-induced apoptosis. Both ban activation and increased protection from IR required receptor tyrosine kinase Tie, which was identified in a genetic screen for modifiers of ban. tie mutants are hypersensitive to radiation, and radiation sensitivity of tie mutants was rescued by increased ban gene dosage. It is proposed that dying cells activate ban in surviving cells through Tie to make the latter cells harder to kill, thereby preserving tissues and ensuring organism survival. The protective effect reported in this study differs from classical radiation bystander effect in which neighbors of irradiated cells become more prone to death. The protective effect also differs from the previously described effect of dying cells that results in proliferation of nearby cells in Drosophila larval discs. If conserved in mammals, a phenomenon in which dying cells make the rest harder to kill by IR could have implications for treatments that involve the sequential use of cytotoxic agents and radiation therapy (Bilak, 2014).

    In metazoa where cells exist in the context of other cells, the behavior of one affects the others. The consequences of such interactions include not just cell fate choices but also life and death decisions. In wing imaginal discs of Drosophila melanogaster larvae, dying cells release mitogenic signals. Signaling from dying cells, or dying cells kept alive by the caspase inhibitor p35 (the so-called 'undead' cells), in wing discs operate through activation of Wingless (Drosophila Wnt) and JNK, and through repression of the tumor suppressor Salvador/Warts/Hippo pathway. A crosstalk between JNK and Hpo has also been reported. The consequences on the neighbors include increased number of cells in S phase and activation of targets of Yki, a transcription factor that is normally repressed by Hpo signaling. Mitogenic signals from dying cells results in increased proliferation of neighbors, which is proposed to compensate for cell loss and help regenerate the disc (Bilak, 2014).

    A target of Yki is bantam microRNA, but ban was not examined in above-described studies. ban was first uncovered in a genetic screen for promoters of tissue growth when overexpressed in Drosophila. Further study found a role for ban in both preventing apoptosis and promoting proliferation. A key target of ban in apoptosis is hid, a Drosophila ortholog of mammalian SMAC/Diablo proteins. These proteins antagonize DIAP1 to liberate active caspases and allow apoptosis. Hid is pro-apoptotic; repression of Hid by ban via binding sites in hid 3′UTR curbs apoptosis (Bilak, 2014).

    Since the initial characterization of ban, the role of this miRNA has expanded to include coordinating differentiation and proliferation in neural and glial lineages, cell fate decisions in germ line stem cells, in circadian rhythm, and in ecdyson hormone production. In these and other contexts, ban is regulated by a number of transcriptional factors and signaling pathways including, Hpo/Yki, Wg, Myc, Mad, Notch and Htx. The regulatory region of ban gene is likely to be complex and substantial; p-element insertions more than 10 kb away from ban sequences produce ban phenotypes (Bilak, 2014).

    The experimental evidence in Drosophila that dying cells promote proliferation presaged by several years the experimental evidence for a similar but mechanistically different phenomenon in mammals. A response called 'Phoenix Rising' occurs in mice after cell killing by ionizing radiation. Here, the activity of Caspase 3 and 7 is required in dying cells and mediates the release of prostaglandin E2, a stimulator of cell proliferation. These signals act non-autonomously to stimulate proliferation and tissue regeneration. A follow-up study in mice found a requirement for Caspase 3 in tumor regeneration after radiation treatment. Not all consequences on neighboring cells are protective or mitogenic. In the classical 'radiation bystander effect', seen in cell culture and in mice, the effect of irradiated cells on the neighbors is destructive, making the latter more prone to death. There is evidence for a soluble signal; media from irradiated cells can induce the bystander effect on naïve cells. Inhibitors of the bystander effect include antioxidants, suggesting that oxidative stress and energy metabolism may be involved in radiation bystander effect (Bilak, 2014).

    It has been shown previously that ban activity increased after exposure to ionizing radiation (IR) in wing imaginal discs of Drosophila larvae (Jaklevic, 2008). IR-induced increase in ban activity required caspase activity: expression of a viral caspase inhibitor, p35, or mutations in p53 that reduced and delayed the onset of caspase activation attenuated ban activation. It is noted that while IR-induced cell death is scattered throughout the disc, ban activation is homogeneous. This suggested a non-cell-autonomous component in activation of ban. The current study came out of efforts to understand how ban is activated in response to IR. Drosophila tie, which encodes a receptor tyrosine kinase of VGFR/PDGFR family, was identified as an important mediator of IR-induced changes in ban. Previous knowledge of Tie function in Drosophila was limited to long range signaling for border cell migration during oogenesis (Wang, 2006). This study reports that Tie was needed to activate ban in response to cell death. One consequence of ban activation was that remaining cells were harder to kill by IR (Bilak, 2014).

    This study has documented a previously unknown phenomenon in wing imaginal discs of Drosophila larvae; dying cells protected nearby cells from death. Killing cells by any one of three methods -- ptc-GAL4-driven expression of dE2F1RNAi or pro-apoptotic genes hid and rpr, exposure to ionizing radiation (IR) and clonal induction of Hid/Rpr -- activated an anti-apoptotic microRNA, bantam. Death by ptc-GAL4 or clonal expression of Hid/Rpr also made surviving cells more resistant to killing by IR. The protective effect was sensitive to ban gene dosage. This phenomenon was named 'Mahakali effect', after the Hindu goddess of death who protects her followers. Mahakali effect differs from classical radiation 'bystander effect' in which byproducts from cell corpses make surviving cells more prone to death. The Mahakali effect appears to operate in a non-cell-autonomous fashion. Disc-wide protection by ptc4>Rpr and Hid/Rpr that included even cells in the P compartment that did not express ptc, provides the strongest evidence for non-autonomy. This idea is supported by the finding that IR-induced caspase activation was reduced in cells outside Hid/Rpr flip-out clones (Bilak, 2014).

    A recent paper describes a non-autonomous induction of apoptosis by apoptotic cells. These results do not necessarily contradict what is reported in this study. Most of the experiments in the published work used undead cells kept alive by p35; Mahakali effect is seen without p35. Non-autonomous apoptosis was assayed at, typically, 3-4 days after induction of undead cells; this study detected Mahakali effect 6 hr after cell death induction using similar death-inducing stimuli (Hid/Rpr). It would be interesting to see how long Mahakali effect persists and whether non-autonomous apoptosis, occurring at longer time points, also produces Mahakali effects of its own. Another recent paper describes tissue regeneration after massive cell ablation in wing discs. It would also be interesting to see if the Mahakali effect operates among regenerating cells (Bilak, 2014).

    The data shown in this study suggest that the basic components of the Mahakali effect are caspase activity in dying cells (because expression in dying cells of p35, an inhibitor of effector caspases, blocked ban activation), ban (because ban activation resulted from cell death and the protective effect was sensitive to ban gene dosage), and tie (because tie was required to activate ban and the protective effect was sensitive to tie gene dosage). A model is proposed in which caspase activity in dying cells acts through Tie to cause non-autonomous activation of ban and the Mahakali effect. A validated target of ban in apoptosis inhibition is hid, whose 3'UTR includes 4 potential ban binding sites. Previous work has shown that a GFP sensor with hid 3'UTR is reduced after IR (Jaklevic, 2008), reflecting repression of hid by ban. Deletion of two potential ban-binding sites in the hid 3'UTR abolished the IR-induced changes in GFP (Bilak, 2014).

    The Mahakali effect differs in two ways from previously described effects of dead/dying cells in wing discs. First, the Mahakali effect extended further than previously reported signaling from dead/dying cells. In the extreme case of ptc4>Hid/Rpr, the protection reached as far as the edge of the disc. This distance, on of order of 100 or more mm is comparable to the distance of border cell migration, in which Tie is known to function. In contrast, the mitogenic effect that occurs through JNK/Wingless in response to undead cells in the wing disc is seen up to 5 cells away. Activation of proliferation through the Hpo/Yki axis also spans 3-5 cells away. This can be seen as activation of Yki targets such as DIAP1. This result could be reproduced: ptc4>dE2f1RNAi activated a Yki target, DIAP1, but only within or close to the ptc domain. YkiB5 allele, which disrupts cell death-induced proliferation, did not alter the Mahakali effect, further supporting the idea that the two effects are different. Second, ban activation in response to cell death was sensitive to the caspase inhibitor p35. In contrast, the mitogenic effect of dying cells in wing imaginal discs is not sensitive to p35. It is noted that the mitogenic effect of dying cells is inhibited by p35 in the differentiating posterior region of eye imaginal discs, which is similar to what was seen for ban activation in the wing discs (Bilak, 2014).

    This study found that tie was required for IR-induced activation of ban and for larval survival after irradiation. There were similarities as well as differences in the role of ban and tie. tie mutants were IR-sensitive, as are viable alleles of ban (Jaklevic, 2008). Tissue-specific overexpression of ban results in abnormal growth; this study found that 6 independent UAS-tie transgenic lines were lethal when driven by actin-GAL4. Thus, too much ban or tie has consequences. On the other hand, reducing tie or ban gene dosage by half attenuated the Mahakali effect. Thus, too little ban or tie also has consequences. In fact, UAS-ban or UAS-tie without a GAL4-driver was sufficient to rescue ban and tie mutant phenotypes. Thus, intermediate levels of expression may be important for the function of these genes (Bilak, 2014).

    The biggest difference between ban and tie, of course, was that while tie homozygous larvae were viable (this study), ban homozygous larvae are lethal. tie became necessary only after radiation exposure. This suggests that tie was needed to regulate ban not during normal development but after radiation exposure. How is IR and cell death linked to Tie? mRNA for Pvf1, a ligand for Tie in border cell migration, was found to be induced by IR and this induction appeared to be dependent on cell death (abolished in p53 mutants). Pvf1EP1624 mutants that are mRNA and protein null, also showed reduced Mahakali effect. The degree of reduction was significant but not back to the level seen in control discs without ptc4>dE2f1RNAi, suggesting the involvement of additional ligands or mechanisms for Tie activation. In agreement, no ban activation or the Mahakali effect was seen after overproduction of Pvf1. Pvf1 was necessary but insufficient to produce these effects without cell death (Bilak, 2014).

    Tie activated ban, at least in part by increasing ban levels. How IR and caspase activity promotes Pvf1 expression and how Tie activity increases ban levels will be key questions to address in the future. Testing the role of known apoptosis regulators, such as Diap1, and signaling molecules, such as Wg, may help address these questions. The genetic screen that identified Tie will be completed in future studies; it has the potential to identify additional components of the Mahakali effect (Bilak, 2014).

    Pvr, a PDGF/VEGF receptor homolog that function redundantly with Tie in border cell migration, also plays an anti-apoptotic role in embryonic hemocytes. A recent study in wing discs found that Pvr is activated in neighbors of dying cells in a JNK-dependent manner, to result in cytoskeletal changes that allow the engulfment of the dead cell by the neighbor. It is interesting that two PDGF/VEGF receptor homologs that function redundantly in cell migration during oogenesis may also play non-redundant roles in non-autonomous responses to cell death in wing discs (Bilak, 2014).

    Cancer therapy routinely comprises the application of two or more cytotoxic agents (taxol and radiation, for example) to cancer cells. A phenomenon in which cell killing by one agent influence resistance to the second agent is, therefore, of potential clinical significance. The bulk of the current analysis focused on protection from IR-induced cell death. But preliminary evidence indicates that the Mahakali effect can also protect against cell death induced by maytansinol, a microtubule depolymerizing agent with relevance to cancer therapy that we found before to induce cell death in Drosophila wing discs. An important question is whether a phenomenon like Mahakali effect exists in mammals and acts as a survival mechanism in response to cell death. Ang-1, a ligand for mammalian Tie-2, is a pro-survival factor for endothelial cells during serum deprivation and after irradiation in cell culture models. Interestingly, Ang1 is produced not by endothelial cells but by neighbors, at least in cell culture. Based on these data, it is possible that radiation exposure results in Ang1 production by dead/dying cells that promote the survival of endothelial cells via Tie-2. Consistent, an Ang-1 derivative that is a potent activator of Tie-2 has been shown to protect endothelial cells from radiation-induced apoptosis (Bilak, 2014 and references therein).

    Tie-mediated signal from apoptotic cells protects stem cells in Drosophila melanogaster

    Many types of normal and cancer stem cells are resistant to killing by genotoxins, but the mechanism for this resistance is poorly understood. This study shows that adult stem cells in Drosophila melanogaster germline and midgut are resistant to ionizing radiation (IR) or chemically induced apoptosis; the mechanism for this protection was dissected. Upon IR the receptor tyrosine kinase Tie/Tie-2 is activated, leading to the upregulation of microRNA bantam that represses FOXO-mediated transcription of pro-apoptotic Smac/DIABLO orthologue, Hid in germline stem cells. Knockdown of the IR-induced putative Tie ligand, PDGF- and VEGF-related factor 1 (Pvf1), a functional homologue of human Angiopoietin, in differentiating daughter cells renders germline stem cells sensitive to IR, suggesting that the dying daughters send a survival signal to protect their stem cells for future repopulation of the tissue. If conserved in cancer stem cells, this mechanism may provide therapeutic options for the eradication of cancer (Xing, 2015).

    A form of programmed cell death, apoptosis, is characterized as controlled, caspase-induced degradation of cellular compartments to terminate the activity of the cell. Apoptosis plays a vital role in various processes including normal cell turnover, proper development and function of the immune system and embryonic development. Apoptosis is also induced by upstream signals, such as DNA double-strand breaks (DSB), to destruct severely damaged cells. DSB activate ATM checkpoint kinase and Chk2 kinase-dependent p53 phosphorylation and induction of repair genes. However, if DSB are irreparable, p53 activation will result in pro-apoptotic gene expression and cell death. However, aggressive cancers contain cells that show inability to undergo apoptosis in response to stimuli that trigger apoptosis in sensitive cells. This feature is responsible for the resistance to anticancer therapies, as well as the relapse of tumours after treatment, yet the molecular mechanism of this resistance is poorly understood (Xing, 2015).

    As the cell type that constantly regenerates and gives rise to differentiated cell types in a tissue, stem cells share high similarities with cancer stem cells, including unlimited regenerative capacity and resistance to genotoxic agents. Adult stem cells in model organisms such as Drosophila melanogaster, have been utilized to study stem cell biology and for conducting drug screens, thanks to their intrinsic niche, which provides authentic in vivo microenvironment. This study shows that Drosophila adult stem cells are resistant to radiation/chemical-induced apoptosis, and the mechanism for this protection was dissected. A previously reported cell survival gene with a human homologue, pineapple eye (pie) , acts in both stem cells and in differentiating cells to repress the transcription factor FOXO. Elevated FOXO levels in pie mutants lead to apoptosis in differentiating cells, but not in stem cells, indicating the presence of an additional anti-apoptotic mechanism(s) in the latter. We show that this mechanism requires Tie, encoding a homologue of human receptor tyrosine kinase Tie-2, and its target, bantam, encoding a microRNA. The downstream effector of FOXO, Tie and ban, is show to be Hid, encoding a Smac/DIABLO orthologue. Knocking down the ligand Pvf1/PDGF/VEGF/Ang in differentiating daughter cells made stem cells more sensitive to radiation-induced apoptosis, suggesting that Pvf1 from the apoptotic differentiating daughter cells protects stem cells (Xing, 2015).

    This study shows that an anti-apoptotic gene, pie, is required for stem cell self-renewal but not for resistance to apoptosis, indicating a compensatory anti-apoptotic mechanism in stem cells. The cell cycle marker profile of pie GSCs resembles that of InR deficient GSCs, leading to the finding that pie controls GSC, as well as ISC self-renewal/division through FOXO protein levels. Surprisingly, pie targets FOXO as well in differentiating cells, failing to explain why the loss of pie does not induce apoptosis in stem cells. However, while the upregulation of FOXO leads to the upregulation of its apoptotic target Hid in differentiating cells, in adult stem cells Hid is not upregulated. Hence additional regulatory pathway is in place to repress Hid and thereby apoptosis in stem cells. This study identified Tie-receptor as the key gatekeeper for the process in the GSCs. The signal (Pvf1) from the dying daughter cells activates Tie in GSCs to upregulate bantam microRNA that represses Hid, thereby protecting the stem cells. Bantam is known to repress apoptosis and activate the cell cycle. However, while protected from apoptosis in this manner, the stem cells do not activate the cell cycle but rather stay in protective quiescence through FOXO activity. When the challenge is passed, stem cells repopulate the tissue (Xing, 2015).

    The mammalian pie homologue, G2E3 was reported to be an ubiquitin ligase with amino terminal catalytic PHD/RING domains. G2E3 is essential for early embryonic development (Brooks, 2008). Importantly, microarray data show significant enrichment of G2E3 expression levels in human embryonic stem (ES) cell lines. These observations suggest a critical role of G2E3 in embryonic development, potentially in maintaining the pluripotent capacity. Since FOXO is shown to be an important ESC regulator, it will be interesting to test whether defects in G2E3 result in changes in FOXO levels. Furthermore, future studies are required to test whether human ES cells also are protected from apoptosis due to external signals from dying neighbouring cells (Xing, 2015).

    The cell cycle defects of pie mutant stem cells, such as abnormal cell cycle marker profile, can be a consequence of elevated FOXO levels, since FOXO is a transcription factor with wide array of target genes, many of which are involved with cell cycle progress, such as the cyclin-dependent kinase inhibitor p21/p27 (Dacapo in Drosophila). This may be critical when bantam function is considered in the stem cells. Bantam is known to function as anti-apoptotic and cell cycle inducing microRNA. While in GSC bantam is critical through its anti-apoptotic function as a Hid repressor, it has no capacity to induce GSC cell cycle after irradiation. In a challenging situation, such as irradiation, an additional protection mechanism for the tissue is to keep the stem cell in a quiescent state during challenge. bantam's pro-cell division activity may be dampened by FOXO's capacity to upregulate p21/Dacapo (Xing, 2015).

    The FOXO family is involved in diverse cellular processes such as tumor suppression, stress response and metabolism. The FOXO group of human Forkhead proteins contains four members: FOXO1, FOXO3a, FOXO4, and FOXO6. Studies to elucidate their function in various stem cell types in vivo using knockout mice have shown some potential redundancy of FOXO proteins. Recent publications have demonstrated a requirement for some of the FOXO family members in mouse hematopoietic stem cell proliferation, mouse neural stem cells, leukaemia stem cells and human and mouse ES cells in vitro. However, FOXO is shown to be dispensable in the early embryonic development in mouse. Drosophila genome has only one FOXO, allowing a definitive study of FOXO's function in stem cells. This study now demonstrates that tight regulation of FOXO protein levels is essential for in vivo GSC and ISC self-renewal in Drosophila. While the loss of FOXO function generates supernumerary stem cells, inappropriately high level of FOXO results in stem cell loss. Under challenge, such as exposure to irradiation, stem cells depleted of FOXO fail to stay quiescent and become more sensitive to the damage, leading to the loss of GSC population. These data demonstrate the importance of the balanced FOXO expression level for stem cell fate (Xing, 2015).

    Previous studies have shown that multiple adult stem cell types manage to avoid cell death in response to severe DNA damage. This work has studied the mechanisms that stem cells utilize to avoid apoptosis in absence of pie and revealed that apoptosis is protected through a receptor, Tie and its target miRNA bantam that can repress the pro-apoptotic gene Hid. The ligand for Tie is likely secreted from the dying neighbours since Tie is essential in GSC only after irradiation challenge, IR induces Tie's potential ligand Pvf1 expression in cystoblasts and knockdown of Pvf1 in cystoblasts eliminates stem cells' protection against apoptosis. Further studies will reveal whether the same protective pathway is utilized in other stem cells. Community phenomenon have been described previously around dying cells: compensatory proliferation, Phoenix rising, bystander effect and Mahakali. While Bystander effect describes dying cells inducing death in the neighbours, compensatory proliferation, Phoenix rising and Mahakali describe positive effects in cells neighbouring the dying cells. The present work shows that adult stem cell can survive but show no immediate induction of proliferation when neighboured by dying cells. However, since adult stem cells can repopulate the tissue when death signals have passed, it is proposed that in adult stem cells these phenomenon merge. First, the GSCs survive by bantam repressing the apoptotic inducer, Hid, and later repopulate the tissue by activating cell cycle. Recent findings have suggested that p53 might play an important role in re-entry to cell cycle in stem cells51. The results from the current studies shed light on the general understanding of stem cell behaviour in response to surrounding tissue to ensure the normal tissue homeostasis. It is also plausible that cancer stem cells hijack these normal capacities of stem cells (Xing, 2015).

    Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila

    Members of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. The mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. This study reports the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. Seventeen putative modifiers involved in various function or process were isolated: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. The brat gene belongs to a group of suppressors, which is implicated in cell growth, proliferation or death. Other identified genes are involved in carbohydrate metabolism, such as Gpo-1. This result is in agreement with the evidence that Bcl-2 family proteins, in addition to their well characterized function in cell death, also play roles in metabolic processes in particular at the level of energetic metabolism. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation (Colin, 2015).

    Major executioners of programmed cell death by apoptosis are relatively well conserved throughout evolution. However, the control of commitment to apoptosis exhibits some differences between organisms. During mammalian cells apoptosis, various key pro-apoptotic factors are released from the inter-membrane space of mitochondria. These factors include cytochrome c, Apoptosis Inducing Factor (AIF), Endonuclease G, Smac/DIABLO (Second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease Omi/HtrA2. Once released in the cytosol, cytochrome c binds to the WD40 domain of Apaf-1 and leads to the formation of a cytochrome c/Apaf-1/caspase-9 complex called 'apoptosome', in which caspase-9 (a cysteinyl aspartase) auto-activates to initiate a caspase activation cascade that will lead to cell death. Mitochondrial permeabilization is under the control of the Bcl-2 family of proteins. These proteins share one to four homology domains with Bcl-2 (named BH1-4) and exhibit very similar tertiary structures. However, while some of these proteins (such as Bcl-2) are anti-apoptotic, the others are pro-apoptotic and assigned to one of the following sub-classes: BH3-only proteins (such as Bid) and multi-domain proteins (such as Bax). During apoptosis, Bax translocates to the mitochondrial outer membrane, undergoes conformational changes, oligomerizes and finally allows the release of pro-apoptotic factors from the intermembrane space. Anti-apoptotic proteins of the Bcl-2 family oppose this Bax-mediated mitochondrial release of apoptogenic factors while BH3-only proteins can activate Bax or inhibit anti-apoptotic proteins of the family (Colin, 2015 and references therein).

    In C. elegans, activation of the caspase CED-3 requires CED-4, the homologue of Apaf-1 but no cytochrome c. The Bcl-2 family protein CED-9 constitutively interacts with CED-4 and thereby prevents the activation CED-3. This repression of cell death is released upon binding of CED-9 to the BH3-only protein EGL-1, which induces a conformational change in CED-9 that results in the dissociation of the CED-4 dimer from CED-9. Released CED-4 dimers form tetramers, which facilitate auto-activation of CED-3. Although CED-9 appears bound to mitochondria, these organelles seem to play a minor role in apoptosis in C. elegans, contrarily to mammals (Colin, 2015 and references therein).

    The role of mitochondria in Drosophila programmed cell death remains more elusive. Cytochrome c does not seem crucial in the apoptosome activation, which is mediated by the degradation of the caspase inhibitor DIAP1 by proteins of the Reaper/Hid/Grim (RHG) family. The apoptotic cascade appears somehow inverted between flies and worm/mammals. In these two last organisms, apoptosis regulators are relocated from mitochondria to the cytosol. Contrarily, Drosophila apoptosis regulators are concentrated at or around mitochondria during apoptosis. Indeed, targeting the RHG proteins Reaper (Rpr) and Grim to mitochondria seems to be required for their pro-apoptotic activity. Furthermore, Hid possesses a mitochondrial targeting sequence and is required for Rpr recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo (Colin, 2015).

    The important role played in Drosophila by the mitochondria in apoptosis is also suggested by the mitochondrial subcellular localization of Buffy and Debcl, the only two members of the Bcl-2 family identified, so far, in this organism. Buffy was originally described as an anti-apoptotic Bcl-2 family member, but it can also promote cell death. Debcl (death executioner Bcl 2 homolog), is a multidomain death inducer that can be inhibited by direct physical interaction with Buffy. When overexpressed in mammalian cells, debcl induces both cytochrome c release from mitochondria and apoptosis. This protein interacts physically with anti-apoptotic members of the Bcl-2 family, such as Bcl-2 itself, in mammals. In Drosophila, Debcl is involved in the control of some developmental cell death processes as well as in irradiation-induced apoptosis (Colin, 2015).

    Previous studies have shown in Drosophila that mammalian Bcl-2 inhibits developmental and irradiation-induced cell death as well as rpr- and bax-induced mitochondrial membrane potential collapse . Interestingly, bax-induced cell death has been shown to be mitigated by loss-of-function (LOF) mutations in genes encoding some components of the TOM complex which controls protein insertion in the outer mitochondrial membrane. These results suggest that Bax mitochondrial location remains important for its activity in Drosophila. Therefore, flies provide a good animal model system to study Bax-induced cell death in a simple genetic background and look for new regulators of Bcl-2 family members (Colin, 2015).

    This study reports the results of the screening of P[UAS]-element insertion (UYi) library, performed in order to identify modifiers of bax-induced phenotypes in Drosophila. Among 1475 UYi lines screened, 17 putative modifiers were isolated, that include genes involved in various cellular functions. This paper presents a more detailed study of one of these modifiers, UY1039, and shows that glycerophosphate oxidase-1 (Gpo-1) [EC] participates in debcl-induced apoptosis by increasing reactive oxygen species (ROS) production (Colin, 2015).

    This screen provided 17 suppressors of phenotypes induced by the expression of bax under control of the wing specific vg-GAL4 driver (lethality and wing notches). The possibility that these suppressors affect GAL4 synthesis or that the selected insertions titrate the GAL4 transcription factor is unlikely, since the number of suppressors is limited (1.6% of the collection). Moreover, UYi insertions were isolated that were not identified in other screens performed using the same collection and the UAS/Gal4 system. Finally, the specificity of one of the suppressors, UY3010, which corresponds to a gain-of-function of the Ubiquitin activating enzyme-encoding gene Uba1 has been reported. Indeed, Uba1 overexpression allows the degradation of Bax and Debcl, thanks to the activation of the ubiquitin/proteasome pathway. This study also showed that Debcl is targeted to the proteasome by the E3 ubiquitin ligase Slimb, the β-TrCP homologue (Colin, 2015).

    Nine of the bax-modifiers also behaved as suppressors of debcl-induced wing phenotype while 4 showed no significant effect on this phenotype. Three hypotheses could explain this discrepancy. One possibility is that these bax modifiers are context artifacts and do not represent bona fide Bax interactors. The second possible explanation involves the difference in the driver used in each assay (vg-GAL versus ptc-GAL). Indeed, UY3010 did not significantly suppress debcl-induced apoptosis while another Uba1 overexpression mutant (Uba1EP2375) did. Third, although Bax and Debcl, share similarities in their mode of action and regulation, some signaling pathways could be specific of bax-induced apoptosis. Indeed, a LOF of brat mitigates neither debcl -- (this paper) nor hid -- or Sca3-induced cell death(Colin, 2015).

    The brat gene belongs to a group of suppressors, which is implicated in cell growth, proliferation or death. Mutations in this type of genes could compensate cell loss due to ectopic apoptosis induction. Results observed for this group of modifiers can generally be easily interpreted with the literature data. UY1131 corresponds to an insertion in the brat (for brain tumor) gene that could allow the expression of a truncated form of the protein. To check whether this insertion leads to a LOF or a GOF of brat, the effect of the characterized LOF allele bratk0602 on bax-induced phenotypes was tested. This mutation strongly suppressed the wing phenotype showing that UY1131 is a LOF of brat. Brat belongs to the NHL family of proteins, represses translation of specific mRNAs and is a negative regulator of cell growth. The suppression of bax-induced phenotypes by a LOF of brat could suggest that this gene also regulates cell death, which seems unlikely according to its inability to suppress other cell death pathways. Alternatively brat could regulate somehow compensatory proliferation in this system (Colin, 2015).

    Some candidate suppressors encode proteins involved in secretion or components of the extra-cellular matrix. The effect of these genes could rely on cell signaling. Change in levels of secreted proteins could modify cell-extracellular matrix interactions and thus affect viability via processes similar to anoikis (Colin, 2015).

    Several suppressors are implicated in pathfinding (comm, comm3, hat, scratch and lola). Two hypotheses can be formulated. Either neurons are of particular importance in bax-induced phenotypes or a more general role of these proteins in signaling is responsible for these suppressions. If the neuronal death could explain the decreased survival of bax expressing flies, it could hardly explain the wing phenotypes. Therefore, these suppressor genes may have a more general role in signaling and in particular in cell death regulation. For example, UY2669 corresponds to a GOF mutant of scratch (scrt). This gene is a Drosophila homologue of C. elegans ces-1, which encodes a snail family zinc finger protein involved in controlling programmed death of specific neurons. Interestingly, a mammalian homologue of scratch, named Slug, is involved in a survival pathway that protects hematopoietic progenitors from apoptosis after DNA damage. Slug also antagonizes p53-mediated apoptosis by repressing the bcl-2-family pro-apoptotic gene puma. More recently, a regulatory loop linking p53/Puma with Scratch has been described in the vertebrate nervous system, not only controlling cell death in response to damage but also during normal embryonic development (Colin, 2015).

    Another possibility is that these modifiers could affect some extracellular survival and/or death factors. For example, sugarless, which was found twice in the screen, has been shown to interact with several survival pathways such as Wingless, EGF and FGF pathways that can play a role in defining shape and size of tissues and organs. This result can be paralleled with the suppressive effect of mutations in hephaestus and lola, both of which interact with the Notch/Delta signaling. Notably, lola, a gene encoding a Polycomb group epigenetic silencer, has been shown to be required for programmed cell death in the Drosophila ovary. Lola has also been identified for its role in normal phagocytosis of bacteria in Drosophila S2 cells and as a component of the Drosophila Imd pathway that is key to immunity. In contrast, Lola is required for axon growth and guidance in the Drosophila embryo. This indicates that lola could play a role in cell adhesion and motility. Accordingly, when coupled with overexpression of Delta, misregulation of pipsqueak and lola induces the formation of metastatic tumors associated with a downregulation of the Rbf (Retinoblastoma-family) gene (Colin, 2015).

    Bcl-2 family proteins, in addition to their well characterized function in cell death, also play roles in metabolic processes in particular at the level of energetic metabolism. In particular, Bcl-2 regulates mitochondrial respiration and the level of different ROS through a control of cytochrome c oxidase activity. Study of heterologous bax expression in yeast has provided clues on Bax function in relation to ROS and yeast LOF mutants of genes involved in oxidative phosphorylation show increased sensitivity to Bax cytotoxicity. In agreement, Bcl-xL complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism. Furthermore, both the anti-apoptotic effect of LOF mutations in Gpo-1 and the GOF in transketolase genes can be related to a protective effect against oxidative stress. This result suggests that the cell death process induced by Bax involves, at least in part, the modulation of different ROS levels (Colin, 2015).

    Indeed, this study reports that the suppressor effect of a null allele of Gpo-1 is associated with a decreased ability of Debcl to induce ROS production. This result is in agreement with the observation that 70% of the total cellular H2O2 production was estimated to stem from Gpo-1 in isolated Drosophila mitochondria. This enzyme has also been implicated in ROS production in mammalian brown adipose tissue mitochondria when glycerol-3-phosphate was used as the respiratory substrate and, more recently, in prostate cancer cells. In this latter case, ROS production seems to be beneficial to cancer cells, whereas this study show that it favors cell death in Drosophila wing disc cells. This apparent contradiction could be related to the abnormal ROS production occurring during the oncogenic transformation and the shift to a glycolytic metabolism (Colin, 2015).

    In conclusion, this study shows that Gpo-1 contributes to debcl-induced apoptosis by increasing reactive oxygen species (ROS) production and provides a substantial resource that will aid efforts to understand the regulation of pro-apoptotic members of the Bcl-2 family proteins (Colin, 2015).

    Tumor suppressor gene OSCP1/NOR1 regulates apoptosis, proliferation, differentiation, and ROS generation during eye development of Drosophila melanogaster

    OSCP1/NOR1 (Organic solute carrier partner 1/Oxidored-nitro domain-containing protein 1) is a known tumor suppressor protein. OSCP1 has been reported to mediate transport of various organic solutes into cells, however its role during development has not yet been addressed. This study reports the results of studies with dOSCP1 (the Drosophila orthologue of hOSCP1) knockdown flies to elucidate the role of OSCP1/NOR1 during development. Knockdown of dOSCP1 in the eye imaginal discs induces a rough eye phenotype in adult flies. This phenotype results from an induction of caspase-dependent apoptosis followed by a compensatory proliferation and ROS generation in eye imaginal discs. The induction of apoptosis appears to be associated with down-regulation of the anti-apoptotic Buffy gene and up-regulation of the pro-apoptotic Debcl gene. These effects of knockdown of dOSCP1 lead to mitochondrial fragmentation, degradation, and a shortfall in ATP production. It was also found that knockdown of dOSCP1 causes a defect in the cone cell and pigment cell differentiation of pupal retinae. Moreover, mutations in EGFR pathway-related genes, such as Spitz and Drk enhance the rough eye phenotype induced by dOSCP1-knockdown. These results suggest that dOSCP1 positively regulates EGFR signaling pathway. Overall these findings indicate that dOSCP1 plays multiple roles during eye development of Drosophila (Huu, 2015)

    Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye

    Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. This study examined how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. This study investigated here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. Neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response (Kumar, 2015).

    This paper used the Drosophila eye as a model system with which to study how morphogen gradients can be converted into sharply constrained tissue patterns. The action of the Wg morphogen gradient was examined and it was asked how the highest threshold response, the death of the peripheral ommatidia, is orchestrated. Three observations argue that the secondary Wg expressed by the cone cells combines with the primary Wg from the head capsule to generate a sufficient concentration to kill the ommatidia. First, when the Wg pathway is activated in all cone cells (pros->arm* were arm* is an N-terminally truncated form of Armadillo, a constitutive, cell autonomous activator of the Wg transduction pathway) there is an extended zone of apoptosis in the region where the primary Wg source is known to be high. Second, when the secondary Wg (that secreted by the cone cells) is removed the extended band of ommatidial death is lost. Third, when a level of Wg equivalent to that normally found in the peripheral regions is supplied to pros-arm* eyes all ommatidia now die. Thus, this represents a novel gradient read-out mechanism in which the primary morphogen (Wg derived from the head capsule) elicits a secondary morphogen expression (Wg expressed by the cone cells) in the target cells. Thereafter, the two sources unite to generate the high local morphogen concentration needed to direct the appropriate cell behaviors at that position (Kumar, 2015).

    If there is a permissive zone in the periphery (∼3 ommatidial rows) in which the ommatidia will die if cone cell Wg expression occurs, then this raises the question of how the cone cells responses are normally tightly restricted to the peripheral-most row of ommatidia to ensure that only these ommatidia die. The following describes 1he mechanisms likely responsible for this restriction (Kumar, 2015).

    (1) The high threshold of the ommatidial response: It is surmised that the cone cells have a high threshold response to the morphogen, and the initial responses to the primary Wg source (diffusing from the head capsule) is restricted to the outermost ommatidia. However, it can be envisioned that the secondary Wg secreted by the outer cone cells could diffuse and elicit the same output in the next ommatidial row, and an extreme view could see a relay mechanism in which even more internal rows of ommatidia could express Wg in their cone cells (Kumar, 2015).

    (2) The role played by Notum:
    The expression of Notum is similar to Snail family transcription factors in that it is expressed in the cone cells and 2°/3° PCs of the outermost ommatidia, and since Notum functions to inhibit the free diffusion of Wg, it likely acts to prevent Wg diffusion into more interior ommatidia. Indeed previous studies have shown that in notum mutant clones the zone of death expanded out into more interior rows. Thus Notum (and other mechanisms for preventing Wg diffusion) is seen as playing a critical role in restricting the ommatidial death to the outermost row of ommatidia (Kumar, 2015).

    (3) Combining the high threshold response with the restriction of Wg diffusion: Consider the primary Wg diffusing from the head capsule. It enters the outer row of ommatidia and is of sufficient concentration to elicit the appropriate responses (the various expressions in the cone cells and 2°/3° PCs) but not at a level high enough to kill the ommatidia. The cone cells of the outermost row now begin to secrete the secondary Wg, but the concomitant expression of Notum by the cone cells and 2°/3° PCs of these ommatidia provide a barrier to the movement of both the primary and secondary sources of Wg. This restriction of Wg movement not only protects the more internal ommatidia, but ensures that the high levels of morphogen are constrained in the outermost ommatidia to provide the requisite signal for apoptosis (Kumar, 2015).

    In addition to uncovering the synergy between the Wg derived from the head capsule and the cone cells, a number of phenomena relating to the behavior of the various cell types have been detected (Kumar, 2015).

    (1) The early cone cell death: Following the collapse of the cone cells, the ommatidial apoptosis program begins with the death of cone cells themselves, followed ∼two hours later by the other ommatidial cells. This precocious cone cell death may represent a lower apoptosis threshold for these cells, but it is noted that they are sources of Wg secretion and likely experience autocrine and paracrine (between cone cells of the same ommatidium) Wg signaling as they collapse, and as such are more likely to reach the critical Wg activation level before the other cells (Kumar, 2015).

    (2) The cone cell immunity to death: In pros-arm* eyes, in which all cone cell nuclei fall to the photoreceptor layer and express Wg, there is a wide swath of extended death at the periphery in which all cells of the ommatidia die (including the cone cells). But upon prevention of the cone cell nuclear fall by the expression of esg RNAi, the cone cells survive while the photoreceptors in the extended peripheral zone still die. In these ommatidia, levels of Wg needed to drive apoptosis are achieved, but the cone cells appear invulnerable to it. Whether this invulnerability results from the absence of Snail family transcription factors needed to prime the cone cells for the death signal, or whether by remaining in the apical location they somehow avoid the full level of Wg exposure remains unclear (Kumar, 2015).

    (3) The fall of the cone cell nuclei: The maintenance of cone cell cell-bodies in the appropriate apical location is seemingly critical for the ommatidial stability and integrity, as their fall leads to the disruption of corneal lens units and delamination of photoreceptors. This fall appears to be directed by their expression of Snail family transcription factors. In pros-arm* eyes, the expression of esg.RNAi prevents the fall, and correspondingly the ectopic expression of esg in otherwise wild type cone cells engenders their nuclear fall (albeit prematurely). It was asked whether the fall of the cone cell nuclei resulted from a wholesale collapse of the apical junctions of the cone cells, but D/E-cadherin staining showed a normal apical junction pattern many hours after the nuclei had migrated basally. Thus it does not appear that the cone cells nuclei move basally because the cells lose their apical attachments, rather it is inferred that expression of the Snail family transcription factors reprograms some other behaviors of the cone cells. Such a behavior could be a switch in cell-type affinity. If cone cells normally maintain an apical location by adhesive differences with the photoreceptors, and if these adhesive differences are switched, then cone cell plasma membranes will then preferentially move to the photoreceptor layer. Since the nucleus defines the site of maximum cell body profile with corresponding maximum membrane area, then the fall of the nuclei may simply result from the cone cells acquiring an adhesive affinity with the photoreceptors. Other mechanisms can also be envisaged, in which, for example, motor machinery of the cell is used to reposition the cone cell nuclei in the more basal location (Kumar, 2015).

    An appropriate Gal4 driver line is not available to activate gene expression selectively in the 1° PCs, and the mechanism of their death remains unresolved. In GMR.wg eyes, their death was observed coincident with the photoreceptors (following the apoptosis of the cone cells) and it is surmised that it is the high level of Wg derived from head capsule and the cone cells that directs their death. However, there are a number of indications from that offer clues to a more nuanced understanding of their behavior. Initially the nuclei of the 1° PCs flank the clustered photoreceptor nuclei in their more apical region, but when the cone cell nuclei fall, those of the 1° PCs are shunted more basally. This movement deeper into the photoreceptor layer may play a role in their death. A similar argument can be made from the analysis of * eyes in which 1° PCs are lost, but when Snail family transcription factors are removed from this background, the cone cell nuclei do not fall, and the 1° PCs do not die. Hence the 1° PCs behave in a similar manner as the cone cells; if their position is maintained they do not die even though ambient Wg concentrations are sufficient for their death. This may indicate a general principle; that cells need to be in the correct topological position to experience the death signal (Kumar, 2015).

    Furthermore, in * eyes, the cone cell nuclear fall is accompanied by the loss of the 1° PCs even though the cone cells themselves do not die. The removal of Wg expression from the pros-arm* cone cells rescues the 1° PCs indicating that their loss is normally triggered by the cone cell Wg expression, and it is suspected that the low-level apoptosis seen in the main body of pros-arm* eyes may represent the death of the 1° PCs. If this is the case, then this suggests that the 1° PCs have a lower threshold Wg response for their apoptosis than the cone cells and photoreceptors (Kumar, 2015).

    The death of the photoreceptors appears to simply require the additive of effects of the two Wg sources to trigger their death. But another feature has emerged from these studies – the idea that chronic exposure to sub-lethal levels of Wg triggers photoreceptor degeneration. Consider pros-arm*/esgRNAi eyes; here the photoreceptor death occurs only at the widened zone of peripheral apoptosis, but in the main body of the adult eyes ommatidia show degenerate rhabdomere-like tissue in the apical retinas. The presence of rhabdomere-like tissue suggests the differentiation and subsequent degeneration of the photoreceptors leaving them alive but in a runtish condition. Since this phenomenon is Wg dependent (it is absent when wgRNAi is additionally included) it is inferred that the persistent Wg expression from the cone cells chronically signals to the photoreceptors. Indeed, when GMR.wg/GMR.P35 eyes (in which the apoptosis mechanism is suppressed and the photoreceptors are therefore subject to chronic Wg exposure), were examined a similar degenerate phenotype occurred. This observation suggests another function for the removal of the outer-most row of ommatidia: if they were not removed, chronic exposure to high levels of Wg emanating from the head capsule would lead them to deteriorate into a runtish condition (Kumar, 2015).

    A striking feature of the peripheral patterning mechanism is the timing aspect. The peripheral ommatidia are exposed to head capsule-derived Wg from the time of their birth. And yet they only respond to this Wg signal at defined times. The first occurs shortly after pupation when ac/da transcription is repressed and hth expression is induced. This corresponds with the surge in ecdysone expression that occurs in the animals at this time. The second response is the death of ommatidia at 42 h APF and this mechanism is closely tied with the large peak of ecdysone expression that occurs in the second day of pupation. Thus, it is speculated that Wg provides the spatial signal for peripheral patterning, but that the hormone system of the fly provides the temporal cue that determines when the spatial information can be utilized (Kumar, 2015).

    It is concluded that the periphery of the fly eye is an excellent model system with which to study how morphogen gradients are decoded into discrete tissue types, and this study has delved into the mechanism that precisely restricts the spatial positioning of one of those tissue types. An intricate mechanism has been uncovered in which initial threshold responses lead to the local boosting of the morphogen signal while at the same time upregulating mechanisms to prevent the spread of the morphogen. Evidence is also provided to support the idea that appropriate spatial, temporal and topological context is required for the peripheral ommatidia to undergo developmental apoptosis (Kumar, 2015).

    The human Bcl-2 family member Bcl-rambo localizes to mitochondria and induces apoptosis and morphological aberrations in Drosophila

    Bcl-2 family proteins play a central role in regulating apoptosis. It has been previously reported that human Bcl-rambo, also termed BCL2L13, localizes to mitochondria and induces apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in Drosophila melanogaster. It was found to mainly localize to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induces apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induces aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induces the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo is partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, it was shown that human Bcl-rambo interacts genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. These data demonstrate that human Bcl-rambo localizes to mitochondria and at least regulates an apoptosis signaling pathway in Drosophila (Nakazawa, 2016).

    Analysis of the function of apoptosis during imaginal wing disc regeneration in Drosophila melanogaster

    Regeneration is the ability that allows organisms to replace missing organs or lost tissue after injuries. This ability requires the coordinated activity of different cellular processes, including programmed cell death. Apoptosis plays a key role as a source of signals necessary for regeneration in different organisms. The imaginal discs of Drosophila provide a particularly well-characterised model system for studying the cellular and molecular mechanisms underlying regeneration. Although it has been shown that signals produced by apoptotic cells are needed for homeostasis and regeneration of some tissues of this organism, such as the adult midgut, the contribution of apoptosis to disc regeneration remains unclear. Using a new method for studying disc regeneration in physiological conditions, this study has defined the pattern of cell death in regenerating discs. The data indicate that during disc regeneration, cell death increases first at the wound edge, but as regeneration progresses dead cells can be observed in regions far away from the site of damage. This result indicates that apoptotic signals initiated in the wound spread throughout the disc. Results are presented that suggest that the partial inhibition of apoptosis does not have a major effect on disc regeneration. Finally, these results suggest that during disc regeneration distinct apoptotic signals might be acting simultaneously (Diaz-Garcia, 2016).

    Different cell cycle modifications repress apoptosis at different steps independent of developmental signaling in Drosophila

    Apoptotic cell death is important for the normal development of a variety of organisms. Apoptosis is also a response to DNA damage and an important barrier to oncogenesis. The apoptotic response to DNA damage is dampened in specific cell types during development. Developmental signaling pathways can repress apoptosis, and reduced cell proliferation also correlates with a lower apoptotic response. However, because developmental signaling regulates both cell proliferation and apoptosis, the relative contribution of cell division to the apoptotic response has been hard to discern in vivo. This study used Drosophila oogenesis as an in vivo model system to determine the extent to which cell proliferation influences the apoptotic response to DNA damage. It was found that different types of cell cycle modifications are sufficient to repress the apoptotic response to ionizing radiation independent of developmental signaling. The step(s) at which the apoptosis pathway is repressed depends on the type of cell cycle modification; either upstream or downstream of expression of the p53-regulated proapoptotic genes. These findings have important implications for understanding the coordination of cell proliferation with the apoptotic response in development and disease, including cancer and the tissue specific responses to radiation therapy (Qi, 2016)

    Autophagy in neurodegeneration: two sides of the same coin

    Autophagy is a bulk lysosomal degradation process important in development, differentiation and cellular homeostasis in multiple organs. Interestingly, neuronal survival is highly dependent on autophagy due to its post-mitotic nature, polarized morphology and active protein trafficking. A growing body of evidence now suggests that alteration or dysfunction of autophagy causes accumulation of abnormal proteins and/or damaged organelles, thereby leading to neurodegenerative disease. Although autophagy generally prevents neuronal cell death, it plays a protective or detrimental role in neurodegenerative disease depending on the environment. This review describes the two sides of autophagy, the ability to protect or impair cell survival depending on the physiological and pathological environment (Lee, 2009. Full text of article).

    Genes involved in autophagic cell death

    Programmed cell death (PCD), important in normal animal physiology and disease, can be divided into at least two morphological subtypes, including type I, or apoptosis, and type II, or autophagic cell death. This study reports the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo. During Drosophila metamorphosis, the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade. To identify and analyze the genes expressed, wild-type patterns of gene expression were examined in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE). 1244 transcripts, including genes involved in autophagy, defense response, cytoskeleton remodeling, noncaspase proteolysis, and apoptosis, were expressed differentially prior to salivary gland death. Expression was detected of the steroid hormone 20-hydroxyecdysone (ecdysone)-induced primary response genes E74, E75, and E93 and the cell death genes ark, dronc, crq, rpr, and iap2. Mutant expression analysis has indicated that several of these genes are regulated by E93, a gene required for salivary gland cell death. These analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis, and that there are important differences (Gorsky, 2003).

    Multiple ecdysone-induced genes were detected. Abundantly expressed were members of the L71, or Eig71E, late gene family. The function of the L71 genes has not been established, but they are reported to be induced in late third instar larvae. Their abundance at 16 hr APF and decline by 23 hr APF is consistent with a role during the early larval ecdysone pulse. Eip63F-1, a calcium binding EF-hand family member, and Eip71CD (or Eip28), a protein-methionine-S-oxide reductase, both peak in gene expression at 20 hr APF, similar to the profile observed for E74 and E75. While Eip63F-1 has been implicated in calcium-dependent salivary gland glue secretion during earlier stages of salivary gland development, a role for Eip63F-1 or Eip71CD in salivary glands at the prepupal-pupal stage transition has not been described. Similarly, a role for Hormone-receptor-like in 78 (Hr78) at this stage has not been characterized (Gorsky, 2003).

    The findings indicate that transcriptional regulators other than the known ecdysone-induced factors may be involved in autophagic cell death regulation. Transcription factors with an expression profile similar to E74 and E75 (i.e., upregulated at 20 hr APF) include bunched (bun), a RNA polymerase II, and EP2237, a transcriptional activator implicated in sensory organ development. Also upregulated was Drosophila maf-S, a gene similar to a v-maf musculoaponeurotic fibrosarcoma oncogene family member in humans . Another upregulated transcription factor, CG3350, has no previous associated function (Gorsky, 2003).

    Expression of genes implicated in multiple different signal transduction pathways was detected, emphasizing the likely complex interplay of signaling pathways in autophagic cell death. One gene highly induced was A kinase anchoring protein 200 (akap200). In general, Akaps function in cyclic AMP-dependent protein kinase (PKA) signal transduction, targeting bound PKA to docking sites in organelles or the cytoskeleton. Redistribution of the cytoskeleton is a feature of autophagic cell death, and it is possible that Akap200 plays a role in cytoskeleton remodeling. Genetic studies in Drosophila have also implicated akap200 as a negative regulator of Ras pathway signaling, and thus it may regulate PCD via this pathway. Another gene significantly upregulated was Darkener of apricot (Doa), a dual specificity LAMMER kinase that is involved in the differentiation of a wide variety of cell types. These findings indicate that Doa, in addition to several other differentially expressed kinases and phosphatases identified, may also be involved in regulating autophagic cell death (Gorsky, 2003).

    Detection of members of the Drosophila defense response pathways (i.e., Toll pathway and imd/TNFα-like pathway) suggests that these pathways or some of their components may play a role in developmentally regulated autophagic cell death. In mammals, TNFα signaling can lead to NFκB activation or to apoptosis and has been linked to a possible autophagic type of cell death in T-lymphoblastic leukemic cells. In Drosophila, the TNFα-like pathway functions in both apoptosis and the immune response, and these results indicate that it may also be involved in autophagic cell death (Gorsky, 2003).

    Multiple genes involved in apoptotic cell death are also expressed during autophagic cell death, supporting the notion that these two processes can utilize common pathways or pathway components. In addition to the previously identified cell death genes expressed in the salivary gland, additional genes associated, in other tissues, with apoptotic cell death were identifed. Besides dronc, a second caspase, dcp-1, is upregulated transcriptionally in predeath stage salivary glands. In addition to the CD36-related scavenger receptor crq, upregulation was detected of three other CD36-related scavenger receptor genes whose function has not yet been characterized. The expression of additional cell death-related genes, death executioner Bcl-2 homolog (debcl or dborg-1), buffy/dborg-2, iap-1, dredd, and sickle, was detected in salivary glands and showed low level changes or no changes in expression levels. It is possible that these genes play a role in salivary gland death but are regulated primarily at the protein level. Given the overlap of genes involved in autophagic and apoptotic cell, it is reasonable to expect that some of the novel autophagic cell death-associated genes identified in this study may also be associated with apoptotic cell death (Gorsky, 2003).

    The results suggest that genes associated with the process of autophagy (i.e., bulk cellular degradation) can be regulated transcriptionally and this regulation is likely integral to the mechanism of autophagic cell death. Known genes involved in autophagy have been defined largely by genetic screens in yeast and include at least 16 autophagy-defective (apg) genes and 6 autophagy (aut) genes, with overlap between the two groups. Putative Drosophila orthologs of at least ten of the apg/aut genes were identified and evidence of expression was found for at least nine of these. Strikingly, CG6194 was induced prior to cell death and is one of two Drosophila genes similar to apg4/aut2, a yeast gene encoding a novel cysteine endoprotease required for autophagy. CG6194 encodes a functional homolog of APG4/AUT2 and interacts genetically with several members of the Notch signaling pathway. Results of real-time RT-PCR analyses have indicated upregulated expression of other apg/aut-like genes including CG1643 (apg5-like), CG10861 (apg12-like), and CG5429 (apg6-like). In addition to apg/aut-like genes, evidence was found for upregulated expression of Drosophila rab-7, one of several rab gene family members implicated in autophagy in yeast and humans (Gorsky, 2003).

    The terminal phase of autophagy involves autolysosome formation by fusion of the autophagosome with a lysosome and subsequent degradation of sequestered cellular components. Lysosomal components with upregulated transcripts in predeath stage salivary glands include lysozyme, β-galactosidase, and cathepsins B, D, E, F, and L. Multiple components involved in autophagy are conserved in Drosophila and likely play a role in ecdysone-induced autophagic cell death in the salivary glands (Gorsky, 2003).

    To identify the genes with differential expression that are most likely associated with the autophagic cell death process, E93 mutant analyses was carried out. E93 expression appears to specifically foreshadow steroid-induced cell death, and E93 mutant salivary glands display morphological features indicative of a block in the early stages of autophagic cell death. Further, the ecdysone-induced genes BR-C, E74, and E75 and the cell death genes rpr, hid, crq, and dronc are all transcribed at reduced levels in E93 mutant salivary glands. E93 encodes a novel nuclear protein that binds to multiple sites on larval salivary gland polytene chromosomes. The map position of crq correlates with an E93 binding site and it may thus be regulated directly by E93. To identify other genes that may be regulated transcriptionally by E93 in salivary gland death, all differentially expressed genes were screened for those with a map position corresponding to E93 binding sites. Forty-three upregulated genes were identified and forty-one downregulated genes corresponding to 39 of the 65 known E93 binding sites. To test further whether these genes may be regulated directly by E93, transcription profiles were analyzed in E93 mutant salivary glands. Since previous studies indicated a role for E93 as a positive regulator of cell death gene expression, genes upregulated significantly at 23 hr APF were tested. Of 18 confirmed upregulated genes tested, all but one (Sox14) exhibited a reduction in the fold-difference in expression in the E93 mutant background compared to control genes. These results indicate that these 17 genes are regulated by E93, indirectly or directly, and that their expression is thus likely associated specifically with autophagic cell death (Gorsky, 2003).

    This study represents the first comprehensive analysis of genes associated with autophagic cell death in vivo. Autophagic cell death is shown to be associated with the induction of genes that participate in protein synthesis, transcription, multiple signal transduction pathways, and two ubiquitin-like pathways required for autophagy. Multiple genes involved in apoptotic cell death also appear to be regulated in autophagic cell death, supporting the view that these two processes can utilize common pathways or pathway components. Further, many genes were implicated for the first time in cell death and represent candidate markers and/or mediators of autophagic cell death and, possibly, apoptotic cell death. In addition to similarities, likely differences were revealed between these two morphological forms of cell death. In particular, genes similar to those involved in autophagy (i.e., bulk cellular degradation) are upregulated in dying salivary gland cells, and these may prove to be useful molecular markers for the autophagic cell death process (Gorsky, 2003).

    Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila

    Autophagy is involved with the turnover of intracellular components and the management of stress responses. Genetic studies in mice have shown that suppression of neuronal autophagy can lead to the accumulation of protein aggregates and neurodegeneration. However, no study has shown that increasing autophagic gene expression can be beneficial to an aging nervous system. This study demonstrates that expression of several autophagy genes is reduced in Drosophila neural tissues as a normal part of aging. The age-dependent suppression of autophagy occurs concomitantly with the accumulation of insoluble ubiquitinated proteins (IUP), a marker of neuronal aging and degeneration. Mutations in the Atg8a gene (autophagy-related 8a) result in reduced lifespan, IUP accumulation and increased sensitivity to oxidative stress. In contrast, enhanced Atg8a expression in older fly brains extends the average adult lifespan by 56% and promotes resistance to oxidative stress and the accumulation of ubiquitinated and oxidized proteins. These data indicate that genetic or age-dependent suppression of autophagy is closely associated with the buildup of cellular damage in neurons and a reduced lifespan, while maintaining the expression of a rate-limiting autophagy gene prevents the age-dependent accumulation of damage in neurons and promotes longevity (Simonsen, 2008).

    Macroautophagy (henceforth referred to as autophagy) is a highly conserved pathway that involves sequestering cytoplasmic material into double-membrane vesicles that fuse with lysosomes where the internal cargo is degraded. Autophagy occurs in response to starvation and environmental stress and has been well characterized in yeast. Recent studies in higher eukaryotes have shown that autophagy is involved in several complex cellular processes including cell death and immune response pathways. In mice, suppression of basal autophagy in the nervous system results in the accumulation of ubiquitinated proteins and neural degeneration, indicating that the continuous turnover of long-lived proteins is essential for nerve cell survival. In addition, the pathway is suppressed by insulin/ insulin-like growth factor-1 (IGF-1) signaling (through TOR kinase) and is enhanced when animals are placed on a caloric restricted diet (a well known anti-aging regime), suggesting that activation of autophagy may facilitate the removal of damaged macromolecules and organelles that accumulate during cellular aging. Protein turnover and electron microscopy studies have suggested that a functional decline in macroautophagy does occurs in older liver cells (Simonsen, 2008).

    However, age-related changes in autophagy gene expression patterns have not been well studied in an organism that permits the genetic dissection of pathway function. This report addressed the role of autophagy during Drosophila aging; the overall level of autophagy gene expression is reduced by age. The age-related reduction in autophagic activity is correlated with an increased accumulation of cellular damage (build up of IUP). Further this study investigated the effect of decreased or elevated levels of Drosophila Atg8a, a member of the Atg8/LC3 protein family, on the aging fly nervous system. Atg8a mutant flies have shorter lifespans, show a dramatic accumulation of IUP and increased sensitivity to oxidative stress. In contrast, the data show that elevating the Atg8a protein in older neurons maintains the basal rates of autophagy, which is reflected in an inverse correlation with accumulation of cellular damage and a positive correlation with Drosophila longevity (increased average lifespan) (Simonsen, 2008).

    The expression of select autophagy genes is downregulated in older Drosophila. To examine age-related changes in autophagy gene expression, mRNA levels of the Atg1, Atg2, Atg5, Atg8a, Atg18 and blue cheese (bchs) genes were analyzed using quantitative real-time PCR (qRT-PCR) across the entire age range of adult Drosophila lifespan and compared to message levels detected in one-day old flies. These genes represent a broad spectrum of gene function and participate at multiple stages in the pathway. The expression profiles of autophagy genes were stable (Atg1 and Atg5) or decreased significantly (Atg2, Atg8a, Atg18, bchs) by 3-weeks and remained suppressed (up to 75%) over the 9-week testing period. In contrast, the message level of the proteasome subunit rpn6 increased between 2 to 6-fold with age, in line with previous studies showing that proteasomal activity maybe upregulated with age. Together these data reveal that the expression of several essential autophagy genes decline in fly neural tissues as a normal part of aging and indicate that autophagic activity may decrease in older Drosophila (Simonsen, 2008).

    Atg8a protein levels decrease in the aging CNS and in Atg8a mutant flies. To ask if there is a link between suppressed autophagy and accelerated aging, focus was placed on the Drosophila Atg8a gene, which is essential for the formation of autophagosomes and was found to have possible genetic interactions with a second autophagy protein, Bchs. The amount of Atg8a protein is also down-regulated as much as 60% by 4 weeks of age. Cytosolic Atg8 (Atg8-I) undergoes C-terminal cleavage and activation before being conjugated to lipids (Atg8-II). As a result, Atg8-II remains bound to autophagosomes throughout their formation, transport and fusion with lysosomes and has the potential to become a rate limiting component of the pathway when cellular demand for autophagy is high. Two mutant lines containing P-element insertions in the Atg8a gene (Atg8a1 or EP-UAS-Atg8a and Atg8a2) were used to examine the effects that altered gene expression has on the aging fly nervous system (Simonsen, 2008).

    Atg8a1/Atg8a1 and Atg8a1/Atg8a2 mutants had reduced or absent Atg8a-I protein levels, which was confirmed by similar reductions in the Atg8a mRNA levels. The Atg8b gene is expressed at very low levels in female heads as determined by qRT-PCR, indicating that Atg8b protein level is below the detection limits of Western analysis. To determine if the age-related decline in the Atg8a message and protein could be reversed, the Drosophila Gal4/UAS system was used to drive Atg8a expression in the adult Drosophila CNS. Female flies from the APPL-Gal4 driver line (allows adult pan-neural gene expression) were crossed to males containing a UAS-P-element located in the 5' region of the Atg8a gene (EP-UAS-Atg8a, Atg8a1). While Atg8a mRNA levels were significantly reduced by age in wildtype flies, the Atg8a message remained elevated in Atg8a expressing flies for at least 4 weeks, as determined by qRT-PCR analysis. In addition, Western analysis of F1 offspring showed that the Atg8a protein declined only 20% compared to a 60% reduction in control flies. Therefore, the normal age-dependent decline seen in both the Atg8a message and protein levels in normal flies can be repressed using the APPL-Gal4 driver (Simonsen, 2008).

    The accumulation of ubiquitinated proteins and aggregates in nerve cells has been observed in many human neurodegenerative diseases that are associated with aberrant protein folding and in neural tissues with suppressed autophagy. It was therefore asked whether IUP profiles change in wildtype flies as they age. Canton-S (wildtype) flies were collected at day one and at weekly intervals and their heads were processed by sequential detergent extraction. This technique allows the differential extraction of proteins based on their solubility properties in non-ionic (Triton-X) and ionic (SDS) detergents. Ubiquitinated proteins frequently accumulate in the insoluble (SDS) fraction in age-dependent neurodegenerative disorders. Western blots of SDS soluble proteins were sequentially hybridized with anti-ubiquitin and anti-actin antibodies. While young wildtype flies (day one to 3 weeks) exhibit low IUP levels, older flies (4 to 8 weeks) show a dramatic accumulation of IUP. The IUP build up is preceded by the age-dependent decrease in the expression of autophagy genes, suggesting that the progressive loss of autophagic function is a significant factor leading to compromise protein turnover by this pathway (Simonsen, 2008).

    Since Atg8a levels are significantly reduced in Atg8a1/Atg8a2 mutants at week one, these flies were used to examine the effect that loss of Atg8a has on Drosophila longevity. Atg8a- (Atg8a1/Atg8a2) and control (CS) flies were, aged at 25oC and lifespan profiles determined for each genotype. Female Atg8a- flies have a 53% decrease in longevity when compared to wildtype and genotype controls. To determine whether Atg8a mutants also develop neuronal aggregates, brains of 15 day old wildtype and Atg8a- (Atg8a1/Atg8a2) flies were dissected, stained for ubiquitin and examined using confocal microscopy. Control flies had a uniform pattern of ubiquitin staining throughout the adult brain, whereas age-matched Atg8a- mutants showed formation of ubiquitinated protein inclusions in many CNS regions, including the optic lobe (OL) and subesophageal ganglia. Transmission electron microscopy analysis of brain tissue from one week-old Atg8a- flies also showed the appearance of electron dense protein aggregates or granules in the cytoplasm of neurons. These structures were primarily surrounded by a single membrane layer, but were also found without obvious membrane limitations. Microtubule-like structures could be observed that assemble with the membrane free aggregates. Similar structures are rarely seen in brains from age-matched controls. The development of protein deposits and the formation of abnormal intracellular structures are reminiscent of the CNS pathology of mice with disruption of either the Atg5 or Atg7 genes. Since suppression of autophagy is known to effect protein turnover, the IUP profiles of Atg8a mutants were examined. While young control flies (CS) had low IUP levels in SDS soluble extracts, Atg8a mutants (Atg8a1/Atg8a2 and Atg8a2) showed a significant accumulation of IUP beginning as early as one week. These data indicate that the elimination of cellular material is no longer efficient in flies with suppressed autophagy, leading to the build up of proteins and neural inclusions (Simonsen, 2008).

    To assess whether enhanced Atg8a expression has an effect on the aging CNS, the lifespan profiles of F1 females and control flies maintained under standard culture conditions were examined. Elevated neuronal expression of Atg8a produces a dramatic extension of adult longevity (Simonsen, 2008).

    Maximal lifespan was extended from 88 to 96 days and the average lifespan is increased 56% above that of controls. Similar results were obtained when an independent transposable construct encoding the GFP-Atg8a protein is expressed in the brains of both male and female flies. Lifespan extension was not seen when Atg8a was expressed using an early pan-neural driver line. Expression of two other autophagy genes (Atg2 and bchs) or other proteins associated with enhanced longevity (Hsc70 and GST) using the APPL-Gal4 driver did not extend adult Drosophila lifespan to the same extent as the Atg8a protein. The difference between the APPL-Gal4 and ELAV-Gal4 expression of Atg8a is likely related to the age-dependent expression differences of each Gal4-driver, suggesting that the timing of Atg8a expression in the aging CNS is critical for its ability to enhance longevity. Elevated Atg8a expression is also protective when flies are maintained at higher temperatures (29oC), under conditions known to accelerate Drosophila aging. Since wild type Drosophila have a dramatic increase in IUP profiles starting at 4 weeks and Atg8a mutants show accelerated IUP accumulation, it was asked whether increased neuronal expression of Atg8a could prevent the buildup of IUP that naturally occurs with age. Control flies (CS), Atg8a1/Atg8a1 (Atg8a-) and Atg8a expressing flies (Atg8a+) were aged for 4 weeks and IUP levels from SDS head extracts were examined by Western analysis. Control (CS) and Atg8a- fliesshowed a significant accumulation of IUP that is typical for both genotypes at this age. In contrast, age-matched Atg8a+ animals showed a 12-fold reduction in IUP levels. These data clearly show that the decrease in autophagy normally occurring with age correlates with IUP accumulation and suggests that elevated levels of a rate-limiting component of autophagy can facilitate the clearance of ubiquitinated or aggregate-prone proteins later in life (Simonsen, 2008).

    As a consequence of a normal aerobic metabolism cells are exposed to reactive oxygen species (ROS), which can cause direct damage to macromolecules. There is also an increase in oxidative damage associated with age and age-related neurodegenerative diseases. To determine if autophagy affects the acute oxidative stress response in the Drosophila nervous system, control, Atg8a1/Atg8a2 mutant or Atg8a expressing (APPL-Gal4/EP-UAS-Atg8a) flies were placed on to media containing 1.5% H2O2 and analyzed their lifespan profiles. While suppression of autophagy resulted in a shortened lifespan, Atg8a expressing flies exhibited longer lifespans than controls in the presence of oxidants. One potential mechanism for autophagy to regulate macromolecular damage caused by oxidant exposure involves the direct removal of ROS damaged proteins. Previous studies have measured damage by examining the accumulation of IUP or carbonylated protein levels in neural tissues. Therefore, both parameters were examined after exposing duplicate sets of control, Atg8a mutant and Atg8a expressing female flies to normal media (-) or media containing 1.5% H2O2 (+) for 24 hours. IUP levels increased on average 20% following H2O2 exposure in control flies. Atg8a mutants show a dramatic 126% increase in IUP, whereas flies with elevated neuronal Atg8a have a marked reduction in IUP accumulation relative to control flies. In a parallel study, control and Atg8a mutant flies showed a pronounced accumulation of several carbonylated proteins. In contrast, upregulating Atg8a dramatically lowers the level of damaged proteins following H2O2 treatment. Taken together, these data indicate that autophagic activity is inversely correlated with lifespan and accumulation of ROS-modified proteins following exposure to oxidative stress (Simonsen, 2008).

    This study has demonstrate for the first time that maintaining the bulk clearance pathway of macroautophagy in a mature nervous system promotes longevity and reduces markers of cellular aging like IUP. This work also demonstrates that several key pathway members are suppressed at the level of gene transcription as a normal part of Drosophila aging. The age-dependent decrease in autophagy gene expression is paralleled by a pronounced accumulation of IUP (Simonsen, 2008).

    Consistent with the hypothesis that the progressive loss of autophagic function results in the accumulation of aging markers, Atg8a mutant flies also have a reduced lifespan, increased sensitivity to oxidative stress and morphological phenotypes consistent with premature or accelerated aging. Both mutational loss and an age-dependent decline in autophagy decreases the pathway's ability to serve as the bulk clearance mechanism for cellular damage, which can go on to further impair the long-term function of neurons. The loss-of-function phenotypes seen in mutant Drosophila have striking similarities to those characterized in some of the most common human neurodegenerative disorders associated with misfolded protein, and in mouse models in which basal autophagy is suppressed in the brain. This diverse data underscores the functional conservation of the pathway and suggests that the age-dependent suppression of autophagy may be a contributing factor for human disorders (Simonsen, 2008).

    Insulin/IGF-1 signaling and caloric restriction have been shown to be major determinants of aging. Most studies examining the link between aging and Insulin/IGF-1/CR-mediated signaling have focused on downstream mediators such as the forkhead transcription factors and sirtuins. However, a recent study in C. elegans revealed that the enhanced longevity phenotype of an insulin-signaling mutant is negated by decreased expression of the beclin-1/Atg6 gene, suggesting that caloric restriction and the insulin/TOR signaling may also affect lifespan via autophagic pathways. This study has demonstrated that circumventing upstream signaling pathways and directly maintaining the expression of an essential autophagy gene (At8ga) in the aging nervous system leads to a dramatic extension of lifespan and resistance to oxidative stress. This information and the placement and function of Atg8/LC3 within the pathway and its degradation by the lysosome suggest it may become a rate-limiting by directly enhancing Atg8a expression. These results suggest that upregulation and the supplementation of rate-limiting components of the autophagic pathway may also be beneficial for the health and maintenance of the human nervous system under a wide variety of stressful conditions that involve oxidant exposure, misfolded proteins and simply old age (Simonsen, 2008).

    Atg1-mediated myosin II activation regulates autophagosome formation during starvation-induced autophagy

    Autophagy is a membrane-mediated degradation process of macromolecule recycling. Although the formation of double-membrane degradation vesicles (autophagosomes) is known to have a central role in autophagy, the mechanism underlying this process remains elusive. The serine/threonine kinase Atg1 has a key role in the induction of autophagy. This study shows that overexpression of Drosophila Atg1 promotes the phosphorylation-dependent activation of the actin-associated motor protein myosin II. A novel myosin light chain kinase (MLCK)-like protein, Spaghetti-squash activator (Sqa), was identified as a link between Atg1 and actomyosin activation. Sqa interacts with Atg1 through its kinase domain and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the formation of autophagosomes under starvation conditions. In mammalian cells, it was found that the Sqa mammalian homologue zipper-interacting protein kinase (ZIPK) and myosin II had a critical role in the regulation of starvation-induced autophagy and mammalian Atg9 (mAtg9; see Drosophila Atg9) trafficking when cells were deprived of nutrients. These findings provide evidence of a link between Atg1 and the control of Atg9-mediated autophagosome formation through the myosin II motor protein (Tang, 2011).

    Myosin II is a conventional two-headed myosin composed of two heavy chains, two essential light chains, and two regulatory light chains. Myosin II activation is regulated by the phosphorylation of its regulatory light chain via MLCKs. Rho GTPase and Rho kinase have been implicated in the regulation of myosin activation. However, this study found that neither RNA-mediated knockdown of dRok nor mutations in Rho1 or dRhoGEF2 could suppress the Atg1-induced wing defects. Instead, it was found that depletion of Sqa rescued Atg1-induced wing defects. This epistasis analysis showed that Sqa functioned downstream of Atg1. Moreover, it was found that Sqa but not Atg1 could directly phosphorylate Spaghetti squash (Sqh) in the in vitro kinase assay, suggesting that Atg1 stimulates myosin activity via Sqa. Importantly, Atg1 phosphorylates and interacts with Sqa, indicating that Atg1-Sqa functions in a kinase cascade to regulate myosin II activation. Moreover, Atg1 has been found to have a critical role in the regulation of autophagy induction under stress conditions in yeast, Drosophila, and mammalian cells. These results provide the first evidence that nutrient starvation stimulates myosin II activation in an Atg1-Sqa-dependent manner. Most significantly, a dramatic decrease was found in the size and number of autophagosomes in cells expressing Sqa-T279A, Sqa-RNAi, and SqhA20A21 on nutrient deprivation, indicating that Atg1-Sqa-mediated actomyosin activation has a critical role in autophagy (Tang, 2011).

    The kinase domain of Sqa is also highly homologous to that of the mammalian DAPK family proteins. Recent studies have indicated that DAPK1 regulates autophagy through its association with MAP1B and Beclin1, or by modulating the Tor signalling pathway. As DAPK family proteins also regulate myosin II phosphorylation, one might speculate that Sqa may be the Drosophila counterpart of DAPK protein. Indeed, although overexpression of Sqa does not induce cell death, Sqa shares several characteristics with DAPK3/ZIPK. First, unlike MLCK family proteins, both Sqa and ZIPK contain an amino-terminal kinase domain that has 42% sequence identity and 61% similarity. Moreover, like ZIPK, recent sequence analysis from FlyBase identified a Sqa isoform that also contains a leucine-zipper domain. Second, as phosphorylation of Thr-265 in ZIPK is essential for its kinase activity, this study found that Atg1 phosphorylates Sqa at the corresponding Thr-279, and is critical for Sqa activity. Third, just as Sqa specifically associates with kinase-inactive Atg1, the results indicate a similar interaction between ZIPK and Ulk1. Importantly, depletion of Sqa and ZIPK resulted in autophagic defects in response to nutrient deprivation. These findings together suggest that ZIPK may act as a mammalian homolog of Sqa during starvation-induced autophagy. Further investigation is needed to determine whether the mammalian Atg1 (Ulk1) directly phosphorylates ZIPK at Thr-265, and the role of this regulation in autophagy (Tang, 2011).

    In autophagy, the source of the autophagosomal membrane and dynamics of autophagosome formation are fundamental questions. Studies in yeast and mammalian cells have identified several intracellular compartments as potential sources for the PAS (also termed isolation membrane/phagophore). Formation of PI(3)P-enriched ER subdomains (omegasomes) has been reported during nutrient starvation and autophagy induction, and a direct connection has been observed between ER and the phagophore using the 3D electron tomography. In addition, recent studies in yeast cells have suggested Atg9 and the Golgi complex have a role in the formation of autophagosomes. It has been proposed that the integral membrane protein Atg9 may respond to the induction signal in promoting lipid transport to the forming autophagosomes. The mAtg9 has been found to localize on the TGN and the endosomes in nutrient-rich conditions and translocate to LC3-positive autophagosomes on nutrient deprivation. Although several proteins, including Ulk1, mAtg13, and p38IP, have been found to regulate starvation-induced mAtg9 trafficking, the molecular motor that controls the movement of mAtg9 between different subcellular compartments remains unknown (Tang, 2011).

    The finding that myosin II redistributes from peripheral to the perinuclear region of cells on starvation suggests that myosin II has a role in membrane trafficking. In fact, it has been reported that myosin II is required for the trafficking of major histocompatibility complex (MHC) class II molecules and antigen presentation in B lymphocytes. Myosin II has also been found to be involved in the protein transport between ER and Golgi. This study has shown that there here is a molecule link between mAtg9 and the actomyosin network, indicating that myosin II may function as a motor protein for mAtg9 trafficking during early autophagosome formation. In conclusion, this work has unravelled a regulatory mechanism between Atg1 activity and the Atg9-mediated formation of autophagosomes. Further studies are needed to determine the involvement of this signalling process in other stress-induced or developmentally regulated autophagy (Tang, 2011).

    Atg9 Interacts with dTRAF2/TRAF6 to Regulate Oxidative Stress-Induced JNK Activation and Autophagy Induction

    Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes. Atg9 is the only integral membrane protein among autophagy-related (Atg) proteins thought to carry the membrane source for forming autophagosomes. This study shows that Drosophila Atg9 interacts with Drosophila tumor necrosis factor receptor-associated factor 2 (dTRAF2: TNF-receptor-associated factor 6) to regulate the c-Jun N-terminal kinase (JNK) signaling pathway. Significantly, depletion of Atg9 and dTRAF2 compromised JNK-mediated intestinal stem cell proliferation and autophagy induction upon bacterial infection and oxidative stress stimulation. In mammalian cells, mAtg9 interacts with TRAF6, the homolog of dTRAF2, and plays an essential role in regulating oxidative stress-induced JNK activation. Moreover, it was found that ROS-induced autophagy acts as a negative feedback regulator of JNK activity by dissociating Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. These findings indicate a dual role for Atg9 in the regulation of JNK signaling and autophagy under oxidative stress conditions (Tang, 2013).

    Macroautophagy (hereafter autophagy) is a conserved catabolic pathway in which double membrane vesicles called autophagosomes engulf macromolecules or organelles. Subsequently, autophagosomes fuse with lysosomes to form autolysosomes where degradation occurs. Autophagy is involved in cytoprotective responses to environmental stresses, stem cell maintenance and differentiation, tumorigenesis, and programmed cell death. There have been more than 30 autophagy-related (Atg) genes essential for autophagy process identified through genetic screens in yeast. Atg9 is the only one identified as a transmembrane protein, and it has been thought to promote lipid transport to the forming autophagosomes. Mammalian Atg9 (mAtg9) localizes on the trans-Golgi network and endosomes under nutrient-rich conditions, whereas it translocates to forming autophagosomes under starvation conditions. The recycling of mAtg9 during autophagy is regulated by several proteins including Ulk1, ZIPK, mAtg13, and p38IP. Interestingly, one recent study has reported that mAtg9 modulates innate immune response in an autophagy-independent manner . However, the physiological functions of Atg9 remain elusive (Tang, 2013).

    Reactive oxygen species are highly reactive free radicals that can cause irreversible oxidative damage to proteins, lipids, or nucleotides in cells. Excessive production of ROS or depletion of antioxidants causes oxidative stress that often leads to cell dysfunction and diseases such as neurodegeneration, cancer, and aging. More importantly, ROS also plays critical roles in host defense and in the regulation of various cellular signaling pathways The ROS-induced signaling pathways include several mitogen-activated protein (MAP) kinase cascades involving the c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase. The JNK signaling pathway regulates diverse biological functions, including apoptosis, cytoprotection, metabolism, and epithelial homeostasis in response to several cytokines and environmental stresses. Depending on the duration and magnitude of exposure, ROS-induced JNK activation may lead to the promotion of either cell survival or apoptosis. In Drosophila, JNK signaling was found to protect cells from oxidative stress and extend lifespan of adult flies. It has been shown that the JNK pathway is required for intestinal epithelium renewal during bacterial infection-induced ROS/oxidative stress. One of the mechanisms that JNK meditates to protect flies against acute oxidative insults is the activation of autophagy. In response to oxidative stress, JNK signaling stimulates the expression of several ATG genes. Several recent studies have reported that overexpression of ATG genes and activation of autophagy are sufficient to extend lifespan and confer stress resistance in Drosophila (Tang, 2013).

    How does ROS/oxidative stress trigger JNK activation? It has been shown that signaling molecules, including apoptosis signal-regulating kinase (Ask1), glutathione S-transferase Pi (GSTp), and Src kinase can function as molecular links between ROS and JNK. Ask1 is a MAPKKK that activates JNK by phosphorylating MKK4/7. Under normal physiological conditions, Ask1 is inhibited by forming a complex with the redox regulatory protein thioredoxin. Upon exposure to ROS/oxidative stress, the oxidized thioredoxin dissociates from Ask1 and results in the activation of Ask1 signaling pathway. GSTp has been identified as a JNK inhibitor. Under oxidative conditions, GSTp forms oligomers and dissociates from JNK, leading to JNK activation. A number of reports have also shown the involvement of Src and its downstream targets in H2O2-induced JNK activation, although the underlying molecular mechanism remains elusive. Recently, tumor necrosis factor receptor-associated factors (TRAFs) have been found to be involved in ROS-mediated JNK activation. In mammals, the TRAF family consists of seven members and functions as scaffold proteins that link cell surface receptors to the downstream effectors. Among them, TRAF2 and TRAF6 are found to associate with Ask1 and form the active Ask1 signalsome in response to ROS stimulation. Moreover, the involvement of TRAF4 in oxidative activation of JNK via its interaction with the NAD(P)H oxidase p47phox has been demonstrated. The Drosophila TRAF2 (dTRAF2), a homolog of human TRAF6, was found to mediate Eiger/Wegen (tumor necrosis factor/tumor necrosis factor receptor [TNF/TNFR])-induced JNK signaling. However, the role of dTRAF2 in ROS-mediated JNK activation remains unclear (Tang, 2013).

    This study has identified a biological function of Atg9 in regulation of JNK signaling pathway. Drosophila Atg9 can activate JNK signaling through its interaction with dTRAF2. Depletion of Atg9 compromised oxidative stress-induced JNK activation, the JNK-mediated epithelium renewal, and autophagy induction. In mammalian cells, mAtg9 was found to be essential for JNK activation in response to ROS/oxidative stress, indicating a highly conserved role of Atg9 in regulating JNK activity. It was further found that ROS-induced autophagy negative feedback regulates JNK activity through the dissociation of Atg9/mAtg9 from dTRAF2/TRAF6 in Drosophila and mammalian cells, respectively. These findings provide insights into the crosstalk between autophagy and JNK signaling pathway in response to oxidative stress (Tang, 2013).

    The Atg9 transmembrane protein has been shown to play an essential role in autophagy pathway in yeast and mammals. In this study, Drosophila Atg9 was also found to be required for autophagy induction upon nutrient deprivation or under oxidative stress conditions. More importantly, a role was uncovered for Atg9 in regulating the JNK signaling pathway. Upon bacterial infection, Atg9 interacts with dTRAF2 to activate JNK-mediated autophagy induction and epithelium renewal in Drosophila gut cells. The role of Atg9 in activating JNK signaling was also observed in mammalian cells. Moreover, this study found that ROS-induced autophagy in turn inhibits JNK signaling via a negative feedback mechanism by dissociation of Atg9 from dTRAF2 and TRAF6 in Drosophila and mammalian cells, respectively (Tang, 2013).

    Atg9 is a highly conserved and the only multi-spanning transmembrane Atg protein essential for the formation of autophagosomes. In yeast, Atg9 cycles between the preautophagosomal structure (PAS) and peripheral cytoplasmic structures. Recently, using single particle tracking, Yeast Atg9 exists as highly motile vesicles that contribute to PAS formation. In mammalian cells, mAtg9 is localized mainly to the trans-Golgi network and endosomes. However, upon nutrient starvation, mAtg9 is enriched in endosomal pools and undergoes a dynamic interaction with forming autophagosomes. The current study found that Drosophila Atg9 not only distributed in cytoplasm, but also concentrated at cell-cell junctions, suggesting Atg9 may have additional roles besides its function in autophagy. For example, it has been reported that mAtg9 can function as a regulator for dsDNA-triggered innate immune response (Tang, 2013).

    The involvement of Atg1/Ulk1 in Atg9 trafficking has been described in yeast and mammalian cells. Consistent with these findings, the current study found that Drosophila Atg9 redistributed from peripheral pools to forming autophagosomes in an Atg1-dependent manner. A previous reported that overexpression of Drosophila Atg1 induces cell death. Interestingly, this study found that overexpression of Atg1 did not induce JNK activation and the Atg1-induced cell death could not be rescued by inhibition of JNK signaling. The current findings highlight that, in addition to its role in autophagy, Atg9 plays a role in the regulation of JNK activation in response to oxidative stress (Tang, 2013).

    The JNK signaling pathway is one of the mitogen-activated protein kinase (MAPK) cascades involved in stress responses. Activation of the JNK pathway has been implicated in a number of biological processes including cell proliferation, survival, apoptosis, and migration. The involvement of JNK in both proapoptotic and anti-apoptotic activities indicates a complex function of the JNK pathway, whereas the molecular mechanism that regulates JNK to mediate both processes remains elusive. This study study has shown that ectopic expression of Atg9 in the developing wing and eye leads to JNK activation and apoptotic cell death. Moreover, the results provided evidence that, upon ROS stimulation, Atg9, but not Atg12, is required for JNK-mediated intestinal stem cell proliferation and autophagy induction in Drosophila. These results indicate that Atg9 may play a critical role in regulating JNK-mediated cell survival and apoptosis. It was further shown that Atg9 regulates JNK signaling via its association with dTRAF2 and TRAF6 in Drosophila and mammals, respectively. GST-pull down assay revealed that the C terminus of Drosophila Atg9 can interact with dTRAF2. Surprisingly, Atg9 lacking the C-terminal region can still promote JNK activation and cell death. One possibility is that Atg9 may interact with dTRAF2 through multi-regions. On the other hand, yeast Atg9 has been shown to self-interact through the C terminus, and Atg9 self-association is critical for its function in autophagy. Sequence analysis revealed that Drosophila Atg9 also contains the conserved self-interacting motif (VGNVC) between amino acids 560 and 564. It is possible that Atg9ΔC may exert its function in regulating JNK activity by interacting with the endogenous Atg9 (Tang, 2013).

    TRAF6 functions as a RING-domain containing ubiquitin ligase involved in a variety of biological processes including adaptive and innate immunity, bone metabolism and tissue development. TRAF6 is required for interleukin-1 (IL-1) and transforming growth factor-β-mediated JNK activation. In Drosophila, dTRAF2 plays a role in Eiger/Wegen (TNF/TNFR)-induced JNK signaling. How does Atg9 regulate TRAF-mediated JNK activation? One mechanism may be that Atg9 associate with TRAF6 to modulate its ubiquitin ligase activity. Indeed, a recent study indicates that Atg9 interacts and promotes TRAF6 ubiquitination. Alternatively, because Atg9 is a membrane protein with diverse subcellular localization, Atg9 may bind and target TRAF6 to peripheral membrane regions in response to bacterial infection and oxidative stress. These two mechanisms need not be mutually exclusive and can occur together (Tang, 2013).

    Recent studies suggested there to be a complex relationship between the JNK pathway and autophagy. On the one hand, under nutrient starvation conditions, JNK has been found to phosphorylate Bcl-2, leading to the dissociation of Bcl-2 from beclin 1 and the activation of autophagy. JNK signaling also activates autophagy via the upregulation of ATG gene expression in response to oxidative stress and oncogenic transformation. On the other hand, JNK can act as a negative regulator of FoxO-dependent autophagy in neurons. It is interesting to note that, although Atg9 overexpression activates JNK, the current data showed that Atg9 overexpression could not induce autophagy in the larval fat body. Because Atg9 promotes JNK activation through its association with dTRAF2, dTRAF2 may not be expressed in the fat body. Indeed, RNA expression analysis reveals that dTRAF2 expresses in the fat body at a relatively low level . Alternatively, it has been reported that JNK overexpression activates autophagy independently of Atg1 and nutrient signal. However, the current results showed that Atg9 interacts with Atg1 and is required for starvation-induced autophagy. Overexpression of JNK may induce a noncanonical autophagy that is independent of 'core Atg proteins.' (Tang, 2013 and references therein).

    This current study also demonstrates that autophagy can act as a negative feedback regulator for JNK activation upon oxidative stress. Inhibition of autophagy in flies fed with Ecc15 or paraquat resulted in a substantial increase in JNK activity, which led to increased ISC proliferation and cell death in adult Drosophila midgut. In mammalian cells, depletion of Atg5 led to prolonged JNK activation during hydrogen peroxide-induced oxidative stress. Moreover, activation of autophagy by rapamycin effectively blocked the interaction between Atg9 and TRAF6 and inhibits ROS-induced JNK activity. Considered together, these findings together indicate an important role of autophagy in restricting JNK activity by modulating the interaction between Atg9 and TRAF6 in response to oxidative stress. In conclusion, this work establishes a regulatory mechanism between Atg9, autophagy, and the JNK signaling pathway during oxidative stress conditions (Tang, 2013).

    Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

    Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, chromatin immunoprecipitation-sequencing analysis was performed with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling (Bai, 2013).

    Insulin/IGF-1 signaling modulates longevity in many animals. Genetic analysis in C. elegans and Drosophila shows that insulin/IGF-1 signaling requires the DAF-16/FOXO transcription factor to extend lifespan, while in humans several polymorphisms of FoxO3A are associated with exceptional longevity. Although many downstream effectors of FOXO have been identified through genome-wide studies, the targets of FOXO responsible for longevity assurance upon reduced insulin signaling are largely unknown. This study found 273 genes targeted by Drosophila FOXO using ChIP-Seq with two long-lived insulin mutant genotypes. Focused was placed on daw, an Activin ligand, which is transcriptionally repressed by FOXO upon reduced insulin/IGF signaling. Inactivation of daw and of its downstream signaling partners babo and Smox extend lifespan. These results are reminiscent of observations from C. elegans where reduced TGF-β/dauer signaling extends longevity. Notably, the lifespan extension of TGF-β/dauer mutants (e.g. daf-7 (e1372) mutants) can be suppressed by daf-16 mutants, suggesting that TGF-β signaling intersects with the insulin/IGF-1 pathway for longevity in C. elegans. In phylogenetic analysis, DAF-7, Daw and mammalian Activin-like proteins share common ancestry. Activin signaling, in response to insulin/IGF-1, may thus represent a taxonomically conserved longevity assurance pathway (Bai, 2013).

    Longevity benefits of reduced Activin (TGF-β/dauer) in C. elegans were resolved only when the matricide or 'bagging' (due to progeny hatching within the mother) was prevented by treating daf-7(e1372) mutants with 5-fluorodeoxyuridine (FUdR) to block progeny development. This approach made it possible to distinguish the role of Activin in somatic aging from the previously recognized influence of BMP (Sma/Mab signaling) upon reproductive aging in C. elegans. Activin, of course, is a somatically expressed regulatory hormone of mammalian menstrual cycles that induces follicle-stimulating hormone (FSH) in the pituitary gland. In young females, FSH is suppressed within a cycle when maturing follicles secrete the related TGF-β hormone Inhibin. In mammalian reproductive aging, the effect of Activin in the pituitary becomes unopposed as the stock of primary follicles declines, thus inducing elevated production of FSH. This study now found that reduced Activin but not BMP signaling favors somatic persistence in Drosophila. These parallels between reproductive and somatic aging among invertebrate models and humans suggest that unopposed Activin signaling is pro-aging while favoring reproduction (Bai, 2013).

    Reduced insulin/IGF signaling extends lifespan through interacting autonomous and non-autonomous actions. Reducing IIS in some distal tissues has been shown to slow aging because this reduces insulin secretion from a few neurons: reducing IIS by increasing dFOXO in fat body or muscle extends Drosophila fly lifespan while decreasing IPC production of systemically secreted DILP2. This study has identified Activin as a direct, downstream target of insulin/dFOXO signaling within muscles that has the capacity to non-autonomously regulate lifespan. Knockdown of Activin in muscle but not in fat body is sufficient to prolong lifespan. RNAi for muscle Activin signaling led to decreased circulating DILP2 and increased peripheral insulin signaling. Muscle is thus proposed to produce a signaling factor, a myokine, which impacts organism-wide aging and metabolism (Bai, 2013).

    Aging muscle may produce different myokine-like signals in response to their physiological state. Aged muscles degenerate in many ways including changes in composition, mitochondria, regenerative potential and within-cell protein homeostasis. Protein homeostasis is normally maintained, at least in part, by autophagy. Loss of macroautophagy and chaperone-mediated autophagy with age will accelerate the accumulation of damaged proteins. Expression of Atg8a in Drosophila CNS is reported to extend lifespan by 56% (Simonsen, 2008), while recent studies find elevated autophagy in long-lived mutants including those of the insulin/IGF-1 signaling pathway. The current results show that insulin/IGF signaling can regulate autophagy through its control of Activin via dFOXO. Poly-ubiquitinated proteins accumulate in aging Drosophila while lysosome activity and macroautophagy decline. Muscle performance with age (flight, climbing) was preserved by inactivating Activin within this tissue. This genetic treatment also reduced the accumulation of protein aggregates. These effects are mediated by blocking the transcription factor Smox, which otherwise represses Atg8a. Smox directly regulates Atg8a through its conserved Smad binding motif (AGAC AGAC). These results, however, contrast with an observation where TGF-β1 promotes autophagy in mouse mesangial cells (Bai, 2013).

    Insulin/IGF-1 signaling is a widely conserved longevity assurance pathway. The data indicate that reduced insulin/IGF-1 retards aging at least in part through its FOXO-mediated control of Activin. Furthermore, affecting Activin only in muscle is sufficient to slow its functional decline as well as to extend lifespan. Autophagy within aging muscle controls these outcomes, and it is now found that Activin directly regulates autophagy through Smox-mediated repression of Atg8a. If extrapolated to mammals, pharmaceutical manipulations of Activin may reduce age-dependent muscle pathology associated with impaired autophagy, and potentially increase healthy and total lifespan through beneficial signaling derived from such preserved tissue (Bai, 2013).

    Aging and Autophagic Function Influences the Progressive Decline of Adult Drosophila Behaviors

    Multiple neurological disorders are characterized by the abnormal accumulation of protein aggregates and the progressive impairment of complex behaviors. Drosophila studies demonstrate that middle-aged wild-type flies (WT, ~4-weeks) exhibit a marked accumulation of neural aggregates that is commensurate with the decline of the autophagy pathway. However, enhancing autophagy via neuronal over-expression of Atg8a (Atg8a-OE) reduces the age-dependent accumulation of aggregates. This study assessed basal locomotor activity profiles for single- and group-housed male and female WT flies and observed that only modest behavioral changes occurred by 4-weeks of age, with the noted exception of group-housed male flies. Male flies in same-sex social groups exhibit a progressive increase in nighttime activity. Infrared videos show aged group-housed males (4-weeks) are engaged in extensive bouts of courtship during periods of darkness, which is partly repressed during lighted conditions. Together, these nighttime courtship behaviors were nearly absent in young WT flies and aged Atg8a-OE flies. These results and previous results suggest that middle-aged male flies develop impairments in olfaction, which could contribute to the dysregulation of courtship behaviors during dark time periods. As Drosophila age, they develop early behavior defects that are coordinate with protein aggregate accumulation in the nervous system. In addition, the nighttime activity behavior is preserved when neuronal autophagy is maintained (Atg8a-OE flies). Thus, environmental or genetic factors that modify autophagic capacity could have a positive impact on neuronal aging and complex behaviors (Ratliff, 2015).

    Uba1 functions in Atg7- and Atg3-independent autophagy

    Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. This study describes an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. These data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy (Chang, 2013).

    Macroautophagy (autophagy) is a system that is used to transfer cytoplasmic material, including proteins and organelles, to lysosomes by all eukaryotic cells. Autophagy is augmented during cell stress to reduce damage to enable cell survival, and is also associated with the death of animal cells. Although most studies of this process have focused on stress-induced autophagy, such as nutrient deprivation, autophagy is also a normal aspect of animal development where it is required for proper death and removal of cells and tissues. Defects in autophagy lead to accumulation of protein aggregates and damaged organelles, as well as human disorders. Most of the knowledge about the genes controlling autophagy is based on pioneering studies in the yeast Saccharomyces cerevisiae, and it is not clear whether cells that exist in extremely different contexts within multi-cellular organisms could use alternative factors to regulate this catabolic process (Chang, 2013).

    Atg genes that are conserved from yeast to humans are required for autophagy, and include the Atg1 and Vps34 regulatory complexes, as well as two ubiquitin-like conjugation pathways. The two ubiquitin-like molecules, named Atg8 (LC3 and GABARAP in mammals) and Atg12, become associated with the isolation membranes that form autophagosomes through the activity of the E1 enzyme Atg7. Atg3 functions as the E2-conjugating enzyme for Atg8, and Atg10 functions as the E2 for Atg12. Atg12 associates with Atg5 and Atg16 during the formation of the autophagosome, and Atg8 is conjugated to the lipid phosphatidyl-ethanolamine enabling this protein to associate with the isolation membrane and autophagosome. Lipidated Atg8 remains associated with autophagosomes until fusion with lysosomes to form autolysosomes where cargoes are degraded by lysosomal enzymes (Chang, 2013).

    Degradation of the midgut of the Drosophila melanogaster intestine involves a large change in midgut length, has elevated autophagy and markers of caspases associated with it, requires autophagy, and seems to be caspase independent. This study shows that autophagy is required for programmed reduction in cell size at the onset of intestine cell death in Drosophila. Atg genes encoding components of the Atg1 and Vps34 complexes are required for midgut cell autophagy and reduction in size. Surprisingly, although Atg8a is required for autophagy and programmed cell size reduction, the evolutionarily conserved E1-activating enzyme Atg7 and E2-conjugating enzyme Atg3 are not required for these cellular events. This study screened the E1-activating enzymes encoded by the fly genome and identified Uba1 as being required for autophagy and reduction of cell size during midgut cell death. Although the genes that control autophagy are conserved throughout eukaryotes, the current data provide evidence indicating that the core autophagy machinery may not be identical in all cells within an organism (Chang, 2013).

    Autophagy has been shown to influence cell size during growth factor and nutrient restriction in mammalian cells lines, but this study indicates that autophagy controls cell size as part of a normal developmental program. The discovery that Atg7 and Atg3 are not required for autophagy and cell size reduction in dying midgut cells in Drosophila is surprising. Although an Atg5, Atg7- and LC3-independent autophagy pathway has been reported (Nishida, 2009), this study describes autophagy that requires Atg8 (LC3) and does not require Atg7 and Atg3. It has been assumed that components of the core Atg8 (LC3) and Atg12 conjugation pathways are used by all eukaryotic cells, but this study provides evidence that alternative factors can function to regulate autophagy in a cell-context-specific manner (Chang, 2013).

    This study highlights that autophagy may have different regulatory mechanisms in distinct cell types within an animal. Different forms of autophagy could involve either unique regulatory pathways , different amounts and rates of autophagy or alternative cargo selection mechanisms, and these are not mutually exclusive. Another possibility is that differences in cargo selection alone, perhaps based on specific cargo adaptor proteins, could mediate a distinct type of autophagy (Chang, 2013).

    This paper reports that an E1 enzyme other than Atg7 is required for Atg8 and Atg5 puncta formation, and clearance of ubiquitin-binding protein p62 and mitochondria. The studies indicate that Uba1 fails to function in place of Atg7, as expected on the basis of the unique architecture and use of ubiquitin-like proteins and E2-binding domains in these highly divergent E1 enzymes. Although the possibility cannot be excluded that Atg8a is activated by unknown factors, the simplest model to explain the data positions Uba1 function at a different stage of the autophagy process that depends on ubiquitin conjugation. Previous work in a mammalian cell line indicated that Uba1 is required for protein degradation by lysosomes, but this was not because of decreased autophagosome formation (Lenk, 1992). In addition, recent work in Drosophila implicated the de-ubiquitylation enzyme USP36 in autophagy (Taillebourg, 2012). However, the inability of Atg5 knockdown to suppress the USP36 mutant phenotype, as well as the accumulation of both GFP-Atg8a and ubiquitin-binding protein p62 in USP36 mutant cells, suggests a defect in autophagic flux rather than a defect in the formation of autophagosomes. p62 and other ubiquitin-binding proteins are known to facilitate recruitment of ubiquitylated cargoes into autophagosomes. In addition, p62 was recently shown to accumulate at sites of autophagosome formation even when autophagosome formation is blocked (Itakura, 2011. Thus, it is possible that Uba1 promotes cargo recruitment to the sites of autophagosome formation to facilitate autophagy. However, it is also possible that Uba1 could function at multiple stages in the regulation of autophagy (Chang, 2013).

    It is critical to understand the mechanisms that regulate autophagy given the interest in this catabolic process as a therapeutic target for multiple age-associated disorders, including cancer and neurodegeneration. Significantly, these studies illuminate that autophagy has different regulatory mechanisms in distinct cell types within an animal, and highlight the importance of studying core autophagy genes in specific cell types under physiological conditions (Chang, 2013).

    The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila

    Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers (Singh, 2016).

    To investigate the molecular mechanism behind the resistance of CSCs to therapeutics, the death of stem cells with different degrees of quiescence was studied in the adult Drosophila digestive system, including intestinal stem cells (ISCs). Expression of the proapoptotic genes rpr and p53 effectively ablated differentiated cells but had little effect on stem cells (Singh, 2016).

    In mammals, treatment-resistant leukaemic stem cells (LSCs) can be eliminated by a two-step protocol involving initial activation by interferon-α (IFNα) or colony-stimulating factor (G-CSF), followed by targeted chemotherapy. In Drosophila, activation of the hopscotch (also known as JAK)-Stat92E signalling pathway induces hyperplastic stem cells, which are overproliferating, but retain their apico-basal polarity and differentiation ability. A slightly different two-step protocol was conducted in Drosophila stem cells by overexpressing the JAK-Stat92E pathway ligand unpaired (upd) and rpr together. The induction of upd + rpr using the temperature-sensitive (ts) mutant esg-Gal4 (esgts > upd + rpr effectively ablated all of the ISCs and RNSCs through apoptosis within four days. Consistent with this result, expressing a gain-of-function Raf mutant (Rafgof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

    Expressing a constitutively active form of Ras oncogene at 85D (also known as RasV12) in RNSCs and the knockdown of Notch activity in ISCs can transform these cell types into CSC-like neoplastic stem cells, which were not only overproliferating, but also lost their apico-basal polarity and differentiation abilit. It ws found that expressing rpr in RasV12-transformed RNSCs or in ISCs expressing a dominant-negative form of Notch (NDN) caused the ablation of only a proportion of the transformed RNSCs and few transformed ISCs and it did not affect differentiated cells; substantial populations of the neoplastic stem cells remained even seven days after rpr induction (Singh, 2016).

    These results suggest that the activation of proliferation can accelerate the apoptotic cell death of hyperplastic stem cells, but that a proportion of actively proliferating neoplastic RNSCs and ISCs are resistant to apoptotic cell death. Neoplastic tumours in Drosophila are more similar to high-grade malignant human tumours than are the hyperplastic Drosophila tumours (Singh, 2016).

    Vesicle-mediated COPI and COPII are essential components of the trafficking machinery for vesicle transportation between the endoplasmic reticulum and the Golgi. In addition, the COPI complex regulates the transport of lipolysis enzymes to the surface of lipid droplets for lipid droplet usage. In a previous screen, it was found that knockdown of COPI components (including Arf79F, the Drosophila homologue of ADP-ribosylation factor 1 (Arf1)) rather than COPII components resulted in stem-cell death, suggesting that lipid-droplet usage (lipolysis) rather than the general trafficking machinery between the endoplasmic reticulum and Golgi is important for stem-cell survival (Singh, 2016)

    To further investigate the roles of these genes in stem cells, a recombined double Gal4 line of esg-Gal4 and wg-Gal4 was used to express genes in ISCs, RNSCs, and HISCs (esgts wgts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79FRNAi in enterocytes or in differentiated stellate cells in Malpighian tubules did not cause similar marked ablation. These results suggest that Arf79F knockdown selectively kills stem cells and not differentiated cells (Singh, 2016).

    It was also found that expressing Arf79FRNAi in RasV12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79FRNAi in NDN-transformed ISCs ablated all of the cells within one week, but restored differentiated cells to close to their normal levels within one week (Singh, 2016).

    δ-COP- and γ-COP-mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique, and it was found that the COPI complex cell-autonomously regulated stem cell survival. In summary, knockdown of the COPI-Arf79F complex effectively ablated normal and transformed stem cells but not differentiated enterocytes or stellate cells (Singh, 2016)

    In the RNAi screen acyl-CoA synthetase long-chain (ACSL), an enzyme in the Drosophila lipolysis-β-oxidation pathway, and bubblegum (bgm), a very long-chain fatty acid-CoA ligase, were also identified. RNAi-mediated knockdown of Acsl and bgm effectively killed ISCs and RNSCs, but killed HISCs less effectively. Expressing AcslRNAi in RasV12-transformed RNSCs also ablated almost all of the transformed RNSCs in one week (Singh, 2016).

    Brummer (bmm) is a triglyceride lipase, the Drosophila homologue of mammalian ATGL, the first enzyme in the lipolysis pathway. Scully (scu) is the Drosophila orthologue of hydroxy-acyl-CoA dehydrogenase, an enzyme in the β-oxidation pathway. Hepatocyte nuclear factor 4 (Hnf4) regulates the expression of several genes involved in lipid mobilization and β-oxidation. To determine whether the lipolysis-β-oxidation pathway is required for COPI-Arf79F-mediated stem cell survival, upstream activating sequence (UAS)-regulated constructs (UAS-bmm, UAS-Hnf4, and UAS-scu) were also expressed in stem cells that were depleted of Arf79F, β-COP, or ζ-COP. Overexpressing either scu or Hnf4 significantly attenuated the stem cell death caused by knockdown of the COPI-Arf79F complex. Expressing UAS-Hnf4 MARCM clones also rescued the stem cell death phenotype induced by γ-COP knockdown. However, bmm overexpression did not rescue the stem-cell death induced by Arf79F knockdown. Since there are several other triglyceride lipases in Drosophila in addition to bmm, another lipase may redundantly regulate the lipolysis pathway (Singh, 2016).

    To further investigate the function of lipolysis in stem cells, the expression of a lipolysis reporter (GAL4-dHFN4; UAS-nlacZ which consisted of hsp70-GAL4-dHNF4 combined with a UAS-nlacZ reporter gene was investigated. The flies were either cultured continuously at 29°C or heat-shocked for 30 min at 37°C, 12 h before dissection. Without heat shock, the reporter was expressed only in ISCs and RNSCs of mature adult flies, but not in enteroendocrine cells, enterocytes, quiescent HISCs or quiescent ISCs of freshly emerged young adult flies (less than 3 days old. Expressing δ-COPRNAi almost completely eliminated the reporter expression, suggesting that the reporter was specifically regulated by the COPI complex. After heat shock or when a constitutively active form of JAK (hopTum-l) was expressed, the reporter was strongly expressed in ISCs, RNSCs and HISCs, but not in enteroendocrine cells or enterocytes. These data suggest that COPI-complex-regulated lipolysis was active in stem cells, but not in differentiated cells, and that the absence of the reporter expression in quiescent HISCs at 29°C was probably owing to weak hsp70 promoter activity rather than to low lipolysis in these cells (Singh, 2006).

    Lipid storage was futher investigated, and it was found that the size and number of lipid droplets were markedly increased in stem cells after knockdown of Arf79F (Singh, 2016).

    Arf1 inhibitors (brefeldin A, golgicide A, secin H3, LM11 and LG8) and fatty-acid-oxidation (FAO) inhibitors (triacsin C, mildronate, etomoxir and enoximone) were used, and it was found that these inhibitors markedly reduced stem-cell tumours in Drosophila through the lipolysis pathway but had a negligible effect on normal stem cells (Singh, 2016).

    These data together suggest that the COPI-Arf1 complex regulates stem-cell survival through the lipolysis-β-oxidation pathway, and that knockdown of these genes blocks lipolysis but promotes lipid storage. Further, the transformed stem cells are more sensitive to Arf1 inhibitors and may be selectively eliminated by controlling the concentration of Arf1 inhibitors (Singh, 2016).

    These data suggest that neither caspase-mediated apoptosis nor autophagy-regulated cell death regulates the stem-cell death induced by the knockdown of components of the COPI-Arf79F complex. Therefore whether necrosis regulates the stem-cell death induced by knockdown of the COPI-Arf79F complex was investigated. Necrosis is characterized by early plasma membrane rupture, reactive oxygen species (ROS) accumulation and intracellular acidification. Propidium iodide detects necrotic cells with compromised membrane integrity, the oxidant-sensitive dye dihydroethidium (DHE) indicates cellular ROS levels and LysoTracker staining detects intracellular acidification. The membrane rupture phenotype was detected only in esg and the propidium iodide signal was observed only in ISCs from flies that had RNAi-induced knockdown of expression of COPI-Arf79F components, and not in cells from wild-type flies. In the esgts wgts > AcslRNAi flies, all of the ISCs and RNSCs were ablated after four days at 29°C, but a fraction of the HISCs remained, and these were also propidium iodide positive, indicating that the HISCs were dying slowly. This slowness may have been due to either a lower GAL4 (wg-Gal4) activity in these cells compared to ISCs and RNSCs (esg-Gal4) or quiescence of the HISCs. Furthermore, strong propidium iodide signals were detected in transformed ISCs from esgts > NDN + Arf79FRNAi but not esgts flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016).

    Similarly, DHE signals were detected only in ISCs from esgts > Arf79FRNAi flies, indicating that the dying ISCs had accumulated ROS and were intracellularly acidified. Overexpressing catalase (a ROS-chelating enzyme) rescued the stem-cell death specifically induced by the γ-COP mutant clone, and the ROS inhibitor NAC blocked the Arf1 inhibitor-induced death of RasV12-induced RNSC tumours. These data together suggest that knockdown of the COPI-Arf1 complex induced the death of stem cells or of transformed stem cells (RasV12-RNSCs, NDN-ISCs) through ROS-induced necrosis. Although ISCs, RNSCs, and HISCs exhibit different degrees of quiescence, they all rely on lipolysis for survival, suggesting that this is a general property of stem cells (Singh, 2016).

    Cases were noticed where the GFP-positive material of the dying ISCs was present within neighbouring enterocytes, suggesting that these enterocytes had engulfed dying ISCs (Singh, 2016).

    The JNK pathway, autophagy and engulfment genes are involved in the engulfment of dying cells. Therefore, whether these genes are required for COPI-Arf79F-regulated ISC death was investigated. The following was found: (1) ISC death activated JNK signalling and autophagy in neighbouring enterocytes; (2) knockdown of these genes in enterocytes but not in ISCs rescued ISC death to different degrees; (3) the drpr-mbc-Rac1-JNK pathway in enterocytes is not only necessary but also sufficient for ISC death; and (4) inhibitors of JNK and Rac1 could block Arf1-inhibitor-induced cell death of the RasV12-induced RNSC tumours. These data together suggest that the drpr-mbc-Rac1-JNK pathway in neighbouring differentiated cells controls the engulfment of dying or transformed stem cells (Singh, 2016).

    The finding that the COPI-Arf79F-lipolysis-β-oxidation pathway regulated transformed stem-cell survival in the fly led to an investigation of whether the pathway has a similar role in CSCs. WTwo Arf1 inhibitors (brefeldin A and golgicide A) and two FAO inhibitors (triascin C and etomoxir) were tested on human cancer cell lines, and it was found that the growth, tumoursphere formation and expression of tumour-initiating cell markers of the four cancer cell lines were significantly suppressed by these inhibitors, suggesting that these inhibitors suppress CSCs. In mouse xenografts of BSY-1 human breast cancer cells, a novel low-cytotoxicity Arf1-ArfGEF inhibitor called AMF-26 was reported to induce complete regression in vivo in five days. Together, this report and the current results suggest that inhibiting Arf1 activity or blocking the lipolysis pathway can kill CSCs and block tumour growth (Singh, 2016).

    Stem cells or CSCs are usually localized to a hypoxic storage niche, surrounded by a dense extracellular matrix, which may make them less accessible to sugar and amino acid nutrition from the body's circulatory system. Most normal cells rely on sugar and amino acids for their energy supply, with lipolysis playing only a minor role in their survival. The current results suggest that stem cells and CSCs are metabolically unique; they rely mainly on lipid reserves for their energy supply, and blocking COPI-Arf1-mediated lipolysis can starve them to death. It was further found that transformed stem cells were more sensitive than normal stem cells to Arf1 inhibitors. Thus, selectively blocking lipolysis may kill CSCs without severe side effects. Therefore, targeting the COPI-Arf1 complex or the lipolysis pathway may prove to be a well-tolerated, novel approach for eliminating CSCs (Singh, 2016).

    Retromer ensures the degradation of autophagic cargo via maintaining lysosome function in Drosophila

    The retromer is an evolutionarily conserved coat complex that consists of Vps26, Vps29, Vps35 and a heterodimer of sorting nexin (Snx) protein in yeast. Retromer mediates the recycling of transmembrane proteins from endosomes to the trans-Golgi network, including receptors that are essential for the delivery of hydrolytic enzymes to lysosomes. Besides its function in lysosomal enzyme receptor recycling, involvement of retromer has also been proposed in a variety of vesicular trafficking events, including early steps of autophagy and endocytosis. This study shows that the late stages of autophagy and endocytosis are impaired in Vps26 and Vps35 deficient Drosophila larval fat body cells, but formation of autophagosomes and endosomes is not compromised. Accumulation of aberrant autolysosomes and amphisomes in the absence of retromer function appears to be the consequence of decreased degradative capacity, as they contain undigested cytoplasmic material. Accordingly, it was shown that retromer is required for proper cathepsin L trafficking mainly independent of LERP, the Drosophila homolog of the cation-independent mannose 6-phosphate receptor. Finally, it was found that Snx3 and Snx6 are also required for proper autolysosomal degradation in Drosophila larval fat body cells (Maruzs, 2015).

    UTX coordinates steroid hormone-mediated autophagy and cell death

    Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. This study shows that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. It was further shown that salivary gland degradation requires dUTX catalytic activity. These findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development (Denton, 2013).

    UTX function is known to be critical in mammalian embryonic development and somatic and germ cell reprogramming. This study found a novel role for dUTX in steroid hormone-mediated cell death during development. dUTX, together with nuclear hormone receptor EcR/Usp, is capable of regulating gene expression both spatially and temporally in a hormone-dependent manner. UTX gene mutations are frequently observed in malignancies including lethal castration-resistant prostate cancer, although a role for UTX in androgen receptor-mediated transcription has not yet been identified. This study indicates that UTX is a good candidate to extend the investigation to examine the role of UTX in coordinating nuclear hormone receptor-regulated gene expression, particularly in androgen receptor-mediated transcription during mammalian development and hormone-dependent cancers (Denton, 2013).

    The complete degradation of larval salivary glands during metamorphosis utilizes both apoptosis and autophagy and by coordinately controlling the expression of critical genes in these two distinct biological pathways, dUTX ensures timely removal of salivary glands in response to temporal ecdysone pulse. The majority of studies addressing induction of autophagy have focused upon autophagosome formation and protein degradation. The transcriptional regulation of autophagy induction remains poorly understood. Indeed, several Atg genes are transcriptionally upregulated following autophagy induction; however, the molecular pathways are only beginning to be revealed. For example, the master gene controlling lysosomal biogenesis, transcription factor EB, coordinates the expression of both autophagy and lysosomal genes to induce autophagy in response to starvation. Induction of autophagy has been linked to reduced histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF. Downregulation of H4K16 deacetylation was associated with the downregulation of several Atg genes, whereas antagonizing H4K16ac downregulation upon autophagy induction resulted in cell death. The study indicates that a specific histone modification during autophagy modulates the expression of Atg genes, and is important for survival versus death responses upon autophagy induction. This work now describes dUTX as another regulator of autophagy and cell death in the context of developmental PCD and in concert with the steroid hormone response. Future studies to understand the complex nuclear events regulating both repression and induction of autophagy gene expression in response to particular signals will be important (Denton, 2013).

    Despite the opposing roles of H3K27 and H3K4 methylation in transcriptional regulation, UTX has been identified in association with H3K4 methyltransferase and to play demethylase-independent functions. This study suggests that the demethylase activity of dUTX is necessary for hormone-mediated cell death. The nuclear hormone receptor response to ecdysone initiates a hierarchical transcription cascade by induction of transcription factors, including BR-C, E74 and E93. These transcription factors drive expression of downstream genes including cell death genes. The data show that dUTX regulates E93 and suggests that this HDM can regulate cell death both directly, through the transcription of apoptosis and autophagy genes through direct recruitment via EcR/Usp, as well as indirectly through key transcription factor E93. This additional level of regulation through the stage-specific transcription factor E93 may provide temporal control of ecdysone response during metamorphosis (Denton, 2013).

    The role of autophagy in cell death is a matter of considerable debate as autophagy is generally a cell survival mechanism in response to cellular stress and nutrient limitations. Studies in Drosophila have provided perhaps some of the strongest evidence for a role of autophagy in developmental cell death in vivo. The data presented in this paper demonstrating coordinate regulation of both key apoptosis and autophagy genes by a single histone-modifying enzyme further provide genetic and molecular evidence linking autophagy and apoptosis in PCD during metamorphosis (Denton, 2013).

    Hox proteins mediate developmental and environmental control of autophagy

    Hox genes encode evolutionarily conserved transcription factors, providing positional information used for differential morphogenesis along the anteroposterior axis. This study shows that Drosophila Hox proteins are potent repressors of the autophagic process. In inhibiting autophagy, Hox proteins display no apparent paralog specificity and do not provide positional information. Instead, they impose temporality on developmental autophagy and act as effectors of environmental signals in starvation-induced autophagy. Further characterization establishes that temporality is controlled by Pontin, a facultative component of the Brahma chromatin remodeling complex, and that Hox proteins impact on autophagy by repressing the expression of core components of the autophagy machinery. Finally, the potential of central and posterior mouse Hox proteins to inhibit autophagy in Drosophila and in vertebrate COS-7 cells indicates that regulation of autophagy is an evolutionary conserved feature of Hox proteins (Banreti, 2013).

    Autophagy is a cellular process whose induction or inhibition involves multiple levels of regulation, including developmental signals conveyed by the steroid hormone ecdysone, and environmental signals, sensed in the case of amino acid starvation by the InR/dTOR pathways. These regulatory paths do not act independently but seem rather to be interconnected as illustrated by developmentally induced ecdysone-mediated autophagy that acts by repressing the inhibitory function of the InR pathway. This indicates that whereas upstream control is distinct, downstream control may be common (Banreti, 2013).

    This study shows that Drosophila Hox proteins are potent inhibitors of autophagy, with a potent and equivalent impact on both developmental and starvation-induced autophagy, and establish that both converge in the regulation of Hox gene expression. This highlights Hox genes as central regulators of autophagy, acting as a node for mediating autophagy inhibition. In regulating autophagy, Hox proteins act at least through regulation of Atg genes and other autophagy genes. Consistent with a direct transcriptional effect of Hox proteins in controlling Atg genes, Ubx DNA binding was found to be essential for autophagy inhibition, whereas have previously shown that Ubx associates to genomic regions immediately adjacent to Atg5 and Atg7 genes (Banreti, 2013).

    A key aspect underlying Hox-mediated autophagy control is the regulation of Hox gene expression, where Hox downregulation induces autophagy. This aspect is true for both developmental- and starvation-induced autophagy, where the dynamics of Hox proteins respond to ecdysone (developmental autophagy) and to InR/dTOR (starvation) signaling. Signals mediating changes in Hox gene expression result from changes in the expression of Pont, a facultative component of the Brm complex known to act as a global and positive regulator of Hox genes. Although not establishing changes in Brm complex composition at the L3 feeding/L3 wandering transition, the dynamics of Pont expression suggest that a Pont-depleted Brm complex loses its ability to maintain the expression of Hox genes, resulting in the release of Hox-mediated inhibition of autophagy (Banreti, 2013).

    Hox proteins are widely described as providing spatial information required for differential morphogenesis along the A-P axis, within which they largely display paralog-specific activities. However, in regulating autophagy, Hox function is distinct. First, it appears to be generic, with all Hox proteins tested providing inhibitory activity. The need to alleviate global Hox gene function (achieved in this study by impairing the activity of the Brm complex) in order to induce autophagy, further supports their redundant function in inhibiting autophagy. Second, they provide temporal, instead of spatial, information, mediating the temporality of developmental autophagy downstream of ecdysone signaling. Third, in the case of starvation-induced autophagy, Hox genes respond to the InR/dTOR pathways, acting as environmental effectors (Banreti, 2013).

    Investigating the evolutionary conservation of Hox-mediated inhibition of autophagy by exploring the activity of mouse Hox proteins in Drosophila fat body cells as well as in vertebrate COS-7 cells indicates that vertebrate Hox proteins also act as potent autophagy inhibitors. Further studies in vertebrate cells should frame their activity to the multiple physiological and pathological situations that involve autophagy and allow for deciphering the molecular modalities of their regulatory roles (Banreti, 2013).

    In summary, these findings broaden the framework of Hox protein functions, showing that besides providing spatial information during development, they also coordinate temporal processes and, more surprisingly, act as mediators of environmental signals for autophagy regulation (Banreti, 2013).

    Myc-driven overgrowth requires unfolded protein response-mediated induction of autophagy and antioxidant responses in Drosophila melanogaster

    Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. This study shows that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. These data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc (Nagy, 2013).

    Earlier genetic studies have established that Myc is required for proper expression of hundreds of housekeeping genes and is therefore essential for cell growth and proliferation. Myc is a typical example of a nuclear oncogene: a transcription factor that drives tumor progression if its expression is deregulated in mammalian cells. Its mechanisms of promoting cell growth are likely different in many ways from that of cytoplasmic oncogenes such as kinases encoded by PI3K and AKT genes, also frequently activated in various cancers. Overexpression of these drives cell growth in Drosophila as well, but Myc also increases the nuclear:cytoplasmic ratio in hypertrophic cells, unlike activation of PI3K/AKT signaling. PI3K and AKT suppress basal and starvation-induced autophagy, while their inactivation strongly upregulates this process. In contrast, this study shows that both basal and starvation-induced autophagy requires Myc, and that overexpression of Myc increases UPR, leading to PERK-dependent induction of autophagy, and presumably to accumulation of cytoplasmic p62 that activates antioxidant responses. Autophagy deficiency predisposes to cancer at least in part through accumulation of the selective autophagy cargo p62, resulting in activation of antioxidant responses and tumor formation. These analyses show that both of these cytoprotective pathways can be activated simultaneously, and are required in parallel to sustain Myc-induced overgrowth in Drosophila cells (Nagy, 2013).

    Autophagy and antioxidant responses have been considered to act as tumor suppressor pathways in normal cells and during early stages of tumorigenesis, while activation of these processes may also confer advantages for cancer cells. Lack of proper vasculature in solid tumors causes hypoxia and nutrient limitation. These stresses in the tumor microenvironment have been suggested to elevate UPR and autophagy to promote survival of cancer cells. This study demonstrates that genetic alterations similar to those observed in cancer cells (that is, deregulated expression of Myc) can also activate the UPR, autophagy and antioxidant pathways in a cell-autonomous manner in Drosophila. These processes are likely also activated as a consequence of deregulated Myc expression in human cancer cells based on a number of recent reports, similar to the findings in Drosophila presented in this study. First, chloroquine treatment that impairs all lysosomal degradation pathways is sufficient to reduce tumor volume in Myc-dependent lymphoma models. Second, ER stress and autophagy induced by transient Myc expression increase survival of cultured cells, and PERK-dependent autophagy is necessary for tumor formation in a mouse model. Data suggest that UPR-mediated autophagy and antioxidant responses may also be necessary to sustain the increased cellular growth rate driven by deregulated expression of Myc (Nagy, 2013).

    Myc has proven difficult to target by drugs. Myc-driven cancer cell growth could also be selectively prevented by blocking cellular processes that are required in cancer cells but dispensable in normal cells, known as the largely unexplored non-oncogene addiction pathways. Previous genetic studies establish that autophagy is dispensable for the growth and development of mice, although knockout animals die soon after birth due to neonatal starvation after cessation of placental nutrition. Tissue-specific Atg knockout mice survive and the animals are viable, with potential adverse effects only observed in aging animals. Genetic deficiencies linked to p62 are also implicated in certain diseases, but knockout mice grow and develop normally and are viable. Similarly, Nrf2 knockout mice are viable and adults exhibit no gross abnormalities, while these animals are hypersensitive to oxidants. Mice lacking PERK also develop normally and are viable. All these knockout studies demonstrate that these genes are largely dispensable for normal growth and development of mice, and that progressive development of certain diseases is only observed later during the life of these mutant animals. There are currently no data regarding the effects of transient inhibition of these processes, with the exception of the non-specific lysosomal degradation inhibitor chloroquine, originally approved for the treatment of malaria, which is already used in the clinic for certain types of cancer (Nagy, 2013).

    Based on these knockout mouse data, UPR, autophagy and antioxidant responses may be considered as potential non-oncogene addiction pathways: strictly required for Myc-dependent overgrowth (this study) and tumor formation, but dispensable for the growth and viability of normal cells, both in Drosophila and mammals. One can speculate that the transient inactivation of these pathways will have even more subtle effects than those observed in knockout mice, but this needs experimental testing. While it is difficult to extrapolate data obtained in Drosophila (or even mouse) studies to human patients, it is tempting to speculate that specific drugs targeting UPR, autophagy and antioxidant responses may prove effective against Myc-dependent human cancers, perhaps without causing adverse side-effects such as current, less specific therapeutic approaches. Notably, widely used anticancer chemotherapy treatments are known to greatly increase the risk that cancer survivors will develop secondary malignancies. Moreover, the autophagy and antioxidant pathways appear to be required in parallel during Myc-induced overgrowth in Drosophila cells. If a similar genetic relationship exists in Myc-dependent human cancer cells, then increased efficacy may be predicted for the combined block of key enzymes acting in these processes (Nagy, 2013).

    Elucidation of the genetic alterations behind increased UPR, autophagy and antioxidant responses observed in many established human cancer cells may allow specific targeting of these pathways, and potentially have a tremendous benefit for personalized therapies. In addition to non-specific autophagy inhibitors such as chloroquine, new and more specific inhibitors of selected Atg proteins are being developed. Given the dual roles of autophagy during cancer initiation and progression, a major question is how to identify patients who would likely benefit from taking these drugs. For example, no single test can reliably estimate autophagy levels in clinical samples, as increases in autophagosome generation or decreases in autophagosome maturation and autolysosome breakdown both result in accumulation of autophagic structures. Based on this study's data and recent mammalian reports, elevated Myc levels may even turn out to be useful as a biomarker before therapeutic application of inhibitors for key autophagy, UPR or antioxidant proteins in cancer patients (Nagy, 2013).

    The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB

    Increasing evidence reveals that a subset of proteins participates in both the autophagy and apoptosis pathways, and this intersection is important in normal physiological contexts and in pathological settings. This shows that the Drosophila effector caspase, Drosophila caspase 1 (Dcp-1), localizes within mitochondria and regulates mitochondrial morphology and autophagic flux. Loss of Dcp-1 leads to mitochondrial elongation, increased levels of the mitochondrial adenine nucleotide translocase stress-sensitive B (SesB), increased adenosine triphosphate (ATP), and a reduction in autophagic flux. Moreover, SesB was found to suppresses autophagic flux during midoogenesis, identifying a novel negative regulator of autophagy. Reduced SesB activity or depletion of ATP by oligomycin A rescues the autophagic defect in Dcp-1 loss-of-function flies, demonstrating that Dcp-1 promotes autophagy by negatively regulating SesB and ATP levels. Furthermore, it was found that pro-Dcp-1 interacts with SesB in a nonproteolytic manner to regulate its stability. These data reveal a new mitochondrial-associated molecular link between nonapoptotic caspase function and autophagy regulation in vivo (DeVorkin, 2014).

    The results reveal that starvation-induced autophagic flux occurs in both midstage egg chambers that have not entered the degeneration process as well as in those that are undergoing cell death. Furthermore, it was found that the effector caspase Dcp-1 is required for autophagic flux in degenerating midstage egg chambers in addition to its role in cell death. One mechanism of Dcp-1-induced autophagic flux is mediated through SesB. In humans, there are four mitochondrial ANT isoforms, each with a tissue-specific distribution and different roles in apoptosis. Adenine nucleotide translocase family ANT1 and ANT3 were proposed to be proapoptotic, whereas ANT2 and ANT4 were shown to be antiapoptotic (Brenner, 2011). However, the roles of mammalian ANT proteins in autophagy have yet to be characterized. The data show that reduced Dcp-1 leads to increased levels of SesB protein in fed and starvation conditions during Drosophila oogenesis and in Drosophila cultured cells. No significant change was observed in SesB transcript levels in fed conditions or after 4 h of starvation, but a significant increase was observed in cells after 2 h of starvation. This finding suggests that a transcription-related mechanism may play some role in the observed cellular response but is not sufficient to account for all of the observed changes in protein levels. Although Dcp-1 does not cleave SesB, the proform of Dcp-1 interacts with SesB, and it is predicted that this interaction regulates the stability of SesB. It was also found that SesB is required to suppress autophagic flux during midoogenesis even under nutrient-rich conditions, and reduction of SesB in Dcp-1Prev1 flies rescues the autophagic defect after starvation. This is the first study showing that an ANT functions as a negative regulator of autophagy (DeVorkin, 2014).

    The Drosophila genome encodes seven caspases, and to date, only the initiator caspase Dronc and the effector caspase Drice have been shown to localize to the mitochondria (Dorstyn, 2002). In mammalian cells, caspases have been detected at the mitochondria during apoptosis; however, the role of caspases at the mitochondria, especially under nonapoptotic conditions, is poorly understood. The current results demonstrate that Dcp-1 localizes to the mitochondria where it functions to maintain the mitochondrial network morphology. Under nutrient-rich conditions, nondegenerating midstage egg chambers from Dcp-1Prev1 flies contained mitochondria that appeared elongated and overly connected, and ovaries contained increased ATP levels, indicating that Dcp-1 normally functions to negatively regulate mitochondrial dynamics and ATP levels. Consistent with these findings, overexpression of the caspase inhibitor p35 in the amnioserosa suppressed the transition of mitochondria from a tubular to a fragmented state during delamination, further suggesting that inhibition of caspases hinders normal mitochondrial dynamics (DeVorkin, 2014).

    Dcp-1 acts to finely tune the apoptotic process, and cell death only occurs when caspase activity reaches a certain apoptotic threshold. Effector caspases involved in nonapoptotic processes may be restricted in time or space to regulate caspase activity. As Dcp-1 functions not only in autophagy and apoptosis but also at the mitochondria to regulate mitochondrial morphology and ATP levels, one question that remains is to how the activity of Dcp-1 is regulated. As Dcp-1 has autocatalytic activity, perhaps Dcp-1 is sequestered in mitochondria to prevent its full activation. Mitochondrial localized mammalian pro-Caspase 3 and 9 are S-nitrosylated in their catalytic active site, leading to the inhibition of their activity. Perhaps mitochondrial Dcp-1 is also S-nitrosylated, serving to limit Dcp-1's activity. In addition, mammalian Hsp60 and Hsp10 were shown to interact with mitochondrial localized pro-Caspase 3 in which they function to accelerate pro-Caspase 3 activation after the induction of apoptosis. Perhaps Dcp-1 associates with Drosophila Hsp60 or Hsp10 in the mitochondria to regulate its mitochondrial related functions. However, further studies are required to identify upstream regulators of Dcp-1 that regulate its mitochondrial, autophagic, and apoptotic functions (DeVorkin, 2014).

    Effector caspases are the main executioners of apoptotic cell death; however, it is becoming increasingly evident that caspases have nonapoptotic functions in differentiation, proliferation, cytokine production, and cell survival. For example, Caspase 3 was shown to regulate tumor cell repopulation in vitro and in vivo, and it was also shown to be required for skeletal muscle and macrophage differentiation. In Drosophila, the initiator caspase Dronc maintains neural stem cell homeostasis by binding to Numb in a noncatalytic, nonapoptotic manner to regulate its activity (Ouyang, 2011). In addition, Dcp-1 is required for neuromuscular degeneration in a nonapoptotic manner (Keller, 2011). The current results show that Dcp-1 also has a nonapoptotic role during oogenesis, in which it is required to maintain mitochondrial physiology under basal conditions. Loss of Dcp-1 alters this physiology, leading to increased SesB and ATP levels that in part prevent the induction of autophagic flux after starvation. These data support the notion that caspases play a much more diverse role than previously known and that the underlying mechanisms should be better understood to appreciate the full impact of apoptosis pathway modulation for treatment in human pathologies (DeVorkin, 2004).

    An ancient defense system eliminates unfit cells from developing tissues during cell competition

    Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. This study reports that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. Toll-related receptors (TRRs) and the cytokine Spatzle (Spz) lead to NFκB-dependent apoptosis. Null mutations in Toll-3, Toll-8, or Toll-9 suppress elimination of loser cells, increasing loser clone size and cell number per clone, but do not alter control clones. Diverse 'loser' cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. These findings demonstrate a functional repurposing of components of TRRs and NFkappaB signaling modules in the surveillance of cell fitness during development (Meyer, 2014).

    Altogether, these results demonstrate that the conceptual resemblance between cell competition and innate immunity is matched with genetic and mechanistic similarities. Thus, cells within developing tissues that are recognized as mutant or compromised are competitively eliminated via a TRR- and NFκB-dependent signaling mechanism. Although similar core signaling components are activated in both processes, cell competition culminates in local expression of proapoptotic genes rather than systemic induction of antimicrobial genes. Because cell competition is initiated by the emergence of cells of different fitness than their neighbors in a tissue, it is surmised that the initiating signal is common to many competitive contexts. The genetic data leads to a proposal of a model for how this signal is detected and transduced. The results point to a role for Spz in signal detection, as it is a secreted protein that is required for the killing activity of competitive conditioned medium (cCM), is a known ligand for the Toll receptor, and is produced by several tissues in the larva. Thus, it is speculated that Spz functions as a ligand for one or more TRR in cell competition. Because Spz must be activated through a series of proteolytic steps, the relevant proteases may respond directly to the initiating signal in cell competition. It is proposed that the genetic identity or context of the competing populations influences activation of different TRR signaling modules and that the precise configuration of TRRs on loser cells dictates which of the three Drosophila NFκB proteins is activated. How signaling to the NFκBs is restricted to the loser cells is not known, but higher expression of Toll-2, Toll-8, and Toll-9 in loser cells could bias signal transduction. PGRP-LC, a receptor known to bind only bacterial products, also plays a role in Myc-induced competition. As commensal gut microflora is known to influence larval growth, this raises the possibility that it also contributes to the competitive phenotype (Meyer, 2014).

    Throughout evolution, signaling modules have adapted to fulfill different functions even within the same species. This study has provided evidence for adaptation of TRR-NFκB signaling modules in an organismal surveillance system that measures internal tissue fitness rather than external stimuli. It is noteworthy that the killing of WT cells by supercompetitor cells is a potentially pathological form of cell competition that could propel expansion of premalignant tumor cells. If so, activated TRR-NFκB signaling modules in nonimmune tissues could be diagnostic markers, and their competitive functions could serve as therapeutic targets for cancer prevention (Meyer, 2014).

    Elimination of unfit cells maintains tissue health and prolongs lifespan

    Viable yet damaged cells can accumulate during development and aging. Although eliminating those cells may benefit organ function, identification of this less fit cell population remains challenging. Previously, a molecular mechanism, based on 'fitness fingerprints' displayed on cell membranes, was identifed that allows direct fitness comparison among cells in Drosophila. This study reports the physiological consequences of efficient cell selection for the whole organism. The study found that fitness-based cell culling is naturally used to maintain tissue health, delay aging, and extend lifespan in Drosophila. A gene, ahuizotl (azot), was identified that ensures the elimination of less fit cells. Lack of azot increases morphological malformations and susceptibility to random mutations and accelerates tissue degeneration. On the contrary, improving the efficiency of cell selection is beneficial for tissue health and extends lifespan (Merino, 2015).

    Individual cells can suffer insults that affect their normal functioning, a situation often aggravated by exposure to external damaging agents. A fraction of damaged cells will critically lose their ability to live, but a different subset of cells may be more difficult to identify and eliminate: viable but suboptimal cells that, if unnoticed, may adversely affect the whole organism (Merino, 2015).

    What is the evidence that viable but damaged cells accumulate within tissues? The somatic mutation theory of aging proposes that over time cells suffer insults that affect their fitness, for example, diminishing their proliferation and growth rates, or forming deficient structures and connections. This creates increasingly heterogeneous and dysfunctional cell populations disturbing tissue and organ function. Once organ function falls below a critical threshold, the individual dies. The theory is supported by the experimental finding that clonal mosaicism occurs at unexpectedly high frequency in human tissues as a function of time, not only in adults an embryos (Merino, 2015).

    Does the high prevalence of mosaicism in our tissues mean that it is impossible to recognize and eliminate cells with subtle mutations and that suboptimal cells are bound to accumulate within organs? Or, on the contrary, can animal bodies identify and get rid of unfit viable cells (Merino, 2015)?

    One indirect mode through which suboptimal cells could be eliminated is proposed by the 'trophic theory,' which suggested that Darwinian-like competition among cells for limiting amounts of surv ead to removal of less fit cells. However, it is apparent from recent work that trophic theories are not sufficient to explain fitness-based cell selection, because there are direct mechanisms that allow cells to exchange 'cell-fitness' information at the local multicellular level (Merino, 2015).

    In Drosophila, cells can compare their fitness using different isoforms of the transmembrane protein Flower. The 'fitness fingerprints' are therefore defined as combinations of Flower isoforms present at the cell membrane that reveal optimal or reduced fitness. The isoforms that indicate reduced fitness have been called FlowerLose isoforms, because they are expressed in cells marked to be eliminated by apoptosis called 'Loser cells.' However, the presence of FlowerLose isoforms at the cell membrane of a particular cell does not imply that the cell will be culled, because at least two other parameters are taken into account: (1) the levels of FlowerLose isoforms in neighboring cells: if neighboring cells have similar levels of Lose isoforms, no cell will be killed; (2) the levels of a secreted protein called Sparc, the homolog of the Sparc/Osteonectin protein family, which counteracts the action of the Lose isoforms (Merino, 2015 and references therein).

    Remarkably, the levels of Flower isoforms and Sparc can be altered by various insults in several cell types, including: (1) the appearance of slowly proliferating cells due to partial loss of ribosomal proteins, a phenomenon known as cell competition; (2) the interaction between cells with slightly higher levels of d-Myc and normal cells, a process termed supercompetition; (3) mutations in signal transduction pathways like Dpp signaling; or (4) viable neurons forming part of incomplete ommatidia. Intriguingly, the role of Flower isoforms is cell type specific, because certain isoforms acting as Lose marks in epithelial cells are part of the fitness fingerprint of healthy neurons. Therefore, an exciting picture starts to appear, in which varying levels of Sparc and different isoforms of Flower are produced by many cell types, acting as direct molecular determinants of cell fitness. This study aimed to clarify how cells integrate fitness information in order to identify and eliminate suboptimal cells. Subsequently, the physiological consequences were analyzed of efficient cell selection for the whole organism (Merino, 2015).

    In order to discover the molecular mechanisms underlying cell selection in Drosophila, this study analyzed genes transcriptionally induced using an assay where WT cells (tub>Gal4) are outcompeted by dMyc-overexpressing supercompetitors (tub>dmyc) due to the increased fitness of these dMyc-overexpressing cells. The expression of CG11165 was strongly induced 24 hr after the peak of flower and sparc expression. In situ hybridization revealed that CG11165 mRNA was specifically detected in Loser cells that were going to be eliminated from wing imaginal discs due to cell competition. The gene, which was named ahuizotl (azot) after a multihanded Aztec creature selectively targeting fishing boats to protect lakes, consists of one exon. azot's single exon encodes for a four EF-hand-containing cytoplasmic protein of the canonical family that is conserved, but uncharacterized, in multicellular animals (Merino, 2015).

    To monitor Azot expression, a translational reporter was designed resulting in the expression of Azot::dsRed under the control of the endogenous azot promoter in transgenic flies. Azot expression was not detectable in most wing imaginal discs under physiological conditions in the absence of competition. Mosaic tissue was generated of two clonal populations, which are known to trigger competitive interactions resulting in elimination of otherwise viable cells. Cells with lower fitness were created by confronting WT cells with dMyc-overexpressing cells, by downregulating Dpp signaling, by overexpressing FlowerLose isoforms, in cells with reduced Wg signaling, by suppressing Jak-Stat signaling or by generating Minute clones. Azot expression was not detectable in nonmosaic tissue of identical genotype, nor in control clones overexpressing UASlacZ. On the contrary, Azot was specifically activated in all tested scenarios of cell competition, specifically in the cells undergoing negative selection. Azot expression was not repressed by the caspase inhibitor protein P35 (Merino, 2015).

    Because Flower proteins are conserved in mammals, tests were made to see if they are also able to regulate azot. Mouse Flower isoform 3 (mFlower3) has been shown to act as a 'classical' Lose isoform, driving cell elimination when expressed in scattered groups of cells, a situation where azot was induced in Loser cells but is not inducing cell selection when expressed ubiquitously a scenario where azot was not expressed. This shows that the mouse FlowerLose isoforms function in Drosophila similarly to their fly homologs (Merino, 2015).

    Interestingly, azot is not a general apoptosis-activated gene because its expression is not induced upon eiger, hid, or bax activation, which trigger cell death. Azot was also not expressed during elimination of cells with defects in apicobasal polarity or undergoing epithelial exclusion-mediated apoptosis (dCsk) (Merino, 2015).

    azot expression was analyzed during the elimination of peripheral photoreceptors in the pupal retina, a process mediated by Flower-encoded fitness fingerprints. Thirty-six to 38hr after pupal formation (APF), when FlowerLose-B expression begins in peripheral neurons, no Azot expression was detected in the peripheral edge. At later time points (40 and 44hr APF), Azot expression is visible and restricted to the peripheral edge where photoreceptor neurons are eliminated. This expression was confirmed with another reporter line, azot{KO; gfp}, where gfp was directly inserted at the azot locus using genomic engineering techniques (Merino, 2015).

    From these results, it is concluded that Azot expression is activated in several contexts where suboptimal and viable cells are normally recognized and eliminated (Merino, 2015).

    To understand Azot function in cell elimination, azot knockout (KO) flies were generated by deleting the entire azot gene. Next, Azot function was analyzed using dmyc-induced competition. In the absence of Azot function, loser cells were no longer eliminated, showing a dramatic 100-fold increase in the number of surviving clones. Loser cells occupied more than 20% of the tissue 72hr after clone induction (ACI). Moreover, using azot{KO; gfp} homozygous flies (that express GFP under the azot promoter but lack Azot protein), it was found that loser cells survived and showed accumulation of GFP. From these results, it is concluded that azot is expressed by loser cells and is essential for their elimination.

    In addition, clone removal was delayed in an azot heterozygous background (50-fold increase, 15%), compared to control flies with normal levels of Azot. Cell elimination capacity was fully restored by crossing two copies of Azot::dsRed into the azot-/- background demonstrating the functionality of the fusion protein. Silencing azot with two different RNAis was similarly able to halt selection during dmyc-induced competition. Next, in order to determine the role of Azot's EF hands, a mutated isoform of Azot (Pm4Q12) was generated and overexpressed, that carryed, in each EF hand, a point mutation known to abolish Ca2+ binding. Although overexpression of wild-type azot in negatively selected cells did not rescue the elimination, overexpression of the mutant AzotPm4Q12 reduced cell selection, functioning as a dominant-negative mutant. This shows that Ca2+ binding is important for Azot function. Finally, staining for apoptotic cells corroborated that the lack of Azot prevents cell elimination, because cell death was reduced 8-fold in mosaic epithelia containing loser cells (Merino, 2015).

    The role of azot in elimination of peripheral photoreceptor neurons in the pupal retina was examined using homozygous azot KO flies. Pupal retinas undergoing photoreceptor culling (44hr APF) of azot+/+ and azot-/- flies were stained for the cell death marker and the proapoptotic factor. Consistent with the expression pattern of Azot, the number of Hid and TUNEL-positive cells was dramatically decreased in azot-/- retinas compared to azot+/+ retinas (Merino, 2015).

    Those results show that Azot is required to induce cell death and Hid expression during neuronal culling. Therefore, tests were performed to see that was also the case in the wing epithelia during dmyc-induced competition. Hid was found to be expressed in loser cells and the expression was found to be strongly reduced in the absence of Azot function (Merino, 2015).

    Finally, forced overexpression of FlowerLose isoforms from Drosophila were unable to mediate WT cell elimination when Azot function was impaired by mutation or silenced by RNAi (Merino, 2015).

    These results suggested that azot function is dose sensitive, because heterozygous azot mutant flies display delayed elimination of loser cells when compared with azot WT flies. Therefore advantage was taken of the functional reporter Azot::dsRed to test whether cell elimination could be enhanced by increasing the number of genomic copies of azot. Tissues with three functional copies of azot were more efficient eliminating loser cells during dmyc-induced competition and most of the clones were culled 48hr ACI. From these results, it is concluded that azot expression is required for the elimination of Loser cells and unwanted neurons (Merino, 2015).

    Next, it was asked what could be the consequences of decreased cell selection at the tissue and organismal level. To this end, advantage was taken of the viability of homozygous azot KO flies. An increase of several developmental aberrations was observed. Focus was placed on the wings, where cell competition is best studied and, because aberrations, including melanotic areas, blisters, and wing margin nicks, were quantified. Wing defects of azot mutant flies could be rescued by introducing two copies of azot::dsRed, showing that the phenotypes are specifically caused by loss of Azot function (Merino, 2015).

    Next, it was reasoned that mild tissue stress should increase the need for fitness-based cell selection after damage. First, in order to generate multicellular tissues scattered with suboptimal cells, larvae were exposed to UV light and Azot expression was monitored in wing discs of UV-irradiated WT larvae that were stained for cleaved caspase-3, 24hr after treatment. Under such conditions, Azot was found to be expressed in cleaved caspase-3-positive cells. All Azot-positive cells showed caspase activation and 17% of cleaved caspase-positive cells expressed Azot. This suggested that Azot-expressing cells are culled from the tissue. To confirm this, later time points (3 days after irradiation) were examined; the increase in Azot-positive cells was no longer detectable. The elimination of azot-expressing cells after UV irradiation required azot function, because cells revealed by reporter azot{KO; gfp}, that express GFP instead of Azot, persisted in wing imaginal discs from azot-null larvae. Tests were performeed to see if lack of azot leads to a faster accumulation of tissue defects during organ development upon external damage. azot-/- pupae 0 stage were irradiated, and the number of morphological defects in adult wings was compared to those in nonirradiated azot KO flies. It was found that aberrations increased more than 2-fold when compared to nonirradiated azot-/- flies (Merino, 2015).

    In order to functionally discriminate whether azot belongs to genes regulating apoptosis in general or is dedicated to fitness-based cell selection, whether azot silencing prevents Eiger/TNF-induced cell death was exanubed. Inhibiting apoptosis (UASp35) or eiger (UASRNAieiger) rescued eye ablation, whereas azot silencing and overexpression of AzotPm4Q12 did not. Furthermore, azot silencing did not impair apoptosis during genitalia rotation or cell death of epithelial precursors in the retina. These results highlight the consequences of nonfunctional cell-quality control within developing tissues (Merino, 2015).

    The next part of the analysis demonstrated that the azot promoter computes relative FlowerLose and Sparc Levels. Epistasis analyses were performed to understand at which level azot is transcriptionally regulated. For this purpose, the assay where WT cells are outcompeted by dMyc-overexpressing supercompetitors was used. It was previously observed that azot induction is triggered upstream of caspase-3 activation and accumulates in outcompeted cells unable to die. Then, upstream events of cell selection were genetically modified. Silencing fweLose transcripts by RNAi or overexpressing Sparc both blocked the induction of Azot::dsRed in WT loser cells. In contrast, when outcompeted WT cells were additionally 'weakened' by Sparc downregulation using RNAi, Azot is detected in almost all loser cells compared to its more limited induction in the presence of endogenous Sparc. Inhibiting JNK signaling with UASpuc did not suppress Azot expression (Merino, 2015).

    The activation of Azot upon irradiation was examined. Strikingly, it was found that all Azot expression after irradiation was eliminated when Flower Lose was silenced and also when relative differences of Flower Lose where diminished by overexpressing high levels of Lose isoforms ubiquitously. On the contrary, Azot was not suppressed after irradiation by expressing the prosurvival factor Bcl-2 or a p53 dominant negative. These results show that Azot expression during competition and upon irradiation requires differences in Flower Lose relative levels (Merino, 2015).

    Finally, the regulation of Azot expression in neurons was analyzed. Silencing fwe transcripts by RNAi blocked the induction of Azot::dsRed in peripheral photoreceptors. Because Wingless signaling induces FlowerLose-B expression in peripheral photoreceptors, tests were performed to see if overexpression of Daxin, a negative regulator of the pathway, affected Azot levels. Axin overespression completely inhibited Azot expression. Similarly, overexpression of the cell competition inhibitor Sparc also fully blocked Azot endogenous expression in the retina. Finally, ectopic overexpression of FlowerLose-B in scattered cells of the retina was sufficient to trigger ectopic Azot activation. These results show that photoreceptor cells also can monitor the levels of Sparc and the relative levels of FlowerLose-B before triggering Azot expression (Merino, 2015).

    These results suggest that the azot promoter integrates fitness information from neighboring cells, acting as a relative 'cell-fitness checkpoint.'

    To test if fitness-based cell selection is a mechanism active not only during development, but also during adult stages, WT adult flies were exposed to UV light and monitor Azot and Flower expression were monitored in adult tissues. UV irradiation of adult flies triggered cytoplasmic Azot expression in several adult tissues including the gut and the adult brain. Likewise, UV irradiation of adult flies triggered Flower Lose expression in the gut and in the brain. Irradiation-induced Azot expression was unaffected by Bcl-2 but was eliminated when Flower Lose was silenced or when relative differences of Flower Lose where diminished in the gut. This suggests that the process of cell selection is active throughout the life history of the animal. Further confirming this conclusion, Azot function was essential for survival after irradiation, because more than 99% of azot mutant adults died 6 days after irradiation, whereas only 62.4% of WT flies died after the same treatment. The percentage of survival correlated with the dose of azot because adults with three functional copies of azot had higher median survival and maximum lifespan than WT flies, or null mutant flies rescued with two functional azot transgenes (Merino, 2015).

    The next part of the study addressed the role of cell selection during aging. Lack of cell selection could affect the whole organism by two nonexclusive mechanisms. First, the failure to detect precancerous cells, which could lead to cancer formation and death of the individual. Second, the time-dependent accumulation of unfit but viable cells could lead to accelerated tissue and organ decay. We therefore tested both hypotheses (Merino, 2015).

    It has been previously shown that cells with reduced levels for cell polarity genes like scrib or dlg are eliminated but can give rise to tumors when surviving. Therefore this study checked if azot functions as a tumor suppressing mechanism in those cells. Elimination of dlg and scrib mutant cells was not affected by RNAi against azot or when Azot function was impaired by mutation, in agreement with the absence of azot induction in these mutant cells. However, azot RNAi or the same azot mutant background efficiently rescued the elimination of clones with reduced Wg signaling (Merino, 2015).

    Moreover, the high number of suboptimal cells produced by UV treatment did not lead to tumoral growth in azot-null background. Thus, tumor suppression mechanisms are not impaired in azot mutant backgrounds, and tumors are not more likely to arise in azot-null mutants (Merino, 2015).

    Also tests were performed to see whether the absence of azot accelerates tissue fitness decay in adult tissues. Focused was placed on the adult brain, where neurodegenerative vacuoles develop over time and can be used as a marker of aging. The number was compared of vacuoles appearing in the brain of flies lacking azot (azot-/-), WT flies (azot+/+), flies with one extra genomic copy of the gene (azot+/+; azot+), and mutant flies rescued with two genomic copies of azot (azot-/-;azot+/+). For all the genotypes analyzed, a progressive increase was observed in the number and size of vacuoles in the brain over time. Interestingly, azot-/- brains showed higher number of vacuoles compared to control flies (azot+/+ and azot-/-;azot+/+) and a higher rate of vacuole accumulation developing over time. In the case of flies with three genomic copies of the gene (azot+/+; azot+), vacuole number tended to be the lowest (Merino, 2015).

    The cumulative expression of azot was analyzed during aging of the adult brain. Positive cells were detected as revealed by reporter azot{KO; gfp}, in homozygosis, that express GFP instead of Azot. A time-dependent accumulation of azot-positive cells was observed (Merino, 2015).

    From this, it is concluded that azot is required to prevent tissue degeneration in the adult brain and lack of azot showed signs of accelerated aging. This suggested that azot could affect the longevity of adult flies. Flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 7.8 days, which represented a 52% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 18 days, 25% less than WT flies (azot+/+). This effect on lifespan was azot dependent because it was completely rescued by introducing two functional copies of azot. On the contrary, flies with three functional copies of the gene (azot+/+; azot+) showed an increase in median survival and maximum lifespan of 54% and 17%, respectively (Merino, 2015).

    In conclusion, azot is necessary and sufficient to slow down aging, and active selection of viable cells is critical for a long lifespan in multicellular animals (Merino, 2015).

    The next part of the study demonstrates that death of unfit cells is sufficient and required for multicellular fitness maintenance. The results cited above show the genetic mechanism through which cell selection mediates elimination of suboptimal but viable cells. However, using flip-out clones and MARCM, this study found that Azot overexpression was not sufficient to induce cell death in wing imaginal discs. Because Hid is downstream of Azot, it was wondered whether expressing Hid under the control of the azot regulatory regions could substitute for Azot function (Merino, 2015).

    In order to test this hypothesis, the whole endogenous azot protein-coding sequence was replaced by the cDNA of the proapoptotic gene hid (azot{KO; hid}) flies. In a second strategy, the whole endogenous azot protein-coding sequence was replaced by the cDNA of transcription factor Gal4, so that the azot promoter can activate any UAS driven transgene (azot{KO; Gal4} flies. The number of morphological aberrations was compared in the adult wings of six genotypes: first, homozygous azot{KO; Gal4} flies that lacked Azot; second, azot{KO; hid} homozygous flies that express Hid with the azot pattern in complete absence of Azot; third, azot+/+ WT flies as a control; and finally three genotypes where the azot{KO; Gal4} flies were crossed with UAShid, UASsickle, another proapoptotic gene, or UASp35, an apoptosis inhibitor. In the case of UASsickle flies, a second azot mutation was introduced to eliminate azot function. Interestingly, the number of morphological aberrations was brought back to WT levels in all the situations where the azot promoter was driving proapoptotic genes (azot{KO; hid}, azot{KO; Gal4} × UAShid, azot{KO; Gal4} × UASsickle with or without irradiation. On the contrary, expressing p35 with the azot promoter was sufficient to produce morphological aberrations despite the presence of one functional copy of azot. Likewise, p35-expressing flies (azot{KO; Gal4}/azot+; UASp35) did not survive UV treatments, whereas a percentage of the flies expressing hid (26%) or sickle (28%) in azot-positive cells were able to survive (Merino, 2015).

    From this, it is concluded that specifically killing those cells selected by the azot promoter is sufficient and required to prevent morphological malformations and provide resistance to UV irradiation (Merino, 2015).

    The next part of the study demonstrated that death of unfit cells extends lifespan It was asked whether the shortened longevity observed in azot-/- flies could be also rescued by killing azot-expressing cells with hid in the absence of Azot protein. It was found that azot{KO; hid} homozygous flies had dramatically improved lifespan with a median survival of 27 days at 29°C, which represented a 125% increase when compared to azot-/- flies, and a maximum lifespan of 34 days, 41% more than mutant flies (Merino, 2015).

    Similar results were obtained at 25°C. It was found that flies lacking azot (azot-/-) had a shortened lifespan with a median survival of 25days, which represented a 24% decrease when compared to WT flies (azot+/+), and a maximum lifespan of 40 days, 31% less than WT flies (azot+/+). On the contrary, flies with three functional copies of the gene (azot+/+; azot+) or flies where azot is replaced by hid (azot{KO; hid} homozygous flies) showed an increase in median survival of 54% and 63% and maximum lifespan of 12% and 24%, respectively (Merino, 2015).

    Finally, the effects of dietary restriction on longevity of those flies was tested. It was found that dietary restriction could extend both the median survival and the maximum lifespan of all genotypes. Interestingly, dietary restricted flies with three copies of the gene azot showed a further increase in maximum lifespan of 35%. This shows that dietary restriction and elimination of unfit cells can be combined to maximize lifespan (Merino, 2015).

    In conclusion, eliminating unfit cells is sufficient to increase longevity, showing that cell selection is critical for a long lifespan in Drosophila (Merino, 2015).

    This study has shown that active elimination of unfit cells is required to maintain tissue health during development and adulthood. The gene (azot), whose expression is confined to suboptimal or misspecified but morphologically normal and viable cells. When tissues become scattered with suboptimal cells, lack of azot increases morphological malformations and susceptibility to random mutations and accelerates age-dependent tissue degeneration. On the contrary, experimental stimulation of azot function is beneficial for tissue health and extends lifespan. Therefore, elimination of less fit cells fulfils the criteria for a hallmark of aging (Merino, 2015).

    Although cancer and aging can both be considered consequences of cellular damage, no evidence was found for fitness-based cell selection having a role as a tumor suppressor in Drosophila. The results rather support that accumulation of unfit cells affect organ integrity and that, once organ function falls below a critical threshold, the individual dies (Merino, 2015).

    Azot expression in a wide range of 'less fit' cells, such as WT cells challenged by the presence of 'supercompetitors,' slow proliferating cells confronted with normal proliferating cells, cells with mutations in several signaling pathways (i.e., Wingless, JAK/STAT, Dpp), or photoreceptor neurons forming incomplete ommatidia. In order to be expressed specifically in 'less fit' cells, the transcriptional regulation of azot integrates fitness information from at least three levels: (1) the cell's own levels of FlowerLose isoforms, (2) the levels of Sparc, and (3) the levels of Lose isoforms in neighboring cells. Therefore, Azot ON/OFF regulation acts as a cell-fitness checkpoint deciding which viable cells are eliminated. It is proposed that by implementing a cell-fitness checkpoint, multicellular communities became more robust and less sensitive to several mutations that create viable but potentially harmful cells. Moreover, azot is not involved in other types of apoptosis, suggesting a dedicated function, and - given the evolutionary conservation of Azot - pointing to the existence of central cell selection pathways in multicellular animals (Merino, 2015).

    Transcriptional pausing controls a rapid antiviral innate immune response in Drosophila

    Innate immune responses are characterized by precise gene expression whereby gene subsets are temporally induced to limit infection, although the mechanisms involved are incompletely understood. This study shows that antiviral immunity in Drosophila requires the transcriptional pausing pathway, including negative elongation factor (NELF) that pauses RNA polymerase II (Pol II) and positive elongation factor b (P-TEFb), which releases paused Pol II to produce full-length transcripts. A set of genes was identified that is rapidly transcribed upon arbovirus infection, including components of antiviral pathways (RNA silencing, autophagy, JAK/STAT, Toll, and Imd) and various Toll receptors. Many of these genes require P-TEFb for expression and exhibit pausing-associated chromatin features. Furthermore, transcriptional pausing is critical for antiviral immunity in insects because NELF and P-TEFb are required to restrict viral replication in adult flies and vector mosquito cells. Thus, transcriptional pausing primes virally induced genes to facilitate rapid gene induction and robust antiviral responses (Xu, 2012).

    The metabolite alpha-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR

    Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. This study shows that alpha-ketoglutarate (alpha-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit beta was identified as a novel binding protein of alpha-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. Alpha-KG was found to inhibit ATP synthase and, similar to ATP synthase knockdown, inhibition by alpha-KG leads to reduced ATP content, decreased oxygen consumption, and increases autophagy in both C. elegans and mammalian cells. Evidence is provided that the lifespan increase by alpha-KG requires ATP synthase subunit beta and is dependent on target of rapamycin (TOR) downstream. Endogenous alpha-KG levels are increased on starvation and alpha-KG does not extend the lifespan of dietary-restricted animals, indicating that alpha-KG is a key metabolite that mediates longevity by dietary restriction. These analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases (Chin, 2014).

    Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced neurodegeneration

    The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, the Drosophila homolog, CG2789/dTSPO, was identified, and the effects of its inactivation was studied by P-element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5-4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to gamma-irradiation or H2O2 exposure, as well as extended male fly lifespan and inhibited Aβ42-induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target (Lin, 2014).

    Age-induced reduction of autophagy-related gene expression is associated with onset of Alzheimer's disease

    Aging is a major risk factor Alzheimer’s disease (AD). Aggregation of amyloid beta (Aβ) in cerebral cortex and hippocampus is a hallmark of AD. Many factors have been identified as causative elements for onset and progression of AD; for instance, tau seems to mediate the neuronal toxicity of Aβ, and downregulation of macroautophagy (autophagy) is thought to be a causative element of AD pathology. Expression of autophagy-related genes is reduced with age, which leads to increases in oxidative stress and aberrant protein accumulation. This study found that expression of the autophagy-related genes atg1, atg8a, and atg18 in Drosophila melanogaster is regulated with aging as well as their own activities. In addition, the level of atg18 is maintained by dfoxo (foxo) and dsir2 (sir2) activities in concert with aging. These results indicate that some autophagy-related gene expression is regulated by foxo/sir2-mediated aging processes. It was further found that reduced autophagy activity correlates with late-onset neuronal dysfunction caused by neuronal induction of Aβ. These data support the idea that age-related dysfunction of autophagy is a causative element in onset and progression of AD (Omata, 2014).

    This study shows that expression of autophagy-related genes is regulated by age-related signaling. dsir2 (a Drosophila SIRT1 homolog) and dfoxo are required to maintain atg18 expression during aging, suggesting that, among autophagy-related genes, this gene specifically is regulated by foxo/sir2 activity. Interestingly, aging seems to affect expression of all autophagy-related genes tested, suggesting that aging and foxo/sir2 may act at different levels to regulate autophagy-related gene expression (Omata, 2014).

    Previous studies show that sir2, foxo and 4E-BP are involved in regulating the Drosophila lifespan. Data from this study, however, indicate that 4E-BP antagonizes expression of autophagy-related genes. 4E-BP is believed to be controlled by TOR signaling. Therefore, the negative effect of 4E-BP on autophagy-related gene expression may be mediated through the effect of TOR signaling pathway, which also seems to antagonize autophagy-related gene expression (Omata, 2014). 

    Autophagy is highly correlated with lysosomal activity, and the autophagy-lysosome pathway is thought to be involved in many cellular processes. Earlier studies indicate that lysosomal activity affects expression of autophagy-related genes. The lysosome nutrient sensing (LYNUS) machinery is responsible for sensing whether there are sufficient nutrients. Under a sufficient nutrient status, the mammalian target of rapamycin complex 1 (mTORC1, a member of the LYNUS machinery) phosphorylates transcription factor EB (TFEB) on the lysosomal surface and inhibits its nuclear localization. In this way, TFEB is unable to induce expression of lysosomal and autophagy-related genes under nutrient sufficient conditions. These results suggest that the level of autophagy-related genes might be regulated by the state of lysosome formation and autophagy itself. Here, expression of autophagy-related genes is affected by the activity of other autophagy-related genes as well as their own activity, suggesting that auto-feedback regulation is part of the mechanism used to maintain expression of autophagy-related genes in Drosophila (Omata, 2014). 

    It was observed that reducing the expression of autophagy-related genes strongly enhances the neuronal toxicity caused by Aβ expression. Furthermore, reducing atg1 expression using the Df(atg1)/+ heterozygote shows a more severe enhancement of Aβ-dependent neuronal toxicity than reducing atg18 expression using the Df(atg18)/+ heterozygote. Interestingly, atg1 also demonstrates strong auto-feedback regulation, as reducing expression of atg1 results in further defects in expression of atg genes. Therefore, it is possible that a drastic reduction in expression of many atg genes may contribute to the neuronal toxicity of Aβ42, and that aging and autophagy may be determinants of AD onset (Omata, 2014).

    P62 plays a protective role in the autophagic degradation of polyglutamine protein oligomers in polyglutamine disease model flies

    Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer's disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases since it can degrade even large oligomers. Although p62/sequestosome1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. This study elucidates the role of p62 in such pathogenic conditions in vivo using Drosophila models of neurodegenerative diseases. p62 was shown to predominantly co-localize with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function resulted in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of Autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. It was also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model, as well as ALS model flies. It is therefore concluded that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases, and possibly for other neurodegenerative diseases (Saitoh, 2014).

    Huntingtin functions as a scaffold for selective macroautophagy

    Selective macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. This study demonstrates that Huntingtin, the Huntington disease gene product, functions as a scaffold protein for selective macroautophagy but is dispensable for non-selective macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with Lys-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress was attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which released ULK1 from negative regulation by mTOR. This data uncovers an important physiological function of Huntingtin and provides a missing link in the activation of selective macroautophagy in metazoans (Rui, 2015).

    Homozygous flies lacking the single htt homologue (dhttko) are fully viable with only mild phenotypes. In a genetic screen for the physiological function of Htt, ectopic expression of a truncated form of the microtubule-binding protein Tau (Tau-ΔC; truncated after Val 382) induced a prominent collapse of the thorax in dhttko flies due to severe muscle loss not observed by Tau expression alone, and accelerated decline in mobility and lifespan. These phenotypes were fully rescued by the dhtt genomic rescue transgene (‘dhttRescue’), suggesting that dhtt protects against Tau-induced pathogenic effects (Rui, 2015).

    Although heterozygous dhttko/+ flies expressing Tau (A​Tau; ​dhttko/+) seem normal, removing a single copy of the fly LC3 gene, atg8a (atg8ad4 mutant), in these flies also induces a collapsed thorax and muscle loss, which can be phenocopied by expressing Tau in homozygous atg8ad4−/− flies alone. Four additional components of the early steps of the autophagy pathway, atg1 (ULK1), atg7 and atg13, and an adaptor for the selective recognition of autophagic cargo, also exhibit strong genetic interactions with dhtt. Consistent with its pivotal role in autophagy initiation, loss of atg1 induces the strongest defect, and Tau expression can induce a mild muscle loss phenotype even in heterozygous null atg1Δ3d. Collectively, these genetic interaction studies suggest a role for dhtt in autophagy (Rui, 2015).

    By using the mCherry–GFP–​Atg8a fusion reporter to directly measure autophagic flux in adult dhttko−/− brains, this study found similar number of red fluorescent punctae (acidic autolysosomes originating from autophagosome/lysosome fusion) in young mutant and control flies, but the number of punctae were reduced in old dhttko−/− brains when compared with age-matched controls. As autophagosome accumulation (co-localized green and red puncta) was not observed, it was concluded that the absence of dhtt in older animals was associated with reduced autophagosome formation. The fact that levels of Ref(2)P are significantly higher in old dhttko−/− brains compared with brains from age-matched wild-type controls suggests a possible preferential compromise in selective autophagy in these animals (Rui, 2015).

    Consistent with the role of basal autophagy in quality control in non-dividing cells, it was found that brains from 5-week-old ​dhttko−/− contained almost double the amount of ubiquitylated proteins, a marker of quality control failure, compared with wild-type flies. As genetic interaction analysis and specific ubiquitin proteasome system (UPS) reporters all failed to reveal a functional link between ​dhtt and the UPS pathway, the study proposes that the defects in autophagic activity are the main cause of diminished quality control and increased accumulation of ubiquitylated proteins in dhttko−/− mutants (Rui, 2015).

    Selective autophagy is induced in response to proteotoxic stress. The truncated Tau-ΔC used in genetic experiments in this study is preferentially degraded through autophagy in cortical neurons, serving as a model of proteotoxicity when ectopically expressed. The lower stability of Tau-ΔC compared with full-length Tau in wild-type flies and in UPS mutants was confirmed, but significantly higher levels of Tau-ΔC when expressed in atg8a and in dhttko−/− mutant flies were found, suggesting that autophagy is essential for the clearance of Tau-ΔC also in flies and that dhtt plays a role in this clearance (Rui, 2015).

    In contrast, loss of ​dhtt does not affect flies’ adaptation to nutrient deprivation, which typically induces robust ‘in bulk’ autophagy. Fat bodies of early third instar larvae expressing mCherry–​Atg8, where starvation-induced autophagy can be readily detected, fail to reveal any significant difference between wild-type and dhttko−/− flies; these flies die at the same rate as wild-type flies when tested for starvation resistance. Thus, although dhtt is necessary for selective autophagy of toxic proteins such as Tau-ΔC, it is dispensable for starvation-induced autophagy in flies (Rui, 2015).

    Expression of human Htt (hHTT) in dhttko−/− null flies rescues both the mobility and longevity defects of dhttko−/− mutants and partially rescues the Tau-induced morphological and behavioural defects of dhttko−/− flies. hHTT also suppresses almost all of the autophagic defects observed in dhttko−/−, including decreased levels of autolysosomes, increased levels of ​Ref(2)P and of total ubiquitylated proteins, and accumulation of ectopically expressed ​Tau-ΔC, suggesting that the involvement of dhtt in autophagy is functionally conserved. In fact, confluent mouse fibroblasts knocked down for Htt (Htt(−)) exhibit significantly lower basal rates of long-lived proteins’ degradation than control cells, which are no longer evident on chemical inhibition of lysosomal proteolysis or of macroautophagy, thus confirming an autophagic origin of the proteolytic defect. Htt(−) fibroblasts also exhibit higher p62 levels and accumulate ubiquitin aggregates even in the absence of a proteotoxic challenge. As in dhttko−/− flies, Htt knockdown in mammalian cells does not affect degradation of CL1–GFP (a UPS reporter), β-catenin (a UPS canonical substrate) or proteasome peptidase activities. Reduced autophagic degradation in ​Htt(−) cells is not due to a primary lysosomal defect, as depletion of ​Htt does not reduce lysosomal acidification, endolysosomal number (if anything, an expansion of this compartment was observed) or other lysosomal functions such as endocytosis (for example, transferrin internalization). In fact, analysis of the lysosomal degradation of LC3-II reveals that autophagic flux and autophagosome formation are preserved and even enhanced in Htt(−) fibroblasts at basal conditions (Rui, 2015).

    Cardiac deficiency of single cytochrome oxidase assembly factor scox induces p53-dependent apoptosis in a Drosophila cardiomyopathy model

    The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the oxidative phosphorylation (OXPHOS) system are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated and left ventricular noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defects in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors such as Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, this study interfered with scox (the single Drosophila Sco orthologue) expression in the heart. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reduction in COX activity and they undergo a metabolic switch from OXPHOS to glycolysis, mimicking the clinical features found in patients harbouring Sco mutations. The major cardiac defects observed are produced by a significant increase in apoptosis, which is dp53-dependent. Genetic and molecular evidence strongly suggest that dp53 is directly involved in the development of the cardiomyopathy induced by scox deficiency. Remarkably, apoptosis is enhanced in the muscle and liver of Sco2 knock-out mice, clearly suggesting that cell death is a key feature of the COX deficiencies produced by mutations in Sco genes in humans (Martínez-Morentin, 2015).

    Cardiomyopathies are a collection of myocardial disorders in which the heart muscle is structurally and functionally abnormal. In the past decade, it has become clear that an important proportion of cases of hypertrophic and dilated cardiomyopathies are caused by mutations in genes encoding sarcomeric or desmosomal proteins. In addition, cardiomyopathies (both hypertrophic and dilated) are frequently associated to syndromic and non-syndromic mitochondrial diseases. The importance of oxidative metabolism for cardiac function is supported by the fact that 25–35% of the myocardial volume is taken by mitochondria. The current view of mitochondrial involvement in cardiomyopathy assumes that ETC malfunction results in an increased ROS production, triggering a “ROS-induced ROS release” vicious circle which in turn perpetuates ETC dysfunction via damage in mtDNA and proteins involved in electron transport. Under this view, accumulated mitochondrial damage would eventually trigger apoptosis through mitochondrial permeability transition pore (mPTP) opening other mechanisms. Under normal circumstances, damaged mitochondria would be eliminated through mitophagy. Excessive oxidative damage is supposed to overcome the mitophagic pathway resulting in apoptosis. Nevertheless, although several potential mechanisms have been suggested, including apoptosis deregulation, oxidative stress, disturbed calcium homeostasis or impaired iron metabolism, the molecular basis of the pathogenesis of mitochondrial cardiomyopathy is virtually unknown (Martínez-Morentin, 2015).

    Pathogenic mutations in human SCO1 and SCO2 have been reported to cause hypertrophic cardiomyopathy, among other clinical symptoms. However, the molecular mechanisms underlying this cardiac dysfunction have yet to be elucidated. This study reports the first cardiac-specific animal model to study human SCO1/2-mediated cardiomyopathy. Cardiac-specific scox KD in Drosophila provokes a severe dilated cardiomyopathy, as reflected by a significant increase in the conical chamber size, due to mitochondrial dysfunction. It presents a concomitant metabolic switch from glucose oxidation to glycolysis and an increase in ROS levels, leading to p53-dependent cell death. Interestingly, previous studies on patients and rat models have shown that mitochondrial dysfunction is associated with abnormalities in cardiac function and changes in energy metabolism, resulting in glycolysis optimization and lactic acidosis. Furthermore, in the Sco2KI/KO mouse model, where no evidence of cardiomyopathy has been described, partial loss of Sco2 function induces apoptosis in liver and skeletal muscle. In flies scox KD causes a significant reduction in FS and in the DI, as well as cardiac myofibril disorganization. This degenerative process is most likely due to mitochondrial dysfunction rather than to a developmental defect and moreover, the dilated cardiomyopathy developed by flies resembles that caused by mitochondrial fusion defects in flies (Martínez-Morentin, 2015).

    The ETC is the major site of ROS production in cells, and aging and many neurodegenerative diseases have been linked to mitochondrial dysfunction that results in excessive oxidative stress. Interestingly, there is an increase in ROS formation associated with oxidative DNA damage in human Sco2−/− cells. Accordingly, it was found that cardiac-specific knockdown of scox increases oxidative stress, although it could not be distinguished whether this increase in free radical accumulation arises from the mitochondria or whether it comes from non-mitochondrial sources due to a loss of cellular homeostasis, as reported in yeast and in a neuro-specific COX-deficient Alzheimer disease mouse model (Martínez-Morentin, 2015).

    Sco2 expression is known to be modulated by p53, a transcription factor that participates in many different processes, including cancer development, apoptosis and necrosis. p53 regulates homeostatic cell metabolism by modulating Sco2 expression and contributes to cardiovascular disorders. In addition, p53 activation in response to stress signals, such as increased oxidative stress or high lactic acid production, is well documented. Data from this study, showing that p53 is upregulated in response to scox KD, but not in response to KD of another Complex IV assembly factor, Surf1, suggest a specific genetic interaction between dp53 and scox. This is corroborated by the dramatic effects observed in the heart structure and function when dp53 is overexpressed in scox KD hearts. Furthermore, the functional and structural defects seen in scox KD hearts can be rescued in dp53-DN OE or dp53 null backgrounds, indicating that the scox-induced defects are mediated by increased p53 expression. Interestingly, opposed to scox KD, the heart structure defects induced by dp53 OE can be fully rescued by heart-specific Surf1 KD, further confirming the specificity of the genetic interaction between dp53 and scox (Martínez-Morentin, 2015).

    It has recently been shown that SCO2 OE induces p53-mediated apoptosis in tumour xenografts and cancer cells. Furthermore, SCO2 KD sensitizes glioma cells to hypoxia-induced apoptosis in a p53-dependent manner and induces necrosis in tumours expressing WT p53, further linking the SCO2/p53 axis to cell death. In Drosophila, there is a dp53-mediated upregulation of Reaper, Hid and Grim in response to scox KD. This, coupled with the observation that Reaper overexpression in the adult heart enhances the structural defects caused by cardiac-specific scox KD, suggests that scox normally prevents the triggering of dp53-mediated cell death in cardiomyocytes in stress response. Indeed, it was found that there is massive cell death in the skeletal muscle and liver of Sco2KI/KO mice, supporting the hypothesis that Sco proteins might play this role also in mammals (Martínez-Morentin, 2015).

    The study provides evidence that scox KD hearts exhibit partial loss of COX activity, with cardiomyocytes undergoing apoptosis. There is evidence from vertebrate and invertebrate models that partial inhibition of mitochondrial respiration promotes longevity and metabolic health due to hormesis. In fact, it has recently been shown that mild interference of the OXPHOS system in Drosophila IFMs preserves mitochondrial function, improves muscle performance and increases lifespan through the activation of the mitochondrial unfolded pathway response and IGF/like signalling pathways. This study speculates that cell death, rather than mitochondrial dysfunction itself, is likely to be the main reason for the profound heart degeneration observed in TinCΔ4-Gal4>scoxi flies. Expression of dominant negative dp53 in scox KD hearts rescues dysfunction and cardiac degeneration, and, most importantly, scox KD in dp53−/− animals causes no apparent heart defects, which could attribute the rescue observed to blockade of the p53 pathway. Indeed, inhibiting apoptosis by p35 or Diap1 OE almost completely rescues the morphological scox KD phenotype. As scox KD in the absence of dp53 causes no symptoms of heart disease, coupled with the inability of p35 and Diap1 to completely rescue the morphological phenotype, suggests that, in addition to inducing apoptosis, dp53 plays a key role in the development of cardiomyopathy (Martínez-Morentin, 2015).

    The fact that heart-specific Surf1 KD neither upregulates p53 nor induces apoptosis supports the idea that the partial loss of scox function itself triggers dp53 upregulation and apoptosis, rather than it being a side effect of COX dysfunction and the loss of cellular homeostasis. In this context, it is noteworthy that SCO2 interference in mammalian cells induces p53 re-localization from mitochondria to the nucleus. It is therefore tempting to hypothesize that scox might play another role independent of its function as a COX assembly factor, perhaps in redox regulation as suggested previously and that it may act in conjunction with dp53 to fulfil this role. Another issue deserves further attention, the possibility of this interaction being a tissue-specific response. It may be possible that the threshold of COX deficiency tolerated by the heart might be lower than in other tissues, thus the scox/dp53 genetic interaction may be a tissue-dependent phenomenon or the consequence of a tissue-specific role of scox. In fact, mitochondrial dysfunction in mice is sensed independently from respiratory chain deficiency, leading to tissue-specific activation of cellular stress responses. Thus, more work is necessary to test these hypotheses and try to understand how the partial lack of scox induces cell death through dp53 (Martínez-Morentin, 2015).

    Although the role of mitochondria in Drosophila apoptosis remains unclear, there is strong evidence that, as in mammals, mitochondria play an important role in cell death in flies. The localization of Rpr, Hid and Grim in the mitochondria is essential to promote cell death, and fly mitochondria undergo Rpr-, Hid- and Drp1-dependent morphological changes and disruption following apoptotic stimulus. Moreover, the participation of the mitochondrial fission protein Drp1 in cell death is conserved in worms and mammals. It has been proposed that p53 plays a role in the opening of the mPTP that induces necrotic cell death. According to this model, p53 translocates to the mitochondrial matrix upon ROS stimulation, where it binds cyclophilin D (CypD) to induce mPTP opening independent of proapoptotic Bcl-2 family members Bax and Bak, and in contrast to traditional concepts, independent of Ca2+ (Martínez-Morentin, 2015).

    Apoptotic and necrotic pathways have a number of common steps and regulatory factors, including mPTP opening that is thought to provoke mitochondrial swelling and posterior delivery of necrotic factors, although Drosophila mPTP activation is not accompanied by mitochondrial swelling. Interestingly, although the p53 protein triggers mitochondrial outer membrane permeabilization (MOMP) in response to cellular stress in mammals, releasing mitochondrial death factors, MOMP in Drosophila is more likely a consequence rather than cause of caspase activation and the release of mitochondrial factors does not appear to play a role in apoptosis. Thus, in cardiac-specific scox KD flies, dp53 might induce mPTP opening to trigger cell death, which in the absence of mitochondrial swelling would result in apoptosis instead of necrosis, as occurs in mammals. Drosophila mPTP has been shown to be cyclosporine A (CsA)-insensitive in vitro, although CsA administration ameliorates the mitochondrial dysfunction with a severely attenuated ATP and enhanced ROS production displayed by collagen XV/XVIII mutants. Interestingly, mice lacking collagen VI display altered mitochondrial structure and spontaneous apoptosis, defects that are caused by mPTP opening and that are normalized in vivo by CsA treatment (Martínez-Morentin, 2015).

    p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB

    The selective autophagy receptor p62/sequestosome 1 (SQSTM1) interacts directly with LC3 and is involved in oxidative stress signaling in two ways in mammals. First, p62 is transcriptionally induced upon oxidative stress by the NF-E2-related factor 2 (NRF2) by direct binding to an antioxidant response element (ARE) in the p62 promoter. Secondly, p62 accumulation, occurring when autophagy is impaired, lead to increased p62 binding to the NRF2 inhibitor KEAP1 resulting in reduced proteasomal turnover of NRF2. This gives chronic oxidative stress signaling through a feed forward loop. This study shows that the Drosophila p62/SQSTM1 orthologue, Ref(2)P, interacts directly with DmAtg8a via a LC3-interacting region (LIR) motif, supporting a role for Ref(2)P in selective autophagy. The ref(2)P promoter also contains a functional ARE that is directly bound by the NRF2 orthologue, CncC which can induce ref(2)P expression along with the oxidative stress associated gene gstD1. However, distinct from the situation in mammals, Ref(2)P does not interact directly with DmKeap1 via a KEAP1-interacting region (KIR) motif. Neither does ectopically expressed Ref(2)P, nor autophagy deficiency, activate the oxidative stress response. Instead, DmAtg8a interacts directly with DmKeap1, and DmKeap1 is removed upon programmed autophagy in Drosophila gut cells. Strikingly, CncC induced increased Atg8a levels and autophagy independent of TFEB/MitF in fat body and larval gut tissues. Thus, these results extend the intimate relationship between oxidative stress sensing NRF2/CncC transcription factors and autophagy, and suggests that NRF2/CncC may regulate autophagic activity in other organisms too (Jain, 2015).

    Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

    Autophagy is a process essential for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, this study identified CG11148, whose product is orthologous to GIGYF1 (GRB10 interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; the gene is hereafter refered to as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved (Kim, 2015).

    β-Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP-activated protein kinase-dependent increase in autophagy

    Previous studies have demonstrated that AMP-activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc-51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β-guanidinopropionic acid (β-GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β-GPA and aging remains elusive. In this study, it was hypothesized that feeding β-GPA to adult Drosophila produces the lifespan extension via activation of AMPK-dependent autophagy. It was found that dietary administration of β-GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, it was found that Atg8 protein, the homolog of microtubule-associated protein 1A/1B-light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β-GPA treatment, indicating that autophagic activity plays a role in the effect of β-GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β-GPA on lifespan extension was abolished. Moreover, it was found that AMPK was also involved in this process. β-GPA treatment significantly elevated the expression of phospho-T172-AMPK levels, while inhibition of AMPK by either AMPK-RNAi or compound C significantly attenuated the expression of autophagy-related proteins and lifespan extension in Drosophila. Taken together, these results suggest that β-GPA can induce an extension of the lifespan of Drosophila via AMPK-Atg1-autophagy signaling pathway (Yang, 2015).

    Tousled-like kinase mediated a new type of cell death pathway in Drosophila

    Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. This study found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 is likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, these results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity (Zhang, 2015).

    Ceramides and stress signalling intersect with autophagic defects in neurodegenerative Drosophila blue cheese (bchs) mutants

    Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. This study demonstrates that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death (Hebbar, 2015).

    deubiquitinating enzyme UBPY is required for lysosomal biogenesis and productive autophagy in Drosophila

    Autophagy is a catabolic process that delivers cytoplasmic components to the lysosomes. Protein modification by ubiquitination is involved in this pathway: it regulates the stability of autophagy regulators such as BECLIN-1 and it also functions as a tag targeting specific substrates to autophagosomes. In order to identify deubiquitinating enzymes (DUBs) involved in autophagy, a genetic screen was performed in the Drosophila larval fat body. This screen identified Ubiquitin carboxy-terminal hydrolase L5 ortholog (Uch-L3), Usp45, Usp12 and Ubpy (Ubiquitin specific protease 8). This paper shows that Ubpy loss of function results in the accumulation of autophagosomes due to a blockade of the autophagy flux. Furthermore, analysis by electron and confocal microscopy of Ubpy-depleted fat body cells revealed altered lysosomal morphology, indicating that Ubpy inactivation affects lysosomal maintenance and/or biogenesis. Lastly, shRNA mediated inactivation of UBPY in HeLa cells affects autophagy in a different way: in UBPY-depleted HeLa cells autophagy is deregulated (Jacomin, 2015).

    Drosophila Mitf regulates the V-ATPase and the lysosomal-autophagic pathway

    An evolutionary conserved gene network regulates the expression of genes involved in lysosome biogenesis, autophagy and lipid metabolism. This study reports that the lysosomal-autophagy pathway is controlled by Mitf gene in Drosophila. Mitf regulates the expression of genes encoding V-ATPase subunits as well as many additional genes involved in the lysosomal-autophagy pathway. Reduction of Mitf function leads to abnormal lysosomes and impairs autophagosome fusion and lipid breakdown during the response to starvation. In contrast, elevated Mitf levels increase the number of lysosomes, autophagosomes and autolysosomes, and decrease the size of lipid droplets. Inhibition of Drosophila MTORC1 induces Mitf translocation to the nucleus, underscoring conserved regulatory mechanisms between Drosophila and mammalian systems. Furthermore, Mitf-mediated clearance of cytosolic and nuclear expanded ATXN1 (ataxin 1) was demonstrated in a cellular model of spinocerebellar ataxia type 1 (SCA1). This remarkable observation illustrates the potential of the lysosomal-autophagy system to prevent toxic protein aggregation in both the cytoplasmic and nuclear compartments. It is anticipated that the genetics of the Drosophila model and the absence of redundant MIT transcription factors will be exploited to investigate the regulation and function of the lysosomal-autophagy gene network (Bouche, 2016).

    Necrotic pyknosis is a morphologically and biochemically distinct event from apoptotic pyknosis

    Classification of apoptosis and necrosis by morphological difference has been widely used for decades. However, this method has been seriously doubt in recent years, mainly due to lack of functional and biochemical evidence to interpret the morphology changes. To address these questions, this study devised genetic manipulations in Drosophila to study pyknosis, a process of nuclear shrinkage and chromatin condensation occurred in apoptosis and necrosis. By following the progression of necrotic pyknosis, a transient state was surprisingly observed of chromatin detachment from the nuclear envelope (NE), followed with the NE completely collapsed onto chromatin. This phenomenon lead to the discovery that phosphorylation of barrier-to-autointegration factor (BAF) mediates this initial separation of NE from chromatin. Functionally, inhibition of BAF phosphorylation suppressed the necrosis in both Drosophila and human cells, suggesting necrotic pyknosis is conserved in the propagation of necrosis. In contrast, apoptotic pyknosis did not show a detached state of chromatin from NE and inhibition of BAF phosphorylation had no effect on apoptotic pyknosis and apoptosis. This research provides the first genetic evidence supporting morphological classification of apoptosis and necrosis by pyknosis (Hou, 2016).

    The defender against apoptotic cell death 1 gene is required for tissue growth and efficient N-glycosylation in Drosophila melanogaster

    How organ growth is regulated in multicellular organisms is a long-standing question in developmental biology. It is known that coordination of cell apoptosis and proliferation is critical in cell number and overall organ size control, while how these processes are regulated is still under investigation. This study found that functional loss of a gene in Drosophila, named Drosophila defender against apoptotic cell death 1 (dDad1), leads to a reduction of tissue growth due to increased apoptosis and lack of cell proliferation. The dDad1 protein, an orthologue of mammalian Dad1, was found to be crucial for protein N-glycosylation in developing tissues. Loss of dDad1 function activates JNK signaling and blocking the JNK pathway in dDad1 knock-down tissues suppresses cell apoptosis and partially restores organ size. In addition, reduction of dDad1 triggers ER stress and activates unfolded protein response (UPR) signaling, prior to the activation of JNK signaling. Furthermore, Perk-Atf4 signaling, one branch of UPR pathways, appears to play a dual role in inducing cell apoptosis and mediating compensatory cell proliferation in this dDad1 knock-down model (Zhang, 2016).

    Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster

    Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. Bioinformatic analyses was used to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy (M'Angale, 2016).

    In vivo biosensor tracks non-apoptotic caspase activity in Drosophila

    Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. This study illustrates the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals (Tang, 2016).

    Cellular aspects of gonadal atrophy in Drosophila P-M hybrid dysgenesis

    Gonadal atrophy is the most typical and dramatic manifestation of intraspecific hybrid dysgenesis syndrome leading to sterility in Drosophila melanogaster dysgenic progeny. The P-M system of hybrid dysgenesis is primarily associated with germ cell degeneration during the early stages of Drosophila embryonic development at elevated temperatures. This study has have defined the phase of germ cell death as beginning at the end of embryogenesis immediately following gonad formation. However, the temperature-dependent screening of germ cell developmental patterns in the dysgenic background showed that early germ cells are susceptible to the hybrid dysgenesis at any Drosophila life-cycle stage, including in the imago. Electron microscopy of germ cells after dysgenesis induction revealed significant changes in subcellular structure, especially mitochondria, prior to cellular breakdown. The mitochondrial pathology can promote the activation of cell death pathways in dysgenic germ cells, which leads to gonadal atrophy (Dorogova, 2017).

    Selective endosomal microautophagy is starvation-inducible in Drosophila

    Autophagy delivers cytosolic components to lysosomes for degradation and is thus essential for cellular homeostasis and to cope with different stressors. As such, autophagy counteracts various human diseases and its reduction leads to aging-like phenotypes. Macroautophagy (MA) can selectively degrade organelles or aggregated proteins, whereas selective degradation of single proteins has only been described for chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI). These 2 autophagic pathways, are specific for proteins containing KFERQ-related targeting motifs. Using a KFERQ-tagged fluorescent biosensor, this study identified an eMI-like pathway in Drosophila melanogaster. It was found that this biosensor localizes to late endosomes and lysosomes upon prolonged starvation in a KFERQ- and Hsc70-4- dependent manner. Furthermore, fly eMI requires endosomal multivesicular body formation mediated by ESCRT complex components. Importantly, induction of Drosophila eMI requires longer starvation than the induction of MA and is independent of the critical MA genes atg5, atg7, and atg12. Furthermore, inhibition of Tor signaling induces eMI in flies under nutrient rich conditions, and, as eMI in Drosophila also requires atg1 and atg13, these data suggest that these genes may have a novel, additional role in regulating eMI in flies. Overall, this study provides evidence for a novel, starvation-inducible catabolic process resembling endosomal microautophagy in the Drosophila fat body (Mukherjee, 2016).

    Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium
    Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, the contraction of actomyosin cables formed in apoptotic and neighboring cells drive cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. A model according which modulation of tissue tension represents a mechanism of apoptotic cell extrusion, and would further influence biochemical signals of neighboring non-apoptotic cells (Teng, 2016).

    This study reports the temporal sequence of events during apoptotic cell extrusion, with a focus on the remodeling of AJs, the cytoskeleton, and mechanical tension. After caspase-3 starts to be activated in the polyploid larval epithelial cells (LECs), those undergoing apoptosis initiate apical constriction. It was reasoned that the initiation of this constriction could be due to a combination of actomyosin cable formation in the dying cell and the activity of caspase-3, which assists in the upregulation of actomyosin contractility. Indeed, it has been shown in tissue culture that the cleavage of Rho associated kinase by caspase- 3 is involved in phosphorylation and activation of myosin light chain, which regulates actomyosin contractility. It is proposed that the actomyosin cable that forms in apoptotic LECs is responsible for the early stages of apoptotic cell extrusion. During apical constriction, the level of AJ components including E-cad strongly reduced in a caspase-3-dependent manner. In the neighboring non- dying cells, this reduction is found only at the interface between the apoptotic cell and its neighbors. Since caspase-3 is not activated in the neighboring cells, it is speculated that the reduction of E-cad is a consequence of a loss of trans-interactions between E-cad of the neighboring cell, and E-cad of the apoptotic cell, which undergoes caspase-3-dependent cleavage. This often, but not always, leads to plasma membrane separation, which is suggestive of a loosening of AJ-dependent adhesion. It has been reported that anillin organizes and stabilizes actomyosin contractile rings at AJs and its knock-down is associated with a reduction of E-cad and β-Catenin levels at AJs, leading to AJ disengagement. A gradual decrease in the level of E-cad, and a gradual increase in MyoII accumulation in apoptotic cells was observed prior to the strong reduction of E-cad levels. This lead to the hypothesis that mechanical tension exerted on the cell interface between apoptotic LECs and neighboring cells by the contraction of the actomyosin cable, which forms in the apoptotic cell, is large enough to rupture the weakened contacts between plasma membranes at AJs upon the strong reduction of E-cad levels (Teng, 2016).

    Interestingly, and by contrast, there are cases when AJs are not disengaged even after the level of E-cad is reduced. In these cases the cells exhibit a separation of actomyosin cables from the membrane. It is speculated that the state of cell-cell contacts at AJs, i.e., whether they will disengage or remain engaged during apoptosis, is dependent on which of the following links is weaker: The link between two plasma membranes, or the link between the plasma membrane and the actomyosin cable. Both of these links would be weakened by a strong, albeit incomplete, reduction of E-cad levels. When the former is weaker than the latter, the two plasma membranes could be detached. When the former is stronger than the latter, the two plasma membranes could remain in contact, and the actomyosin cable could be detached from the plasma membrane (Teng, 2016).

    In parallel with the reduction of E-cad levels and the associated release of tension, a supra-cellular actomyosin cable begins to form in neighboring cells. These observations prompted a speculation that the release of tissue tension triggers MyoII accumulation in neighboring cells. Subsequent contraction of this outer ring helps to reshape tissue tension, which is transiently released when E-cad is reduced. As a consequence, the neighboring cells are stretched. Upon completion of apical constriction, neighboring non-apoptotic cells form de novo AJs and the stretched cells undergo cell division and/or cell-cell contact rearrangement. These processes allow a relaxation of the high tension associated with the stretching of cells. Finally, measurements of caspase-3 activity, and the observations from caspase inhibition experiments, lead to a conclusion that the characteristics associated with apoptotic cell extrusion reported in this study are the consequences of the apoptotic process, rather than the cause (Teng, 2016).

    In addition to the progressive remodeling of AJs and modulation of tissue tension during apoptosis, the mechanical role was examined of apoptosis 'apoptotic force' in tissue morphogenesis, which has been proposed, demonstrated, and discussed. It was shown that the mechanical force generated by the contraction of actomyosin cables formed when LECs undergo apoptosis, especially boundary LECs, promotes tissue expansion, along with histoblast proliferation and migration. Nonetheless, it cannot be ruled out that this apical contraction is in part driven by a decrease in cell volume, which can be triggered by caspase activation. Intriguingly, it was found that apoptosis of non-boundary LECs did not affect tissue expansion. This raised the possibility that the mechanical influence of apoptosis in neighboring tissues is dependent not only on the physical connections between cells, but also on the mechanical properties of cells, including cell compliance. If a tissue is soft, for instance, the tensile forces generated by apoptotic process could be absorbed by nearest-neighbor cells and would not propagate to cells further than a single cell away. It is speculated that the apoptotic process could mechanically contribute to cell death-related morphogenesis, only when apoptosis takes place at optimal mechanical properties of a tissue (Teng, 2016).

    This study presents a framework for understanding how cell adhesions and tissue tension are progressively modulated during apoptosis in a developing epithelium. It is concluded that tissue tension reshaping, including the transient release of tension upon a reduction in the levels of AJ components, represents a mechanism of apoptotic cell extrusion. It would be important to explore how this transient modulation in mechanical tension would further influence the biochemical nature of neighboring non-apoptotic cells (Teng, 2016).

    Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila

    Autophagy defects lead to the buildup of damaged proteins and organelles, reduced survival during starvation and infections, hypersensitivity to stress and toxic substances, and progressive neurodegeneration. This study shows that, surprisingly, Drosophila mutants lacking the core autophagy gene Atg16 are not only defective in autophagy but also exhibit increased resistance to the sedative effects of ethanol, unlike Atg7 or Atg3 null mutant flies. This mutant phenotype is rescued by the re-expression of Atg16 in Corazonin (Crz)-producing neurosecretory cells that are known to promote the sedation response during ethanol exposure, and RNAi knockdown of Atg16 specifically in these cells also delays the onset of ethanol-induced coma. Atg16 and Crz colocalize within these neurosecretory cells, and both Crz protein and mRNA levels are decreased in Atg16 mutant flies. Thus, Atg16 promotes Crz production to ensure a proper organismal sedation response to ethanol (Varga, 2016).

    CCT complex restricts neuropathogenic protein aggregation via autophagy

    Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC (see Drosophila Tcp1-like). Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. This study shows that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, these findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity (Pavel, 2016).

    Genetic screen in Drosophila muscle identifies autophagy-mediated T-tubule remodeling and a Rab2 role in autophagy

    Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. This study uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. It was shown that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, the screens uncover an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms (Fujita, 2017).

    Differentiated muscle cells, or myofibers, are highly organized in order to coordinate the roles of specialized subcellular structures involved in contraction. Myofibril bundles of sarcomeres provide the contractile force. The power of contraction, however, requires synchronous sarcomere function under control of the 'excitation-contraction coupling' system that includes two membranous organelles, the sarcoplasmic reticulum (SR) and Transverse (T)-tubules (Al-Qusairi, 2011). The T-tubule membrane network is continuous with the muscle cell plasma membrane, with tubulated membranes that invaginate radially inward in a repeated pattern at each sarcomere. With excitation-contraction coupling, neuromuscular action potentials are transmitted along the muscle T-tubule membrane to the SR junction, or dyad/triad, triggering coordinated SR Ca2+ release and synchronous sarcomere contractions (Al-Qusairi, 2011). Formation of organized T-tubule membranes is thus critical for muscle function (Takeshima, 2015). Mechanisms must also remodel the T-tubule membrane network with ongoing myofiber reorganization in response to muscle use, damage, atrophy and aging. However, the extent and mechanisms of T-tubule remodeling remain largely unknown, in part due to challenges with observing T-tubule membrane network dynamics within intact mammalian myofibers (Fujita, 2017).

    The T-tubule network includes both transversal and longitudinal tubular membrane elements that form and mature with myofiber differentiation and growth. In mouse skeletal muscle, mostly longitudinal tubular membranes initially present in embryonic muscle are remodeled postnatally with expansion to predominantly transversal tubular elements. In contrast, both longitudinal and transversal T-tubule elements are maintained in adult mammalian cardiac muscle and in insect muscles. Relatively few molecular factors are known to shape the T-tubule network, and perhaps not surprisingly, all of which so far encode for membrane-associated functions (CAV3, DYSF, BIN1/Amph2, MTM1, DNM2). Mutations in each also are associated with human myopathy and/or cardiomyopathy with T-tubule disorganization, pointing to the critical importance of membrane-mediated mechanisms to maintain the T-tubule membrane network (Fujita, 2017).

    Drosophila is a powerful system for insights into the functional requirements for T-tubule formation and remodeling. The BIN1 BAR-domain protein has a conserved function involved in membrane tubulation required for T-tubule formation that was first described for the single Drosophila homolog, Amphiphysin. The amph null mutant flies lack transversal T-tubule element membranes in myofibers at all developmental stages, corresponding with both larval and adult mobility defects. In contrast, the myotubularin (mtm) fly homolog of mammalian MTM1/MTMR2/MTMR1 subfamily of phosphatidylinositol 3-phosphate phosphatases is required only at later stages in development for T-tubule remodeling. While mtm loss of function has no obvious effects on larval muscle T-tubule organization or function, mtm-depleted post-larval stage muscles lack transversal T-tubule membranes with adult mobility defects in eclosion and flight. Together, the amph and mtm mutant conditions that both lack transversal T-tubule elements in post-larval stage muscle yet different early development requirements underscores that distinct mechanisms are involved in T-tubule formation (amph-dependent) versus maintenance/remodeling (amph- and mtm-dependent) (Fujita, 2017).

    In Drosophila, a set of larval body wall muscles that persist as viable pupal abdominal muscles, called dorsal internal oblique muscles (IOMs), are essential for adult eclosion. During metamorphosis, changes in IOM cell size and myofibril content have been noted. Previous studies have shown that wildtype IOMs undergo dramatic cortical and membrane remodeling with costamere integrin adhesion complex disassembly and reassembly at discrete pupal stages (Ribeiro, 2011). In contrast, the mtm-depleted IOMs exhibited persistent disassembly or a block in reassembly of integrin costameres along with the loss of transversal T-tubule membranes at late pupal stages, but without any precocious cell death (Ribeiro, 2011). A striking feature in the mtm-depleted IOMs was the accumulation of endosomal-like membranes decorated with integrin and T-tubule markers, Amph and Discs large (Dlg1, a PDZ protein). Altogether, these results suggest that T-tubule membranes may undergo disassembly-reassembly with normal myofiber remodeling, including the delivery of disassembled T-tubule membrane into an endomembrane trafficking pathway. The role for a molecular-cellular program in control of T-tubule remodeling that is at least partially distinct from that involved in initial T-tubule formation raises many questions about possible mechanisms, including the regulation of T-tubule organization and dynamics, the membrane fate(s) and source(s) with disassembly-reassembly, respectively, and the specific membrane trafficking routes and effectors involved. Possible hints may come from studies of other specialized dynamic cell membrane invaginations shown to involve endosomal and Golgi membrane trafficking pathways, such as cellularization of Drosophila syncytial embryos and the tubulated demarcation membrane system in megakaryocyte platelet formation (Fujita, 2017).

    Membrane trafficking relies on the large family of Rab GTPases, with over sixty Rabs in humans and thirty in flies. The different Rabs are under distinct spatiotemporal regulation for recruitment, activation and functions at specific membrane compartments or domains. Guanine nucleotide exchange factors (GEFs) convert specific inactive GDP-bound Rabs to an active GTP-bound form. Active Rab-GTP then recruits a range of specific effector proteins to the membrane that mediate key trafficking functions, including cargo selection, vesicle budding, transport, tethering and fusion. Subsequently, GTPase-activating proteins (GAPs) deactivate Rabs by promoting GTP hydrolysis. Many membrane compartments have been defined by well-established localized functions of specific Rabs, for example: ER (Rab1), Golgi (Rab1, Rab6), secretory vesicles (Rab8), early endosomes (Rab5, Rab21), recycling endosomes (Rab11, Rab35), late endosomes (Rab7, Rab9), lysosomes (Rab7) and others. Thus, identifying the specific Rabs required for a cellular process can provide clues to potential underlying membrane trafficking mechanisms involved. However, examples exist of Rabs with multiple known sites of function or yet unknown functions, and conversely, certain cellular processes - like T-tubule remodeling - lack defined roles yet for any Rabs (Fujita, 2017).

    This study utilized the advantages of Drosophila IOMs to screen for Rab GTPases and related membrane trafficking functions required for T-tubule remodeling in intact muscle. The results show that the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and reassembled in IOMs during Drosophila metamorphosis. Autophagy, the membrane trafficking process for degradation of cytoplasmic contents by delivery to lysosomes, is upregulated with IOM remodeling where it plays an indispensable role for progression through T-tubule disassembly to reassembly. Genetic analysis of IOM remodeling also reveals an unexpected and broad role for Rab2 in autophagy in flies and mammals. From these data, it is proposed that Rab2 localizes to autophagosomes where it interacts with the HOPS complex, which in turn, mediates tethering and trans-SNARE complex formation with Rab7-marked lysosomes to promote autophagosome-lysosome fusion. Together, these results show that Drosophila IOM remodeling provides an unprecedented in vivo context for discovery and analysis of T-tubule dynamics with relevance to human myopathy, as well as an ideal system due to high membrane flux to study fundamental trafficking pathways (Fujita, 2017).

    This study has characterized a wildtype myofiber remodeling program by confocal and electron microscopy in intact muscles in vivo. In Drosophila IOMs during metamorphosis, the entire contractile and excitation-contraction coupling system, including T-tubules, are disassembled and then reassembled. This process highlights that myofibers harbor distinct programs for initial T-tubule formation versus regulated T-tubule remodeling. This likely includes additional mechanisms for T-tubule membrane disassembly and renovation, features that reflect those seen with mammalian myofiber atrophy and recovery. The Drosophila body wall muscles provide an unprecedented system permitting a combination of powerful visualization and systematic perturbation analysis, including the first genetic screens, of T-tubule dynamics and organization (Fujita, 2017).

    Autophagy is upregulated with the onset of IOM remodeling during metamorphosis. Further, disruption of autophagy initiation, autophagosome formation or clearance all induced loss of T-tubules with a block in IOM remodeling at/after T-tubule disassembly. This is the first report of a non-cell death role of autophagy in Drosophila metamorphosis. The role of autophagy in IOMs that persist and redifferentiate during metamorphosis is clearly different from its roles in pupal midgut and salivary gland cells that undergo autophagic forms of cell death. There are multiple speculative direct or indirect role(s) for autophagy specifically in T-tubule membrane remodeling: (1) a direct role in T-tubule membrane recycling, as a means to deliver disassembled T-tubule membrane via autophagosomes to lysosomes or related organelles for intracellular storage, then later redeployed to contribute to T-tubule reassembly; (2) an indirect role in cell renovation, including T-tubule membrane clearance, to permit cell space for redifferentiation; or (3) an indirect role in cell metabolism, to support cell survival and/or the energy cost of redifferentiation with starvation during metamorphosis. Most likely, autophagy serves some combination of these roles in IOM remodeling (Fujita, 2017).

    How could autophagy play a direct role in T-tubule remodeling? It was surprising that mCD8:GFP-positive small vesicles accumulated to a similar degree as autophagosome numbers in IOMs when autophagosome-lysosome fusion was blocked. This suggests that mCD8:GFP localizes to autophagosomes during IOM remodeling. It is possible that T-tubule membranes are a source of autophagosomal membrane, at least in part: mCD8:GFP labels the muscle plasma membrane and T-tubules in larval muscle precursor cells of IOMs, and T-tubule disassembly coincides with the upregulation in autophagy early in metamorphosis. Also, disruption of autophagy induction blocked normal progression in disassembly and remodeling of T-tubule-derived mCD8:GFP-marked membranes. In the absence of autophagy initiation, mCD8:GFP-positive stacked membranes were observed, likely retained or partially disassembled T-tubules. It is proposed that T-tubules are remodeled through autophagosomes. It is important to note that T-tubules are not an apparent autophagic cargo, but instead, a possible source of autophagosome membrane. In this scenario, T-tubules are disassembled into autophagosomes and then reassembled from subsequent autolysosome-related structures, both of which successively increased in numbers during wildtype IOM remodeling (Fujita, 2017).

    Alternatively or additionally, other roles for autophagy could indirectly impact T-tubule remodeling. Extensive IOM atrophy with nearly complete disassembly of the contractile and excitation-contraction systems by 1d APF is followed by a rapid re-differentiation within hours after 3.5d APF. Autophagy could be required to simply clear away and degrade the old contraction systems in order to make space to rebuild and realign new systems, as well as permit the normal central repositioning of nuclei away from the myofiber cortex. However, the persistent block in early IOM remodeling with autophagy disruption suggests that the remodeling normally proceeds through a progression of interrelated steps rather than independent programs for disassembly and reassembly. Autophagy also has a well-established role in metabolic homeostasis through the recycling of amino acids and turnover of damaged mitochondria in the lysosome. The current data suggest that mitochondria are a major autophagic cargo with IOM remodeling. In conditions that disrupted autophagy initiation (Atg1, Atg18 RNAi), the cytoplasm was abnormally filled with mitochondria in IOMs at 4d APF. Consistent with that, a significant portion of autophagosomes harbored intact mitochondria when autophagosome-lysosome fusion was blocked (Rab2, Rab7 or Stx17 RNAi). This is different from observations in larval muscle, in which mitochondria were notably absent in autophagosomes that accumulated with a block in autophagy. It is possible that mitophagy, a selective form of autophagy for mitochondrial turnover, is upregulated and could play both metabolic and cell renovation roles in IOM remodeling. Interestingly, the autophagy-blocked IOMs remained viable throughout metamorphosis, suggesting that autophagy is not absolutely required for cell survival through the starvation with metamorphosis (Fujita, 2017).

    Through a systemic screen of all Drosophila Rab GTPases, an unexpected role was uncovered for Rab2 in autophagy. The striking Rab2 RNAi IOM phenotype was shared with RNAi of other functions known to be specifically required for autophagosome-lysosome fusion. Genetic blockade of autophagosome-lysosome fusion resulted in a dramatic phenotype, with massive accumulations of autophagosomes within IOMs. Previously, autophagosome-lysosome fusion was shown to involve the cooperative functions of Rab7, the HOPS tethering complex, and a trans-SNARE complex between Stx17, SNAP29 and VAMP7/8. Among these tethering and fusion functions, it has been shown that Stx17 (a hairpin SNARE) is recruited to autophagosomal membranes, while Rab7 and VAMP7/8 localize to endolysosomal membranes. Stx17 localizes to autophagosomes as well as to the ER and mitochondria, but the HOPS complex directly associates and colocalizes with Stx17 only at autophagosomes (Jiang et al., 2014; Takáts et al., 2014). This suggests that Stx17 is not a sole determinant for HOPS complex recruitment (Fujita, 2017).

    It is proposed that Rab2 is required for the autophagosomal recruitment of the HOPS complex. Rab2 specifically localized to completed autophagosomes, and Rab2 had an affinity with the HOPS complex, as does Stx17. It is envisioned that upon completion of autophagosome biogenesis/maturation, Rab2 and Stx17 are recruited to the outer autophagosomal membrane. Then, the HOPS complex is subsequently recruited to autophagosomes in a Rab2-depedent manner through coincident interactions with both Stx17 and Rab2 (see Hierarchal analysis of Rab2 and factors involved in autophagosome-lysosome fusion). At the same time, the HOPS complex binds Rab7 on lysosomes. In turn, the HOPS complex tethers autophagosomes and lysosomes to promote trans-SNARE complex formation between Stx17, SNAP29 and Vamp7/8 and ultimately autophagosome-lysosome fusion (Fujita, 2017).

    Rab2 role in autophagy discovered in fly muscle relates to a broader autophagy requirement in other cell types and across species. The localization of Rab2 on autophagosomes in Drosophila IOMs was conserved for both Rab2A and Rab2B in mouse embryonic fibroblasts (MEFs). As in flies, the Rab2A/2B double knockout led to a delay or block in autophagy clearance as indicated by accumulation of LC3/Atg8. However, the specific Rab2 loss-of-function phenotypes were not identical. While Rab2 was required for autophagosome-lysosome fusion in fly IOMs, the Rab2A/2B double knockouts in MEFs indicated a requirement at a later step in autophagic clearance. Interestingly, this disparity in autophagy phenotypes across species is also seen with Rab7. In flies and yeast, Rab7/Ypt7 is essential for autophagosome-lyososome/vacuole fusion, while mammalian Rab7 knockdowns more clearly indicate a required role in autolysosome maturation. Other examples indicate that the autophagosome-lysosome fusion machinery is not highly evolutionarily conserved. The Stx17-SNAP29-VAMP7/8 trans-SNARE complex is conserved in Drosophila and mammals, but not in yeast, where no autophagosomal SNARE has been reported so far. Moreover, budding yeast do not encode for Rab2 (Fujita, 2017).

    Altogether, it is plausible that Rab2 is required for autophagosome-lysosome fusion efficiency, and Rab2-dependency is variable across different tissues or species. Two possible models could explain the different Rab2 autophagy requirements in flies and mouse cells. First, it is suggested that autophagosomes sequentially fuse with endosomes then lysosomes to become amphisomes and autolysosomes, respectively. If either of the steps requires Rab2A/2B, then intermediates with partially degraded contents could accumulate in double knockout MEFs. Alternatively, an autophagosome may normally fuse with multiple lysosomes to ensure full degradation of its contents. In the absence of Rab2A/2B in MEFs, autophagosomes could still fuse but not with a sufficient number of lysosomes, resulting in an accumulation of partially digested autolysosomes (Fujita, 2017).

    Rab2 has been previously associated with transport events at the Golgi apparatus, ER-to-Golgi traffic and secretory granule formation, as well as in a C. elegans endocytic/phagocytic pathway. Gillingham et al. systematically explored Rab effectors in Drosophila cultured cells, and found that Rab2 interacts with the HOPS complex besides known Golgi-resident effectors (Gillingham, 2014). The interaction between Rab2 and HOPS complex is also conserved in mammals, and the unexpected Rab2 localization to autophagosomes was found. Thus, it is likely that Rab2 exerts multiple functions through interaction with different effectors at different places. A possible Rab2 function in the endosome-lysosome system that affects autophagic flux cannot be excluded, although no clear lysosomal defects were detected in Rab2A/B knockout MEFs. Several other factors that localize to autophagosomes or late endosomes-lysosomes, including Atg14, PLEKHM1 and EPG5, have been shown to control autophagosome maturation. It is plausible that Rab2 contributes to autophagosome maturation through both a direct role in the fusion mechanism and an indirect role in endo-lysosome maturation, the same as Rab7 and the HOPS complex (Fujita, 2017).

    How Rab2 localizes to autophagosomes remains unclear. Localization of Rab2 on autophagosomes in IOMs did not depend on HOPS complex subunits, Vps39 and Vps41, or on Stx17. Further studies will be needed to determine the identities of the Rab2 guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) that regulate Rab2 GTPase activity in autophagosome-lysosome fusion. A conserved TBC domain protein, OATL1/TBC1D25, is a strong candidate for a Rab2 GAP, given OATL1 localization to autophagosomes and involvement in autophagosome-lysosome fusion. Further, it was reported that OATL1 directly bound to and showed GAP activity for Rab2A (Fujita, 2017).

    Autophagy is critical for the maintenance of myofiber homeostasis in mammalian skeletal muscle. It is known that several myopathies are associated with excess accumulation of autophagic structures in muscle. Further, loss of autophagy in mouse skeletal muscle shows anomalies, including abnormal mitochondria, disassembled sarcomeres and disorganized triads, as also seen in aged muscle. It is established that autophagy is down-regulated during the course of aging. This evidence points to a possible significance of autophagy in myofiber remodeling and in T-tubule maintenance. Jumpy/MTMR14 PI3-phosphatase and Dynamin-2 (DNM2) GTPase, two causative genes of human centronuclear myopathy, are required for not only T-tubule maintenance but also proper progression of autophagy. Based on these reports and the current findings, it is speculated that their roles in T-tubule maintenance are mediated, at least in part, through autophagy (Fujita, 2017).

    Signaling pathways that regulate atrophy and hypertrophy in Drosophila have been identified, however, the mechanisms and direct mediators of muscle remodeling remain largely unknown. IOM remodeling is a good model to study the mechanisms of muscle remodeling, given that the signaling pathways that control muscle remodeling are conserved between Drosophila and mammals. Advantages of the IOM system are not only its genetic tractability, but also its reproducibility and structure. As a relatively giant single cell along the body wall, IOMs enable tracking of a single cell and its subcellular organization during metamorphosis. The results show that studies in IOMs can provide new insights into the mechanisms of muscle remodeling as well as regulation of fundamental membrane trafficking pathways, such as autophagy and endocytosis (Fujita, 2017).

    Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy

    The small GTPase Rab5 promotes recruitment of the Ccz1-Mon1 guanosine exchange complex to endosomes to activate Rab7, which facilitates endosome maturation and fusion with lysosomes. How these factors function during autophagy is incompletely understood. This study shows that autophagosomes accumulate due to impaired fusion with lysosomes upon loss of the Ccz1-Mon1-Rab7 module in starved Drosophila fat cells. In contrast, autophagosomes generated in Rab5 null mutant cells normally fuse with lysosomes during the starvation response. Consistent with that, Rab5 is dispensable for the Ccz1-Mon1-dependent recruitment of Rab7 to PI3P-positive autophagosomes, which are generated by the action of the Atg14-containing Vps34 PI3 kinase complex. Finally, Rab5 was found to be required for proper lysosomal function. Thus, the Ccz1-Mon1-Rab7 module is required for autophagosome-lysosome fusion, whereas Rab5 loss interferes with a later step of autophagy: the breakdown of autophagic cargo within lysosomes (Hegedus, 2016).

    Autophagy ensures the lysosomal degradation of self-material, including cytosol and organelles. During the main pathway, double-membrane autophagosomes serve as the transport vesicles. Endocytosis delivers plasma membrane, including transmembrane receptors, and exogenous substances taken up from the environment to lysosomes. Thus autophagy and endocytosis converge at the level of lysosomes, where degradation of cargo arriving from both routes takes place (Hegedus, 2016).

    A critical event during these transport processes is vesicle maturation: how the newly formed vesicles acquire the molecular characteristics and protein complexes that establish their identity and determine the subsequent vesicle fusion events that often culminate in the lysosomal compartment. Several similarities between endosomes and autophagosomes are known. For example, both autophagosomes and endosomes are positive for phosphatidylinositol-3-phosphate (PI3P) due to localized vacuolar protein sorting 34 (Vps34) PI3 kinase activity, which has been showed to be required for the generation of both types of vesicles in Drosophila larvae. Autophagosomes can also fuse with endosomes to give rise to hybrid organelles termed amphisomes, which then fuse with lysosomes (Hegedus, 2016).

    Small GTPases of the Ras-related protein in brain (Rab) family are critical regulators of membrane trafficking in eukaryotic cells. An active, GTP-bound Rab protein binds to various effectors that usually regulate vesicle motility and fusion with the proper membrane compartment. In the endocytic pathway, Rab5 associates with early endosomes and activates a Vps34-containing phosphoinositide 3-kinase complex that generates PI3P on the surface of these vesicles. PI3P-binding domains such as the Fab-1, YGL023, Vps27, and EEA1 (FYVE) domain promote recruitment to early endosomes. Of importance, several proteins, including the vesicle tethers early endosomal antigen 1 (EEA1) and Rabenosin-5, have both FYVE and Rab5-binding domains, indicating that multiple interactions may play a role in the recruitment of effectors. Similarly, the Rab7 guanine nucleotide exchange factor (GEF) monensin sensitivity protein 1 (Mon1)-caffeine, calcium, and zinc 1 (Ccz1) complex binds to both the GTP-bound form of endosomal Rab5 and PI3P. Rab7 is then activated by this complex and promotes fusion of late endosomes and lysosomes (Hegedus, 2016).

    Recruitment of the soluble N-methylamaleimide-sensitive factor attachment protein receptor (SNARE) Syntaxin 17 is a critical step in autophagosome maturation because these vesicles acquire fusion competence this way. Interaction of Syntaxin 17 with the homotypic fusion and vacuole protein sorting (HOPS) tethering complex ensures efficient fusion between autophagosomes and lysosomes. HOPS is believed to be a Rab7 effector, and Rab7 was indeed found to promote the formation of degradative autolysosomes in cultured cells, although it remains to be established whether this protein is already present on autophagosomes before the fusion with lysosomes. In theory, the binding of HOPS to lysosomal Rab7 and autophagosomal Syntaxin 17 (and other factors, such as phospholipids) may be sufficient for its tethering activity. In addition, autophagy-related gene 14 (Atg14), a Vps34 kinase complex subunit that is important for autophagosome formation, also functions as a tether and promotes autophagosome-lysosome fusion by directly binding to Syntaxin 17 (Hegedus, 2016 and references therein).

    In yeast, the fusion machinery differs somewhat from that of the animal cells because the SNAREs involved are not homologous. Still, autophagosome fusion with the vacuole (the equivalent of the lysosomal system in metazoan cells) requires HOPS, Ypt7/Rab7, and its GEF, the Mon1-Ccz1 complex, and more recently, autophagosome-like structures were found to accumulate in yeast cells lacking the major Rab5 homologue Vps21. Of interest, decreased Rab5 function attenuates the autophagic degradation of the pathogenic, mutant form of huntingtin in cultured human cells. This was attributed to impaired Vps34 lipid kinase activity and reduced formation of the Atg5-Atg12 conjugate, both of which are important for autophagosome formation (Hegedus, 2016).

    Thus the role of the Rab5-Ccz1-Mon1-Rab7 axis during autophagy is not clear. This study set out to address this problem in the popular animal model Drosophila. Fruit flies offer certain advantages for such studies. First, there is only a single fly homologue of Rab5 (unlike in mammalian and yeast cells, which both have three different Rab5 proteins). Second, massive induction of autophagy is seen in the fat and liver tissue-like fat cells of starved larvae. Third, it is straightforward to carry out functional studies in mosaic animals, in which mutant cells are surrounded by control cells in the same tissue of the same animal, which reduces variability because one can compare the phenotype of neighboring control and loss-of-function cells. Using this system, Ccz1, Mon1, and Rab7 are shown to be required for autophagosome-lysosome fusion in fat cells of starved animals independent of Rab5. Of interest, Rab5 was found to function downstream of the Rab7 module by controlling a later step of autophagy: degradation of autophagic cargo within lysosomes (Hegedus, 2016).

    This study showed that the Rab7 module and Rab5 control different steps of auto­phagy. Rab7 mediates autophagosome-lysosome fusion together with its GEF, the Ccz1-Mon1 complex. This is likely achieved by the recruitment of Rab7 to autophagosomes in a Ccz1-Mon1-dependent manner. Although Drosophila Mon1 binds to the active, GTP-locked form of Rab5 as in other organisms, Rab5 is dispensable for the fusion of autophagosomes with lysosomes and for Rab7 localization to autophagosomes and autolysosomes. The question is then: what is the signal that recruits Ccz1-Mon1 and Rab7 to autophagic structures? (Hegedus, 2016).

    Mon1 and Ccz1 bind to phospholipids, including PI3P, in yeast, and this study found that Drosophila Mon1 has similar features. This raises the possibility that the Ccz1-Mon1 complex is recruited to the PI3P-positive surface of autophagosomes through this interaction. Vps34-dependent PI3P generation is required for autophagosome formation and endosome maturation. Vps34 is activated by Rab5. Of interest, the current data suggest that loss of Rab5 inhibits PI3P generation only on endosomes but not on autophagosomes. Loss of UVRAG but not Atg14 inhibits PI3P generation on endosomes, whereas loss of Atg14 leads to complete inhibition of PI3P-positive autophagosome biogenesis. Thus UVRAG is dispensable for Vps34 activity during autophagosome formation, and its loss causes a defect in autolysosomal degradation. Similarly, Rab5 mutant cells showed accumulation of autophagic cargo due to impaired lysosomal degradation (Hegedus, 2016).

    Recently the Rab5-related Vps21 small GTPase was suggested to control the fusion of autophagosome with the vacuole (lysosome) in yeast cells. In this study, clusters of autophagic structures were found to accumulate near the vacuole. However, these vesicles were positive for both the autophagy marker GFP-Atg8 and the vacuolar marker FM4-64, suggesting that some sort of fusion must have occurred in this case, too (Hegedus, 2016).

    On the basis of the current results, the following model is proposed of autolysosome formation in fat cells of starved Drosophila larvae (see The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy). PI3P-positive autophagosomes are generated through the action of an Atg14-containing Vps34 PI3 kinase complex. PI3P attracts Ccz1-Mon1, which promotes Rab7 recruitment to autophagosomes. Both PI3P and Rab7 bind to the HOPS tethering complex, and thus these factors promote the tethering of autophagosomes with late endosomes and lysosomes. The membrane fusion is then executed by the Syx17-Snap29-Vamp7 SNARE complex. Autophagic cargo is broken down in autolysosomes, and their full degradative capacity requires the function of Rab5 and the UVRAG-containing Vps34 complex for the proper delivery of lysosomal proteins, likely including both acidic hydrolases and membrane proteins. This is in line with the finding that simultaneous knockdown of all three Rab5 homologues leads to a collapse of the endolysosomal system in mouse liver cells (Hegedus, 2016).

    It has already been demonstrated that autophagosome-lysosome fusion is mediated by the HOPS tethering complex and the SNAREs Syx17, Snap29, and Vamp7/8. It is not yet clear how these fusion factors are recruited to the autophagosomal membrane. HOPS is known as a Rab7 effector, and according to the current findings, Rab7 is present on autophagosomes. It is proposed that autophagosomal PI3P recruits the Ccz1-Mon1-Rab7 module to facilitate the loading of HOPS and subsequent tethering of vesicles (Hegedus, 2016).

    Vps34 is considered as a bona fide Rab5 effector. Surprisingly, this study found that whereas Rab5 mediates only the generation of PI3P on endosomes mainly through the action of a UVRAG-containing Vps34 complex, it is dispensable for PI3P-positive autophagosome biogenesis, which depends on the Atg14-containing Vps34 complex. Thus the current concept that Vps34 is a Rab5 effector must be revisited: it is true for endocytosis but not applicable for autophagy in fat cells of starved Drosophila larvae (Hegedus, 2016).

    A previous study showed that Rab5 promotes autophagy-mediated huntingtin clearance in cultured human cells and Drosophila eyes. Simultaneous small interfering RNA knockdown of all three Rab5 genes (Rab5a, Rab5b, Rab5c) reduced the level of Atg5-Atg12 conjugate and autophagosome formation. Although no perturbations of autophagosome biogenesis and fusion were seen in Rab5 mutant fat cells, these discrepancies may be due to the different models used. In the current experiments, starvation induces a massive wave of autophagy in larval Drosophila fat cells that entirely relies on the activity of the Rab5-independent Atg14-Vps34 PI3 kinase complex. It is possible that during the basal, nonstarved conditions in a previous study, Rab5 can contribute to autophagosome formation. In fact, UVRAG has also been suggested to control autophagosome formation in cultured cells, which is compatible with this model (Hegedus, 2016).

    In summary, Rab7 is recruited to autophagosomes by the Ccz1-Mon1 complex to promote autophagosome-lysosome fusion. This study show that autophagosome formation and fusion is independent of Rab5 and the UVRAG-containing Vps34 PI3 kinase complex but requires the action of the Atg14-Vps34 complex. Rab5, similar to UVRAG, is necessary for proper lysosomal function by promoting the trafficking of lysosomal proteins (Hegedus, 2016).

    Microenvironmental autophagy promotes tumour growth

    As malignant tumours develop, they interact intimately with their microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How tumours regulate autophagy in vivo and whether autophagy affects tumour growth is controversial. This study demonstrates, using RasV12scrib−/− tumour cells, a well characterized Drosophila melanogaster malignant tumour model, that non-cell-autonomous autophagy is induced both in the tumour microenvironment and systemically in distant tissues. Tumour growth can be pharmacologically restrained using autophagy inhibitors, and early-stage tumour growth and invasion are genetically dependent on autophagy within the local tumour microenvironment. Induction of autophagy is mediated by Drosophila tumour necrosis factor (Eiger) and interleukin-6-like signalling (Unpaired) from metabolically stressed tumour cells, whereas tumour growth depends on active amino acid transport. Dormant growth-impaired tumours from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. It is concluded that transformed cells engage surrounding normal cells as active and essential microenvironmental contributors to early tumour growth through nutrient-generating autophagy (Katheder, 2017).

    EndoA/Endophilin-A creates docking stations for autophagic proteins at synapses

    Synapses are very specialized compartments with high metabolic demand to maintain neurotransmission, an essential step for basic brain function. Neurons are post-mitotic and synapses need to stay functional over time-sometimes over decades. Given that synapses are often at a long distance from the cell body, they must use local mechanisms to regulate protein quality control. This study shows that macroautophagy/autophagy is one of these local processes and found that it is under strict control of the synapse-enriched protein EndoA/Endophilin-A, previously only implicated in endocytosis. Metabolic and neuronal stimulation induce synaptic autophagy and phosphorylation of EndoA by the Parkinson disease kinase Lrrk/LRRK2 is essential to promote the process. EndoA induces membrane curvature in vitro, and, mechanistically, phosphorylated EndoA creates curved membrane-protein docking sites that are capable of recruiting Atg3. This work reveals a synapse-enriched branch of autophagy under the control of EndoA that may be deregulated in Parkinson disease (Soukup, 2017).

    Heparan sulfate proteoglycans regulate autophagy in Drosophila

    Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. The Drosophila NMJ provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. Evidence is provided that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations (Reynolds-Peterson, 2017).

    Rab2 promotes autophagic and endocytic lysosomal degradation

    Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. This study shows that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. This study demonstrates that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. RAB2A is also required for autophagosome clearance in human breast cancer cells. In conclusion, Rab2 has been identified as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes (Lorincz, 2017).

    The two main pathways of lysosomal degradation are endocytosis and autophagy. Double-membrane autophagosomes (generated in the main pathway of autophagy) and endosomes can fuse with each other to generate amphisomes, and mature into degradative endo- and autolysosomes, respectively, by ultimately fusing with lysosomes. One of the main regulators of intracellular trafficking and vesicle fusions are Rab small GTPases. Active, GTP-bound Rab proteins recruit various effectors including tethers and molecular motors, of which Rab7 is the only known direct regulator of both autophagosome-lysosome and endosome-lysosome fusions (Lorincz, 2017).

    The tethering complex homotypic fusion and vacuole protein sorting (HOPS) was identified in yeast, and it simultaneously binds two yeast Rab7 (Ypt7) molecules on its opposing ends. In animal cells, Rab7 binds to RILP, ORPL1, FYCO1, and PLEKHM1 to recruit dyneins and HOPS and ensure the fusion of late endosomes and autophagosomes with lysosomes. This way, HOPS could cross-link two Rab7-positive membranes to prompt tethering and fusio. Rab7 is present on lysosomes, autophagosomes, and endosomes, but it is not clear whether another Rab is involved in degradative auto- and endolysosome formation, which also requires transport of hydrolases from the Golgi (Lorincz, 2017).

    Rab2 is known to control anterograde and retrograde traffic between the ER and Golgi. A recent biochemical screen identified Rab2 as a direct binding partner of HOPS, and active Rab2 was found to localize to Rab7-positive vacuoles in cultured Drosophila melanogaster cells. This study proposes an updated model in which Rab7 and Rab2 coordinately promote the HOPS-dependent degradation of autophagosomes and endosomes via fusion of these as well as biosynthetic vesicles with lysosomes (Lorincz, 2017).

    Rab2 is highly conserved among higher eukaryotes, including Drosophila melanogaster and humans. The HOPS subunits Vps39 and Vps41 directly bind to Ypt7/Rab7 in yeast, whereas their interaction may be indirect in mammalian cells. No binding was detected between Drosophila Rab7 and Vps39 or Vps41, whereas GTP-locked Rab7 bound to its known effector PLEKHM1 in yeast two-hybrid (Y2H) experiments. Vps39 directly bound Rab2GTP in both Y2H and recombinant protein pull-down experiments, and Rab2GTP immunoprecipitated endogenous Vps16A (another HOPS subunit) from fly lysates. Consistently, it has been reported that recombinant mammalian RAB2A pulls down Vps39 but not Vps41 from cell lysates, and human HOPS subunits did not show Rab7 binding in Y2H experiments (Lorincz, 2017).

    To address whether Rab2 functions in autophagy and endocytosis, rab2 was knocked out by imprecise excision of a transposon from the 5' UTR. The resulting rab2d42 allele carries a 2,047-bp deletion, which removes most of the protein coding sequences of both predicted Rab2 isoforms and eliminates protein expression. Rab2 mutant animals die as L2/L3-stage larvae, and their viability is fully rescued by expression of YFP-Rab2 (Lorincz, 2017).

    Larval fat cells are widely used for autophagy analyses because of their massive autophagic potential. Numerous Lysotracker Red (LTR)-positive vesicles appear upon starvation, which represent newly formed autolysosomes with likely increased v-ATPase-mediated acidification in these cells. LTR dot number and size (and signal intensity as a likely consequence) decreased in rab2-null cells compared with controls, which was rescued by expression of YFP-Rab2. RNAi knockdown of Rab2 in GFP-marked fat cell clones also impaired starvation-induced punctate LTR staining compared with surrounding GFP-negative cells (Lorincz, 2017).

    A 3xmCherry-Atg8a reporter that labels all autophagic structures via retained fluorescence of mCherry inside autolysosomes revealed increased number and decreased size of such vesicles in both starved rab2 RNAi and mutant fat cells. A dLamp-3xmCherry reporter of late endosomes and lysosomes showed similar changes in rab2 RNAi or mutant fat cells of starved animals. Tandem tagged mCherry-GFP-Atg8a reporters are commonly used to follow autophagic flux, because GFP is quenched in lysosomes, whereas mCherry signal persists. Knockdown of rab2 prevented the quenching of GFP that is seen in starved control fat cells: dots positive for both GFP and mCherry accumulated, raising the possibility that Rab2 promotes autophagosome-lysosome fusion, similar to HOPS. Colocalization of 3xmCherry-Atg8a with the lysosomal hydrolase cathepsin L (CathL) was examined. The overlap of these markers of autophagic and lysosomal structures strongly decreased in rab2 mutant fat cells compared with controls, and rab2 RNAi also impaired endogenous CathL-positive vesicle formation, suggesting that formation of degradative autolysosomes requires Rab2 (Lorincz, 2017).

    These phenotypes resembled the autophagosome-lysosome fusion defect of mutants for the autophagosomal SNARE syntaxin 17, HOPS, and Rab7. Accordingly, ultrastructural analysis of starved fat cells revealed accumulation of double-membrane autophagosomes and small dense structures likely representing amphisomes, similar to HOPS mutants. Recently, rab2 RNAi was reported to cause accumulation of autophagosomes in Drosophila muscles and enlarged amphisomes in fat cells. Autophagosome accumulation in our rab2-null mutant fat cells is likely caused by a complete loss-of-function condition (Lorincz, 2017).

    Western blots detected increased levels of the selective autophagy cargo p62/Ref2p, along with both free and lipidated autophagosome-associated forms of Atg8a in starved rab2 mutants. Basal autophagic degradation was also impaired in rab2 mutants, based on increased numbers of endogenous Atg8a and p62 dots in well-fed conditions (Lorincz, 2017).

    The importance of Rab2 for autophagic degradation was confirmed in human cells. Knockdown of RAB2B had no effect on endogenous LC3 structures in breast cancer cells, whereas RAB2A or combined siRNA treatment caused accumulation of autophagic vesicles. LC3 accumulated within Lamp1-positive structures upon RAB2A knockdown, which likely represent amphisomes unable to mature into autolysosomes in these cells, consistent with the recently reported role of Rab2 homologs for degradation of autophagic cargo in mouse embryonic fibroblasts (Lorincz, 2017 and references therein).

    To analyze the possible involvement of Drosophila Rab2 in endosomal degradation, dissected nephrocytes were incubated with fluorescent avidin for 5 min. Trafficking of this endocytic tracer was clearly perturbed in rab2 mutant cells, similar to vps41/lt and rab7 mutants. Loss of HOPS leads to enlargement of late endosomes. Similarly, Rab7 endosomes are enlarged in rab2 mutant nephrocytes compared with control or rescued cells. Importantly, fluorescent avidin was trapped in Rab7 endosomes and failed to reach CathL-positive lysosomes after a 30-min chase in rab2 mutants. LTR staining showed the presence of acidic vacuoles in rab2 mutant nephrocytes, which probably include the enlarged late endosomes in rab2 mutant nephrocytes, based on ultrastructural analysis . Aberrant late endosomes accumulated in mutant cells, which were apparently unable to fuse with neighboring acid phosphatase-positive lysosomes. Of note, the number of acid phosphatase-positive lysosomes also decreased in mutant nephrocytes, suggesting that Rab2 promotes both endosome-lysosome fusion and biosynthetic transport to lysosomes (Lorincz, 2017).

    GTP-locked, constitutively active Rab2GTP redistributes from the Golgi onto Rab7 vacuoles in cultured Drosophila cells. Similarly, Rab2GTP colocalized with endogenous Rab7 in starved fat cells, unlike wild-type Rab2. Rab2GTP appeared as large pronounced rings around LTR-positive autolysosomes in starved fat cells, unlike wild-type Rab2. Similarly, Rab2GTP formed rings around lysosomes and autophagic structures marked by dLamp-3xmCherry and 3xmCherry-Atg8a, respectively. Of note, small Rab2GTP dots often closely associated with large Rab2GTP rings in these experiments, raising the possibility that Rab2 vesicles fuse with autolysosomes. Finally, wild-type Rab2 or Rab2GTP modestly overlapped with autophagosomes marked by endogenous Atg8a (Lorincz, 2017).

    These localization and loss-of-function data pointed to Rab2 as a positive regulator of autolysosome formation. Indeed, fat and midgut cells expressing Rab2GTP contained enlarged and brighter 3xmCherry-Atg8a autophagic structures and dLamp-3xmCherry lysosomes compared with surrounding control cells, suggesting that Rab2 controls autolysosome size. Increased lysosomal input or a block of degradation can cause enlargement of autolysosomes. Systemic expression of Rab2GTP did not impair the viability of animals, and Western blots of starved L3 larval lysates revealed no changes in p62 and Atg8a levels, suggesting that autophagic degradation proceeds normally in cells expressing Rab2GTP. Thus, Rab2GTP may increase autolysosome size by accelerating fusions with other vesicles. Importantly, expression of GTP-locked, active Rab7 did not increase the size of autophagic structures. Rab7 is required for autophagosome-lysosome fusion, and its knockdown prevents the formation of large, bright 3xmCherry-Atg8a-positive autolysosomes: these cells contain only small, faint autophagosomes. Similarly, only small, faint 3xmCherry-Atg8a dots appeared in Rab2GTP-expressing fat cells undergoing Rab7 RNAi, indicating that Rab2-dependent fusions also require Rab7 and there is no functional redundancy between them (Lorincz, 2017).

    Eye pigment granules are lysosome-related organelles. Changes in lysosomal transport often lead to eye discoloration caused by pigment granule alterations, such as in HOPS mutants. Rab2GTP expression led to a slight darkening of eyes and appearance of enlarged pigment granules, consistent with the role of Rab2 in promoting lysosomal fusions (Lorincz, 2017).

    Several homo- and hetero-typic fusions occur during endosome and autophagosome maturation into degradative lysosomes. Known metazoan factors acting at lysosomal fusions include HOPS and EPG5 tethers and Rab7 together with its effectors. Because biosynthetic transport to lysosomes also requires input from Golgi, the role of Golgi-associated Rab2 in various lysosomal fusions fits well into this picture. Consistently, Rab2 promotes breakdown of phagocytosed apoptotic bodies and lysosome-related acrosome biogenesis (Lorincz, 2017).

    Accumulation of unfused autophagosomes and enlarged late endosomes in rab2 mutants resembles the fusion defect of rab7 mutant cells. The decreased function of lysosomes in rab2 mutants is unlikely to account for these fusion defects, because we have shown that autophagosome-lysosome fusion proceeds and gives rise to enlarged, nondegrading autolysosomes in fat cells with perturbed acidification or biosynthetic transport to lysosomes (Lorincz, 2017).

    The role of Rab2 in the fusion of lysosomes with other vesicles is also supported by the autolysosomal localization of its active form and by its binding to the Vps39-containing end of HOPS, the tethering complex required for autophagosomal, endosomal, and biosynthetic transport to lysosomes. Consistently, Rab2 recruits HOPS to Rab7-positive vesicles in cultured Drosophila cells. Expression of Rab2GTP increases degradative autolysosome and pigment granule size, suggesting that it is rate limiting during these fusion reactions, unlike Rab7. This is supported by low levels of wild-type Rab2 on these organelles, unlike wild-type Rab7 that is abundant on autophagosomes, late endosomes, and lysosomes. Consistent with this, it has been recently shown that expression of RAB2AGTP also increases Rab7 vesicle size in human cells. Based on binding of Rab2 to one end of HOPS, an updated model is proposed of lysosomal fusions in animal cells. It is hypothesized that GTP-loaded Rab2 is transported on Golgi-derived carrier vesicles toward Rab7 positive vesicles, and its interaction with Vps39 promotes fusions. Vps41 located on the other end of HOPS may bind Rab7 vesicles via adaptors such as PLEKHM1. These interactions help the tethering and fusion of autophagic, endocytic, and lysosomal vesicles to generate degrading compartments. Lysosomal membranes may contain active Rab2 for only a short period of time, and it likely dissociates upon GTP hydrolysis to limit organelle size. Rab asymmetry is also observed during homotypic vacuole fusion in yeast: GTP-bound Ypt7/Rab7 is necessary on only one of the vesicles, and its nucleotide status is irrelevant on the opposing membrane. Importantly, Rab7 directly interacts with both ends of HOPS in the absence of a Rab2 homolog in yeast. This difference may explain why yeast cells contain one large vacuole instead of the many smaller lysosomes seen in animal cells. Collectively, these data indicate that Rab2 and Rab7 coordinately promote autophagic and endosomal degradation and lysosome function (Lorincz, 2017).

    Complement-related regulates autophagy in neighboring cells

    Autophagy degrades cytoplasmic components and is important for development and human health. Although autophagy is known to be influenced by systemic intercellular signals, the proteins that control autophagy are largely thought to function within individual cells. This study reports that Drosophila macroglobulin complement-related (Mcr), a complement ortholog, plays an essential role during developmental cell death and inflammation by influencing autophagy in neighboring cells. This function of Mcr involves the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Interestingly, Mcr function in epithelial cells is required for macrophage autophagy and migration to epithelial wounds, a Draper-dependent process. This study reveals, unexpectedly, that complement-related from one cell regulates autophagy in neighboring cells via an ancient immune signaling program (Lin, 2017).

    Epigenetic regulation of starvation-induced autophagy in Drosophila by histone methyltransferase G9a

    Epigenetics is now emerging as a key regulation in response to various stresses. This study identified the Drosophila histone methyltransferase G9a (dG9a) as a key factor to acquire tolerance to starvation stress. The depletion of dG9a led to high sensitivity to starvation stress in adult flies, while its overexpression induced starvation stress resistance. The catalytic domain of dG9a was not required for starvation stress resistance. dG9a plays no apparent role in tolerance to other stresses including heat and oxidative stresses. Metabolomic approaches were applied to investigate global changes in the metabolome due to the loss of dG9a during starvation stress. The results obtained indicated that dG9a plays an important role in maintaining energy reservoirs including amino acid, trehalose, glycogen, and triacylglycerol levels during starvation. Further investigations on the underlying mechanisms showed that the depletion of dG9a repressed starvation-induced autophagy by controlling the expression level of Atg8a, a critical gene for the progression of autophagy, in a different manner to that in cancer cells. These results indicate a positive role for dG9a in starvation-induced autophagy (An, 2017).

    Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification

    This study shows that Mask, an Ankyrin-repeat and KH-domain containing protein, plays a key role in promoting autophagy flux and mitigating degeneration caused by protein aggregation or impaired ubiquitin-proteasome system (UPS) function. In Drosophila eye models of human tauopathy or amyotrophic lateral sclerosis diseases, loss of Mask function enhanced, while gain of Mask function mitigated, eye degenerations induced by eye-specific expression of human pathogenic MAPT/TAU or FUS proteins. The fly larval muscle, a more accessible tissue, was then used to study the underlying molecular mechanisms in vivo. Mask was found to modulate the global abundance of K48- and K63-ubiquitinated proteins by regulating macroautophagy/autophagy-lysosomal-mediated degradation, but not UPS function. Indeed, upregulation of Mask compensated the partial loss of UPS function. It was further demonstrated that Mask promotes autophagic flux by enhancing lysosomal function, and that Mask is necessary and sufficient for promoting the expression levels of the proton-pumping vacuolar (V)-type ATPases in a TFEB-independent manner. Moreover, the beneficial effects conferred by Mask expression on the UPS dysfunction and neurodegenerative models depend on intact autophagy-lysosomal pathway. These findings highlight the importance of lysosome acidification in cellular surveillance mechanisms and establish a model for exploring strategies to mitigate neurodegeneration by boosting lysosomal function (Zhu, 2017).

    Small chaperons and autophagy protected neurons from necrotic cell death

    Neuronal necrosis occurs during early phase of ischemic insult. However, knowledge of neuronal necrosis is still inadequate. To study the mechanism of neuronal necrosis, a Drosophila genetic model of neuronal necrosis was established by calcium overloading through expression of a constitutively opened cation channel mutant. This study performed further genetic screens and identified a suppressor of neuronal necrosis, CG17259, which encodes a seryl-tRNA synthetase. Loss-of-function (LOF) CG17259 activated eIF2alpha phosphorylation and subsequent up-regulation of chaperons (Hsp26 and Hsp27) and autophagy. Genetically, down-regulation of eIF2alpha phosphorylation, Hsp26/Hsp27 or autophagy reduced the protective effect of LOF CG17259, indicating they function downstream of CG17259. The protective effect of these protein degradation pathways indicated activation of a toxic protein during neuronal necrosis. The data indicated that p53 was likely one such protein, because p53 was accumulated in the necrotic neurons and down-regulation of p53 rescued necrosis. In the SH-SY5Y human cells, tunicamycin (TM), a PERK activator, promoted transcription of hsp27; and necrosis induced by glutamate could be rescued by TM, associated with reduced p53 accumulation. In an ischemic stroke model in rats, p53 protein was also increased, and TM treatment could reduce the p53 accumulation and brain damage (Lei, 2017).

    Zonda is a novel early component of the autophagy pathway in Drosophila

    Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. This study reports that a novel Drosophila immunophilin, named Zonda (CG5482), is critically required for starvation-induced autophagy. Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions, and soon after starvation nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13 and Atg17 but does not require Vps34, Vps15, Atg6 or Atg14. Zonda interacts physically with ATG1 through its kinase domain, as well as with ATG6 and Vps34. It is proposed that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation (Melani, 2017).


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    Zygotically transcribed genes

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